Mycorrhizal Symbiosis, Second Edition

Preface

Thirteen years have elapsed since the publication of the first edition and the statement made in 1983 that it was 'almost impossible for one person or even two to keep up with all the experimental work and speculation' on the subject of mycorrhizas is even more true today There has been such an enormous amount of new work that the book has been essentially rewritten and detailed reference to much early research has had to be severely reduced, although we have attempted to retain a feeling for the way the subject has developed. Again, it has been impossible to review all topics in detail and the new edition once more reflects the interests of the authors and gives our personal views of the subject. There have been at least three important changes of emphasis. Where the first edition explicitly avoided evolutionary discussions, on the grounds that insufficient information was available to make this profitable, we now believe that information from the fossil record and other new work based on molecular phylogeny of the fungi has filled in many gaps and that techniques are now available which will make this a rapidly developing area that can be addressed analytically. A second important change is the increased emphasis on the extraradical mycelium, which is of key significance in all types of mycorrhizas. Thirdly, whereas in the earlier stages of development of the subject the emphasis was on unifying ideas, there is now an increasing awareness of the diversity of structure and function, superimposed on a few basic patterns. Exposing the extent to which this diversity is important will be one of the challenges for the next phase of research. Throughout the book we have emphasized the experimental approaches that have proved useful and that may need to be applied in the future. We have retained the same general structure of the book, with four sections providing general accounts of the main types of mycorrhiza, including information on the identity of the symbionts, structure and development of the mycorrhizas formed by them, as well as their function and ecological significance. The fifth section is devoted to general themes, in which we have integrated ideas and information essential for clear understanding. These chapters build on the material presented earlier, but so that they can be read separately we have deliberately been repetitive and have also tried to improve the cross-referencing in the text. The general themes have replaced the essays in the first edition. There are several reasons for this: some material from the essays is now of central importance and is incorporated into the main chapters; some of the essays have largely served their purpose and have therefore been eliminated, so that readers interested in these topics will need to use the first edition; and not all readers (or reviewers) appreciated the essays, believing them to be too speculative. Throughout the text we

viii

Preface

have tried to indicate where future experimental research might be directed, although we realize that our personal views may have introduced some bias, which will be recognized by those who know us. Many friends and colleagues have helped us in countless ways, especially by discussions and by allowing us to use unpublished results of their work. Many have unstintingly made available original photographs, and in this context Hugues Massicotte and Diane and Jack Robertson deserve special mention. Drafts of some of the chapters have been read by Susan Barker, Gaby Delp, Lindsay Harley, Jonathan Leake, David Lewis, Francis Martin, Larry Peterson, Andrew Smith, Andy Taylor and Judy Tisdall. We are grateful for their critical and helpful comments, many of which have been incorporated into the text. However, we must take responsibility for all the ideas and views expressed whether good or bad; the errors are all ours. We have also received invaluable help in preparation of the manuscript. Kathie Stove laboured valiantly to try to attain consistency in style and presentation and corrected the worst of our grammatical errors. Jayne Young typed many drafts of chapters and produced the final manuscript, and Jan Ditchfield provided essential secretarial assistance. We also wish to thank Jennie Groom, Glyn Woods and David HoUingworth for photographic assistance and Marcus Brownlow for producing some figures. We have used much previously published material in both tables and figures. The sources are acknowledged in each case, but we wish to give special thanks to all the authors and publishers who generously allowed us to use their copyright material. We also thank The Cooperative Research Centre for Soil and Land Management, Adelaide, for providing financial support towards production of the manuscript. Last, but by no means least, our thanks and love go to the 'long-sufferers', Andrew Smith and Chris Williams, who provided invaluable moral (and immoral) support which helped to see us through the many months of largely self-centred devotion to writing. The book is dedicated to the memory of Jack Harley, whose influence upon the development of our subject was so enormous. Amongst his many contributions, two stand out as being of pre-eminent importance. Frustrated in his earlier career as an ecologist by much woolly thinking about the biology of mycorrhiza. Jack determined to subject the topic to rigorous physiological analysis. Over several decades and using as his research material the ectomycorrhizal roots of beech, he and his collaborators evaluated the basic processes whereby nutrients are exchanged between partners in the symbiosis. The second and arguably more important contribution arose out of his skill as a communicator. The Biology of Mycorrhiza, published in 1959, was the first attempt to synthesize the many and sometimes disparate strands of thought which had developed in over 100 years of research. With characteristic incisiveness he cut through much, often pedantic, debate to focus upon those questions which were in need of resolution. The work was of inestimable importance to many who, like ourselves, were struggling to take their first steps in research on this subject. These combined contributions provided impetus to a further expansion of research to the extent that by 1983 a new and even more substantial volume, the first edition of Mycorrhizal Symbiosis, was required. One of us had the privilege to collaborate in that enterprise. The influence of Jack Harley goes on. Although sadly, he has not been here to

Preface

assist us in updating the book, we have both been conscious of his legacy. Without him it is doubtful whether the subject would be in the pre-eminent position it enjoys today, increasingly recognized by physiologists and ecologists alike as being of central importance in plant and fungal biology. It is with some trepidation that we seek to emulate Jack's achievements in the production of this second edition.

INTRODUCTION

The interest in symbioses noted in the Introduction to the first edition has continued unabated in recent years. On the one hand, the importance of symbioses between different prokaryotes in the evolution of eukaryotic cells is now firmly established. On the other hand, there is increasing recognition that symbiosis at the level of more complex organisms is the rule, rather than the exception. Compared with those at the cellular level, the mutually beneficial associations between identifiably different organisms, such as those between fungus and alga in lichens, plant and fungus in most mycorrhizas, alga and coelenterate in corals, and bacteria and plant in N-fixing symbioses are of relatively recent origin, but they play exceedingly influential roles in natural ecosystems and may also be important in man-made ecosystems. The term 'symbiotismus' (symbiosis) was probably first used by Frank (1877) as a neutral term that did not imply parasitism, but was based simply on the regular coexistence of dissimilar organisms, such as is observed in lichens. De Bary (1887), who is often credited with the introduction of the term, certainly used it to signify the common life of parasite and host as well as of associations in which the organisms apparently helped each other. In time the meaning of the terms symbiosis and parasite changed. Symbiosis was used more and more for mutually beneficial associations between dissimilar organisms, and parasite and parasitism came to be almost synonymous with pathogen and pathogenesis. De Bary had pointed out that there was every conceivable gradation between the parasite that quickly destroys its victim and those that 'further and support' their partners, and in recent years biologists have come back to this view. In this book we use 'symbiosis' in the broad sense originally developed by de Bary. Mutualistic symbioses are those in which both partners can benefit from the association, although there is unresolved discussion as to what actually constitutes 'benefit', increased 'fitness' or 'aptness' for particular environmental circumstances. Most mycorrhizal symbioses are now clearly recognized, together with lichens, corals and N-fixing systems, as being common and significant representatives of the mutualistic end of the symbiotic spectrum, whereas biotrophic pathogens such as rusts and mildews are parasitic symbioses. However, as will become clear, not all of those associations that are described as mycorrhizas have been shown to be mutualistic by experimental analysis of nutritional interactions or determination of fitness. If a symbiosis is mutualistic and based on bidirectional exchange of nutrients, then the description of one partner as the 'host' seems inappropriate. In general we have adopted the terms 'plant' and 'fungus' to describe the partners in mycorrhizal symbioses, although we have retained terms such as 'host range' to describe the diversity of

2

Mycorrhizal symbiosis

plant species with which a single fungus associates. Similarly, and responding to forceful pleas by some, we have where possible substituted the (politically correct) term 'colonization' for 'infection', to avoid the suggestion of parasitic attack by the fungus on the plant. Again, where 'infection' has been used as part of an established term, as in 'infection unit', it has been retained. De Bary (1887) believed that there was some degree of common life, i.e. of symbiosis, in all or almost all examples of association between plants and fungi. The associations are often classified as biotrophic or necrotrophic, depending on whether both symbionts remain alive or the death of one was necessary before substances could be absorbed by the other. There is, however, a great range of behaviour between these two apparently clear-cut extremes, not only between different types of association but also at different times or under different environmental conditions in the same association. These variations in function, sometimes also apparent in changes in structural relationships between the symbionts, are seen in mycorrhizal associations as much as in other symbioses. One of the important features of recent research on mycorrhizas is the recognition of the considerable diversity of structure, development and function that exists even within a single mycorrhizal type. The changes that occur during the normal development of a symbiosis may be great, but until recently they have not been given much attention. Furthermore, the identity of the symbionts may have both major and minor influences on the structure (and presumably also function) of mycorrhizas formed between them. Examples include the colonization of roots by Wilcoxina, which forms ectendomycorrhizas with Pinus but ectomycorrhizas with Picea. Mycorrhizas formed by glomalean fungi can also be quite variable, the same fungus forming extensive intracellular coils and rather few arbuscules in some species of plant, and intercellular hyphae and many arbuscules in others. Many of these variations were appreciated by the early workers such as Janse (1897) and Gallaud (1905), but were forgotten as the attention of researchers was drawn to find unifying hypotheses that emphasized the structural and functional similarities of different mycorrhizal types. It is even clearer now than it was in 1983 that there is an immense diversity in what we call mycorrhizas. The description, in structural, developmental and physiological terms, of this diversity and the understanding of its importance in ecosystems are likely to be significant areas of research in the next decades. We take this opportunity to re-emphasize a point made forcibly in the Introduction to the first edition: 'It is clear that since these kinds of variation occur, much of the discussion based on classification and nomenclature is pedantic unless it is helpful in formulating clear questions which will lead to experiments from which answers will be obtained'. Although the term 'mycorrhiza' implies the association of fungi with roots, relationships called mycorrhizal associations, which are involved in the absorption of nutrients from soil, are found between hyphal fungi and the underground organs of the gametophytes of many bryophytes and pteridophytes, as well as the roots of seed plants and the sporophytes of most pteridophytes. Indeed, mycorrhizas, not roots, are the chief organs of nutrient uptake by land plants and recent work has amply confirmed the earlier observations (see Nicolson, 1975) that the earliest land plants, which had no true roots, were colonized by hyphal fungi that formed vesicles and arbuscules strikingly similar to modern vesicular-arbuscular mycorrhizas. The fossil record thus confirms previous speculation that the colonization of

Introduction

3

the land was achieved by symbiotic organisms. The location of the fungal symbiont in the root and its hyphal connections with the soil ensure that it can influence the absorption of soil-derived nutrients, while in many cases obtaining organic C as recent photosynthate from the plant. This bidirectional transfer of nutrients is the basis of mutualism in most mycorrhizal symbioses. One of the important advances in the last decade of research is the increased emphasis on the structure, organization and function of the external mycelium and the role it plays in exploitation of, and nutrient mobilization in, the soil. Mycorrhizal fungi are specialized members of the vast population of microorganisms that colonize the rhizosphere. With a few exceptions mentioned below, they are completely dependent on the plant for organic C. Being independent of the scarce and patchily distributed organic C resources in soil, they are likely to be in a good position to compete with saprotrophs in the mobilization of N, PO^ (phosphate) and other nutrients. Another vital difference between mycorrhizal associations and the general association of organisms with the root surface or rhizosphere lies in the closeness of the relationship and the recognizable and consistent structures formed. In mycorrhizas there is always some penetration of the tissues of the root by the fungus, or a recognizable structure conforming to one of the common types, or both. The difference between the mycorrhizal symbiosis and those symbioses caused by parasites which lead to disease is that the mycorrhizal condition is the normal state for most plants under most ecological conditions. With the increasing awareness of the ecological importance of mycorrhizas and their diversity, research must be directed to experiments and surveys that will elucidate quantitative aspects of the distribution of different types and their contribution to the function of ecosystems, as opposed to simple records of their occurrence or casual speculation. This is one area of research in which tools of molecular biology are already being used to good effect in identification of the fungi present in single mycorrhizal roots by DNA fingerprints. These methods have, for example, enabled confirmation of the way in which plants are linked together by their fungal associates, the same fungus being found in the roots of separate individuals of the same or different species. This approach is proving particularly illuminating for associations involving achlorophyllous plants. These mycoheterotrophic associations are mycorrhizal in the sense that they are normal (indeed essential) for the plant and are important for nutrient acquisition. They differ from most mycorrhizas, in that C movement occurs in the same direction as mineral nutrients, so that strictly the plant is primarily parasitic on its fungal associate and secondarily dependent on another (photosynthetic) mycorrhizal partner. Molecular biology is also contributing to our knowledge of the taxonomy and phylogeny of mycorrhiza-forming fungi and of ways in which changes in gene expression in both symbionts are involved in establishment and function of the symbioses at different stages of development The Introduction to the first edition raised an important point with respect to the taxonomy of the fungi and the variability to be found within a single fungal species. The potential biological significance of genetic and biochemical variations within single fungal taxa has now been appreciated and investigations are in progress which will help to unravel the genetic bases of mycorrhizal development. However, taxonomy and nomenclature constitute continuing problems in the description of mycorrhizas and in understanding their developmental and functional differences.

4

Mycorrhizal symbiosis

For example, the well known mycorrhizal basidiomycete Pisolithus tinctorius should now be called (on grounds of precedence) P. arhizus. The species is notoriously variable in many ways, including its mycorrhiza-forming abilities, and is, we understand, soon to be split into a number of species so that P. arhizus as a name for this complex array of fungi will be short-lived. Consequently, we intend to continue (incorrectly) to use the name P. tinctorius to avoid unnecessary confusion at a later date. There have been many other changes in names of mycorrhizal fungi for reasons of nomenclature, as well as changes resulting from improved understanding of the taxonomy and phylogeny of particular groups. Where the new names are clearly established we have tried to use these, in preference to those in the original papers. In some cases we have indicated both the original names and those in current use. In any event, we are aware of the potential for confusion and apologize for it.

Classification of Mycorrhizas The classification of mycorrhizas adopted here has not changed significantly since the publication of the first edition and is shown in Table 1, which is taken from that edition with minor modifications which reflect advances in knowledge made in the last decade or so. As before, the classification aims to be descriptive and to emphasize problems in need of solutions, rather than to gloss over difficulties. The kinds of mycorrhiza are divided as before, on the basis of their fungal associates, into those involving largely aseptate endophytes in the zygomycetous order Glomales (formerly the Endogonales) and those formed by septate fungi in the Ascomycetes and Basidiomycetes. The plant symbionts in mycorrhizas are so numerous and so taxonomically diverse that a primary classification on that basis would be wholly impractical. We have retained the term 'vesicular-arbuscular mycorrhiza' (VA mycorrhiza) to describe those associations formed by members of the Glomales. The unifying characters of this order are currently defined as the ability to form a mutualistic symbiosis and to produce arbuscules within the cells of compatible plants. Since vesicles are not formed by all members of this order there is a trend, advocated strongly by some, towards adoption of the term 'arbuscular mycorrhiza', to replace the well established VA mycorrhiza (see Berch, 1987; Walker, 1995). The first argument against changing the name was expressed by Walker in 1992. He doubted the wisdom of defining this group of fungi, as Morton and Benny (1990) had done, on the basis of the symbiotic ability of its members. To this we add our own doubts about the assumptions made with respect to the functions of the arbuscule by Morton and Benny (1990). Furthermore, there are problems in extrapolating from structure to function in what now appear to be structurally and possibly also functionally diverse symbioses. The second argument is more pragmatic. The explosion of experimental work in the last decade has led to the appreciation of the potential importance of mycorrhizal symbioses by scientists in a wide range of disciplines and by foresters, horticulturalists and agriculturalists in many parts of the world. They have become used to the term 'VA mycorrhiza', or VAM, and a change of name is likely to cause confusion outside the restricted circle of mycorrhizologists. We therefore retain the general term, which covers a diversity of

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6

Mycorrhizal symbiosis

structures, while recognizing that some fungi never develop vesicles and that the way in which the fungi grow within the roots may vary, especially with respect to the extent of development of coils and arbuscules within the cortical cells. The plant symbionts in this type of mycorrhiza may belong to all phyla: Bryophyta, almost all groups of Pteridophyta, all groups of Gymnospermae and the majority of families in the Angiospermae. This is the most ancient mycorrhizal type, with fossils of Aglaeophyton from the Devonian contaiiung arbuscules and vesicles. As the glomalean fimgi are unculturable it is assumed that they are wholly dependent on a photosynthetic plant, so that the mycorrhizas formed by aseptate fungi in roots of achlorophyllous members of the Burmanniaceae and Gentianaceae, about which very little is known, will be of particular interest with respect to the identity of their fungal symbionts and physiology of the associations. The septate fungi of the remaining kinds of mycorrhiza include members of almost all orders of basidiomycetes and many ascomycetes. The plant associates on which ecto-, ectendo- and ericoid mycorrhizas develop are autotrophic trees, shrubs and, rarely, herbs. Arbutoid mycorrhizas are also formed by trees and shrubs, although some of the plants, such as species of Pyrola, are herbs and are often partially achlorophyllous. The closely related Monotropaceae are all achlorophyllous and are also herbaceous. Members of the Orchidaceae are all achlorophyllous at first, but most are photosynthetic as adults. The type of mycorrhiza formed can be influenced by the identity of both plant and fungus. For example, the same fungus can form arbutoid (monotropoid) and ectomycorrhizas, or ecto- and ectendomycorrhizas, or ecto- and orchid mycorrhizas, depending on the identity of the plant associate, so that there is a plexus of behaviour amongst the species of plant and the septate fungi with regard to mycorrhizal structures that they produce. In ectomycorrhizas the fungus forms a structure called the mantle (or sheath) which encloses the rootlet. Hyphae or rhizomorphs radiate outwards from the mantle into the substrate. Hyphae also penetrate inwards between the cells of the root to form a complex intercellular system, which appears as a network of hyphae in section, called the Hartig net. There is little or no intracellular penetration. In a few plants the development of the Hartig net is slight or absent and in these it is particularly important for experiments to confirm that these associations behave in a typically mycorrhizal manner, as for example in Pisonia. In ectendomycorrhizas the sheath may be reduced or absent; the Hartig net is usually well developed, but the hyphae penetrate into the cells of the plant. As already mentioned, the same species of fungus may form ectomycorrhizas on one species of plant and ectendomycorrhizas on others. Arbutoid mycorrhizas possess sheath, external hyphae and, usually, a well developed Hartig net. In addition, there is extensive intracellular development of hyphal coils in the plant cells. The ectomycorrhizas, ectendomycorrhizas and arbutoid mycorrhizas have several features in common (see Table 1) and it might be supposed that as all the plant symbionts are photosynthetic there are grounds for grouping them all together. However, this approach would hide problems that require investigation. The development of ectendomycorrhizas and the identity of the fungal symbionts that form them is understood much more clearly now than it was in 1983. In these and in arbutoid mycorrhizas, more information on the physiology of the associations and

Introduction

7

on the factors that induce a single fungal species to produce different structures and different extents of intracellular penetration on different plants is now required. Two kinds of mycorrhiza are associated with plants that are totally achlorophyllous for the whole or part of their lives. They differ markedly in structure. The mycorrhizas formed on roots of members of the Monotropaceae are somewhat similar to the three kinds of mycorrhiza just considered, as they have a well developed fungal sheath and Hartig net. In addition, a highly specialized haustorium-like structure (the fungal peg) penetrates the epidermal cells and goes through a complicated developmental pattern as the plant grows and flowers. The fungus also forms ectomycorrhizas on neighbouring autotrophic plants, and the assumption is that organic C is transferred to the monotropoid plant, although the mechanism of transfer and the role of the fungal peg still require experimental investigation. The experimental emphasis has been on organic C as a nutrient for the plant, but the very poor development of roots makes it highly likely that the fungus is also important in mineral nutrition of these plants. In the Orchidaceae the plants are partially or wholly achlorophyllous for some part of their life. They form mycorrhizas with basidiomycetes of various affinities. Some of these are highly effective saprophytes or parasites of other plants and transfer organic C and mineral nutrients to the orchids. There is increasing evidence that some orchids are dependent on fungi that are mycorrhizal with autotrophic plants obtaining organic C from them, as well as mineral nutrients derived from the soil. In many autotrophic members of the Ericaceae and related families the hair-like roots are ermieshed in an extensive weft of hyphae, which also penetrate the cells of the root. No sheath is formed. The fungi certainly identified as forming ericoid mycorrhizas are all ascomycetes. Many ericaceous plants grow in habitats where most of the nutrients in the soil are in organic form and it is becoming increasingly clear that the fungi have a considerable role in mobilizing these nutrients and making them available to the plant. These brief descriptions make it clear that the term 'mycorrhiza' is used to describe many symbiotic associations between fungi and plants and it is important here to try to define what particular characteristics, in combination, make a mycorrhiza. These are constancy of structure, development and presence under natural conditions. Each type of mycorrhiza is likely to have its own characteristic function, and the term 'mycorrhiza' must certainly not be taken to imply that all types have the same function, or that every constant root-fungus association constitutes a mycorrhiza. Associations between fungi and roots are the norm in nature and clearly there must be some defining features of the mycorrhizal condition. At one level, mycorrhizal associations have distinctive structural attributes which are relatively easily recognized. Function is more difficult to ascribe, but some definition is required or any regular or common fungus-root association could indeed be described as mycorrhizal. A unifying feature is likely to be the role of the external hyphae in supplying soil-derived nutrients to the plant. If this criterion is adopted it will be necessary in many cases to confirm experimentally that nutrient uptake is facilitated by the fungus. Such verification should in any event be part of the process of satisfying Koch's postulates, which form the basis of assessing the status of all plant-microbe relationships. Even the definition based on nutrient acquisition may

8

Mycorrhizal symbiosis

need qualification, for some plants which become colonized in a typical manner may not respond to colonization by increases in growth or nutrient status, but by some other aspect involving changes in fitness. In these there may be other, less well established, bases for benefit, such as control of pathogens but, again, only experiments will bring us nearer to the truth. The role of C nutrition as a defining feature in the symbiosis is more difficult to assess because its source varies in the different symbioses. In some mycorrhizas the plant is autotrophic, and in these the symbiosis has the potential to be truly mutualistic with both partners deriving some nutritional benefit from the association. In others the plant is clearly non-photosynthetic and it is doubtful if these symbioses are mutualistic with respect to bidirectional nutrient transfer, although future experiments may reveal other benefits that the fungus derives from the plant. In the following chapters these types of mycorrhizas will be discussed separately in order to emphasize the facts and the outstanding problems known about each. After that, general and comparative aspects of mycorrhizal symbiosis will be discussed. The topics we have chosen are as follows: nutrient transfer between the symbionts because this is central to all mycorrhizal types; the role of mycorrhizas in ecosystems, partly because this topic was covered only briefly in the first edition and also because we now have a better appreciation of how the symbioses may influence community dynamics in natural ecosystems; the applications, actual and potential, of mycorrhizas in agriculture and horticulture, which mainly involve vesicular-arbuscular mycorrhizas; and applications in forestry, together with the impacts of pollutants in systems mainly involving ectomycorrhizal plants.

I The symbionts forming VA mycorrhizas

Introduction Vesicular-arbuscular (VA) mycorrhizas are the most common underground symbiosis. They are formed in the roots of an enormously wide variety of host plants by aseptate, obligately symbiotic fungi in the order Glomales (Zygomycotina). The plants include angiosperms, gymnosperms and pteridophytes, which all have true roots, as well as the gametophytes of some mosses, lycopods and Psilotales, which do not (see Pocock and Duckett, 1984, 1985; Peterson et a/., 1981). It seems highly likely that the fungi had their origins between 353 and 462 million years ago and that the symbiosis is similarly ancient and was probably important in the colonization of land by vascular plants (Simon et al., 1993). The name Vesicular-arbuscular' is derived from characteristic structures, the arbuscules (see Fig. 1.1) which occur within the cortical cells, and vesicles which occur within or between them. A VA mycorrhiza has three important components: the root itself, the fungal structures within the cells of the root and an extraradical mycelium in the soil. The last may be quite extensive under some conditions but does not form any vegetative pseudoparenchymatous structures comparable to the fungal sheath typical of ectomycorrhizas. A few of the fungi, however, do form sporocarps with limited amounts of sterile mycelium (Fig. 1.2; Plate 1). The majority (about 80%) of species presently described form both arbuscules and vesicles. The remainder do not form vesicles and should therefore strictly be called 'arbuscular' mycorrhizal fungi. In some plant species the fungus grows intercellularly and intracellular hyphal coils are restricted to a few cell types only; in others, the same fungi may form abundant coils in the cortical cells of the root and in these mycorrhizas the development of arbuscules may be reduced. Because the characteristic fungal structures develop within the root and changes in the rates of root growth and branching are discernible only by detailed comparison with noncolonized plants, it is usually impossible to tell whether or not a root system is mycorrhizal without staining and microscopic examination. A few plants, such as Allium and Zea, do synthesize a yellow pigment when colonized but this is not sufficiently frequent to be a generally useful diagnostic character.

12

Vesicular-arbuscular mycorrhizas

Figure I.I A mature arbuscule of Glomus mosseae within a cortical cell of Allium porrum (leek). The arbuscule has grown from a well developed intercellular hypha (arrow). From Brundrett et al. (1984), with permission.

VA mycorrhizas were first recognized and described in the last decades of the nineteenth century. Their widespread occurrence and common presence in plants of many phyla in most parts of the world, especially in the tropics, was realized very soon, but very little functional information was learned about them until the mid1950s. The early work was reviewed by Rayner (1927) but she discussed the nature of the fungal symbiont without reaching a convincing conclusion. Indeed, much of the effort put into research on this type of mycorrhiza during the 1920s and 1930s was vitiated by the relative ease with which fungal inhabitants of the root surface and of senescing cells could be isolated into culture and the difficulty, still unsurmounted, of isolating the fungal symbionts themselves. Almost all writings about the identity of the fungus until 1953 may be ignored, except for those of Peyronel who, in 1923, showed that the hyphae of the endophyte might be traced to the sporocarps of species of Endogonaceae in the surrounding soil, and of Butler (1939) who, in an influential review, agreed that the fungi called Rhizophagus were almost certainly imperfect members of the Endogonaceae, which then included all fungi now transferred to the orders Glomales and Endogonales. The work of Mosse (1953), which showed convincingly that mycorrhizal strawberry plants were colonized by a species of Endogone (later transferred to Glomus), may be said to have heralded the modem period. Soon Mosse, Baylis, Gerdemann, Nicolson, and Daft and Nicolson greatly extended these early observations and demonstrated by inoculation that endogonaceous (glomalean) fimgi were symbiotic with many kinds of plants forming the so-called phycomycetous or VA endomycorrhizas. A major milestone was reached in 1974 with a sucessful symposium on endomycorrhizas at which a number of key ideas were developed for the first time. Many of the papers presented at that meeting remain classics (see Sanders et al., 1975). At about this time the first formal Linnean classification of the species was

The symbionts forming VA mycorrhizas

13

developed (Gerdemann and Trappe, 1974) and many general aspects of development and function of this widespread symbiosis were outlined and discussed in the first edition (Harley and Smith, 1983). While more recent work has extended and confirmed the generalizations made at that time, there has also been an increased appreciation of the diversity to be found in VA mycorrhizas and an important new emphasis on the cellular and molecular interactions between the symbionts (see Giovannetti and Gianinazzi-Pearson, 1994; Smith, 1995). In brief, VA mycorrhizal fungi have been recognized as ecologically obligate symbionts of a very wide range of plant species. The symbiosis is biotrophic and normally mutualistic, the long-term compatible interaction being based on bidirectional nutrient transfer between the symbionts. Unlike the biotrophic parasitic symbioses, these associations show a very low degree of taxonomic specificity, a point which will be developed later in this chapter. Because the fungi have very limited capacity for growth from propagules such as spores or from the vesicles or hyphae within root fragments, special methods have had to be adopted to maintain pure strains for experimental or taxonomic purposes. As far as is possible, isolates from single spore types are grown in 'pot culture' on the roots of plants, so that their spore characteristics, mode of colonization and effects on plant growth can be followed by sequential sampling. However, in many cases spore collections from soil form the only basis for taxonomic study.

Fungi Systematics Until relatively recently the causal organisms of VA mycorrhizas were classified in the family Endogonaceae of the order Endogonales. The regular association of the very large spores and sporocarps of members of this family with VA mycorrhizal roots was established long ago by Peyronel (1923). Only later, after the work of Butler (1939) and most especially Mosse (1953, 1956), were they recognized as the chief causal organisms of VA mycorrhizas. This recognition prompted renewed interest in the taxonomy of the family, based mainly on the development, morphology and wall structure of the globose zygospores, azygospores and chlamydospores, and of sporangia (see Fig. 1.2 and Plate 1). Except for zygospores produced by the Endogonaceae {sensu stricto) it is frequently difficult for the type of spore to be determined with confidence and there is disagreement among the experts on the appropriate nomenclature. To avoid such problems we refer simply to 'spores', while recognizing that their development in different taxa varies. Interest in mycorrhizas has led to the realization that members of this family are among the most common soil fungi and that spores or sporocarps can be collected from almost any soil. As increasing numbers of fungi with very large spores (up to 500 |im in diameter) were collected and described, the genus Endogone grew into an unwieldy and variable assemblage of species about which few generalizations could be made. The Linnean classification of the Endogonaceae (Gerdemann and Trappe, 1974, 1975) made no attempt to relate taxonomy to the phylogeny of the group. The family contained seven genera. Endogone sensu lato

14

Vesicular-arbuscular mycorrhizas

Figure 1.2 (a) Spore and subtending hypha of Glomus invermaius. Spore diameter approximately 75 |Lim. (b) Sporocarp of SclerocysHs rubiformis (slightly squashed). Diameter of Individual spores approximately 45 |Lim. From Hall and Abbott (1981), with permission.

was split and only those fungi forming true zygospores were retained within Endogone sensu stricto. Some of these can be cultured and they appear either to be non-mycorrhizal saprophytes or to form ectomycorrhizas; none form VA mycorrhizas. Members of the genera Gigaspora, Acaulospora, Glomus and Sclerocystis form VA mycorrhizas; Glaziella and Modicella were considered at that time to have unknown affinities. Gerdemann and Trappe (1975) regarded their revision of the Endogonaceae as a 'temporary solution to a difficult taxonomic problem', but it was important because it finally put the study of VA mycorrhiza on a firm taxonomic basis. Both dichotomous (Mosse and Bowen, 1968; Hall and Fish, 1979; Hall, 1984) and synoptic (e.g. Trappe, 1982) keys have been produced to help identification. In a revision of the classification, Modicella and Complexipes (a later addition) were removed by Trappe and Schenck (1982), while Glaziella was found to have ascomycetous affinities (see Walker, 1987). By 1993 about 150 species had been described, although early descriptions are in many cases unsatisfactory and revisions are to be expected. The species are widely distributed globally, in accordance with their probable ancient origins. The classification of VA mycorrhizal fungi is at present based almost exclusively on the structure and development of the walls of the spores, so that murographs (Fig. 1.3) are an important component of taxonomic descriptions (Walker, 1983, 1992; Morton and Benny, 1990; Morton and Bentivenga, 1994), irrespective of whether

The symbionts forming VA mycorrhizas

15

iw2 iwl

l ^ ^ Colour A " ^ Spore size Wall thickness

M 1 ^ Colour / y ^ Spore size r ^ Wall thickness Ornamentations

Figure 1.3 Discrete stages in differentiation of subcellular structures, characters and character states in Gigaspora and selected Scutellospora species, (a) Murographic representation of five stages in differentiation of spores of S. heterogama. Spore wall, sw; first inner wall, iwl; second inner wall, i w l ; germination shield, gs; ornamentation character state, 0. Patterns indicate characters of each structure: no pattern indicates the outer layer; vertical dashed lines, laminae; angled lines, flexible layers, (b) Murographs illustrating phenotypes of adult spores. Left, Gigaspora species and right, five species of Scutellospora, Within the genera each species is separated by different character states of the spore walls (arrows). From Morton and Bentivenga (1994), with permission.

identification is the main priority or phylogenetic conclusions are sought (Morton, 1990a). There is no doubt that analyses of DNA sequences and other biochemical characteristics such as fatty acid methyl ester (FAME) profiles will complement the morphological information to a greater and greater extent (Graham et ah, 1995; Morton et al, 1995). The most recent revision of the classification represents another important stage in the development of taxonomy of VA mycorrhizal fungi by taking a phylogenetic perspective (Morton and Benny, 1990). This revised classification separates a new order, Glomales, from the Endogonales. The latter contains a single family of truly zygosporic fungi in the genera Endogone and Sclerogone. The Glomales, as we said before, is now defined as containing only those fungi for which 'C is acquired obligately from their host plants via intraradical dichotomously branching arbuscules' (Morton and Benny 1990). This seems a very restrictive definition as it makes a number of assumptions which have not yet been substantiated physiologically (see Gianinazzi-Pearson et al., 1991a; Smith and Smith, 1996a) and also creates some

16

Vesicular-arbuscular mycorrhizas

practical difficulties. It is assumed that the arbuscule is a key unifying structure and that it is the site of C acquisition by the fungi. This is by no means certain, for there is no a priori reason why intercellular hyphae or intracellular coils should not be sites of C transfer (see Chapter 14). Further, fungi with typical spore development and morphology may exist but have 'atypical' physiology with respect to C transfer. Variation in intraradical development may be induced by host species, or may vary with the age of the plant. Indeed, one of the greatest practical difficulties arising from the use of the arbuscule as the key feature is that in this case all descriptions of fungal species must be accompanied by evidence that the fungi will form arbuscules. This problem has already led Morton (1990a) to include only fungi for which there is visual evidence of their ability to form mycorrhizas (perhaps 40% of described species) in his review of evolutionary relationships. Notwithstanding these concerns, which have also been voiced by others (Hall, 1984; Berch, 1987; Walker, 1992), the revision is an important step forward, as it provides a classification based on particular assumptions about the phylogeny of the group which can be tested through other approaches. Steussy (1992) has emphasized the importance of independent methods, such as nucleic acid sequencing, for determining phylogenetic relationships, bearing in mind particularly the difficulties of determining which characters are primitive and which are advanced in the absence of an extensive fossil record. One point on which all agree is the need for mycorrhizal workers to keep voucher specimens of the fungi used for all investigations, whether they be taxonomic or physiological. The Glomales as currently defined contains two suborders, the Glomineae and the Gigasporineae (Table 1.1). In all members the vegetative mycelium and intraradical structures are aseptate and multinucleate. The spores themselves contain 1000 nuclei, or even more in some species, but the extent of heterokaryosis is unknown at present. The spores germinate and produce limited mycelium in the absence of host plants. At one stage it was thought that nuclear division might not occur until a host plant had been successfully colonized and that this might go some way to explaining the failure of the fungi to grow for prolonged periods in axenic culture (Burggraaf and Beringer, 1987, 1989). Two independent lines of investigation have now made it clear that DNA replication and nuclear division do occur in germ tubes of Gigaspora margarita (Bianciotto and Bonfante, 1992; Becard and Pfeffer, 1993). However, no evidence for the occurrence of nuclear fusion and meiosis has been obtained, so it would appear that the parasexual cycle does not operate at this stage of the life cycle of this fungus, if at all. Zygospore formation has been briefly described for one species only, Gigaspora decipiens, and exchange of nuclei via anastomoses between species and, indeed, between different isolates of the same species, is likely to occur at a low rate despite the fact that the mycelium of a single isolate anastomoses frequently (Tommerup, 1988; Tommerup and Sivasithamparam, 1990). It therefore seems likely that the members of the Glomales may be asexual organisms. We must assume that mutation and possibly heterokaryosis provide the main bases for the variation necessary to permit adaptation to environmental change and continuing evolution. Certainly, variations in DNA sequences have been revealed by amplification of genon\ic DNA using short arbitrary primers (random amplified polymorphic DNA-polymerase chain reaction; RAPD-PCR). These RAPDs show similarities of between 77% and 89% for different spores

17

The symbionts forming VA mycorrhizas

Table I.I

Ordinal and family structure of the Endogonales

Taxon

Characteristics

Order: Endogonales

Reproduction by zygospores, frequendy produced in sporocarps Saprophytic or forming associations with roots similar to ectomycorrhizas

Family Endogonaceae Genera: Endogone (14), Sderogone (I) Order: Glomales Suborder: Glomineae Family: Glomaceae Genera: Glomus (77) Sclerocystis (10)

Family: Acaulosporaceae Genera : Acaulospora (32) Entrophospora (3) Suborder: Gigasporineae

Family: Gigasporaceae Genera: Gigaspora (7); Scutellospora (23)

Obligately biotrophic fungi, obtaining C from their host plants via intraradical arbuscules Vesicles and arbuscules Spores produced terminally or laterally Auxiliary cells absent Spores produced singly or in loose aggregates Sporocarps not as for Sclerocystis Fruiting body a sporocarp composed of spores with lateral walls adhering to one another Base of hyphae sterile Spores formed on or within the neck of a specialized sporiferous saccule Arbuscules only, vesicles absent Spores with bulbous attachment Auxiliary cells formed (usually) on external hyphae Genera separated on: Mode of germination of spores Presence of a flexible wall group in Scutellospora Differences in the ornamentation of auxiliary cells.

The features of the orders are from Morton and Benny (1990) although the inclusion does not indicate agreement (see text). Numbers in parentheses after generic nouns are the numbers of species in each genus taken from Walker and Trappe (1993). Spelling of the names is also from Walker and Trappe (1993).

from the same (Rothamsted) line of Glomus mosseae, 60-76% similarity for spores of the same species from the same geographical region, 44-64% for spores of the same species from different regions and only 9-29% similarity between different species (Wyss and Bonfante, 1993). Considerable variation has also been revealed by isozyme banding patterns, which did not necessarily coincide with species defined by spore morphology (Hepper et al., 1988; Rosendahl, 1989). The relative contributions to this variation made by mutation and exchange of genetic material are not known at present and more work on the potential for exchange, for example within the roots of plants colonized by mixed infections, might be productive. With the rapid adoption of methods to analyse DNA sequences in VA mycorrhizal fungi we are likely to see considerable advances in knowledge of intra- and inter-specific variation and extent of clonality in the near future. It has been argued by Law and Lewis (1983) that in mutualistic symbioses the endobiont (in this case the fungus)

18

Vesicular-arbuscular mycorrhizas

will evolve away from a sexual habit because the selection pressures will be to maintain similarity to - rather than difference from - the parents. This certainly seems to fit with the situation in glomalean fungi: the important ecological niche for C acquisition is the apoplast of the root cortex, in which homeostasis exercised by the plant will be important in maintaining extremely constant environmental conditions compared with the variation likely in the soil. The possibly asexual nature of VA mycorrhizal fungi means that the described taxa cannot be regarded as either biological or ecological species, but rather as phenetic or form species (Morton, 1990b; Walker, 1992). Apart from the use of arbuscules in the current definition of the Glomales, the vegetative structures, together with aspects of spore morphology that vary quantitatively (shape, size, colour), have been excluded from taxonomic or phylogenetic considerations. Characteristics of the vegetative stages, such as form of the entry points and branching of hyphae within the root, are variable between fungal species and can be used for recognition purposes by experienced observers (see Abbott 1982; Lopez-Aguillon and Mosse, 1987). Most of the species of fungi listed above produce 'coarse' colonizations in which intercellular hyphae are 5-10 |im or more in diameter. These can be easily distinguished from 'fine' colonizations, with very narrow hyphae (1-3 |Lim diameter), which have been assigned to Glomus tenuis (Hall, 1977). The small spores of the latter (diameter 10 |xm) were long overlooked and its taxonomic position is still somewhat doubtful. The 'fine endophyte' is, however, extremely common in many soils and the problems that it poses are not only taxonomic but also ecological and physiological.

Fossil History and Phylogeny of Glomalean Fungi Fossils resembling the spores of VA mycorrhizal fungi have long been recognized and date from as early as the Silurian (440-410 million years BP). While these records are doubtful or possibly the result of contamination of samples, the extensive records of both spores and structures from within plant axes or decaying plant material from the famous Rhynie chert flora (Kidston and Lang, 1921) are much more satisfactory and provide compelling evidence for the existence of symbiosis between plants and Glomus-like fungi as early as 410-360 million years BP. Recent re-examination of the Rhynie chert has revealed arbuscules within the protosteles of Aglaophyton major, which should leave us in no doubt that VA or glomalean mycorrhizas had evolved by that time (Remy et al., 1994; see Fig. 1.4a). The significance of these fossils was noted by Nicolson (1975) and subsequently Pirozynski and Dalpe (1989) have provided a critical review of the geological history of the group. This shows continuous occurrence of Glomuslike structures into the quaternary period and the occurrence of Glomus- and SclerocystiS'Vike spores (as well as intraradical colonization, including arbuscules) from silicified peat from the Triassic deposits in the Antarctic (Stubblefield et ah, 1987a,b,c; Fig. 1.4b,c,d). Unfortunately, no reliable fossils of other glomalean taxa have been found which would shed light on phylogenetic origins of the group. Interestingly, the arbuscules in A. major (which seems to have affinities to both bryophytes and vascular plants) are delicate structures with secondary branches of 1-2 |Lim. They are therefore similar to present-day arbuscules, but different

The symbionts forming VA mycorrhizas

19

Figure 1.4 Fossil VA mycorrhizas. (a) Arbuscule (A) in a cell of Aglaeophyton from the Devonian flora of the Rhynie chert. From Remy et al. (1994). Copyright, National Academy of Sciences, USA. (b) Transverse section of a mycorrhlzal root of AntarcticycaSy from the Triassic deposits of Antarctica. Note the colonized central cortex of the root. (c),(d) Details of colonization in Antaraicycas, (c) Dichotomously branched arbuscule (A), with relatively robust branches; (d) vesicle (V). (b),(c),(d) from Stubblefield et al. (1987b), with permission.

from the robust arbuscules found in Antarcticycas from the Triassic (compare Figs 1.1 and 1.4a,c). The first approach to a classification representing phylogentic relationships has been made by Morton (1990a), using cladistic tools and assuming evolutionary significance for 27 characters used in the analysis. The basic assumptions, which as noted earlier may be questionable, are that all glomalean fungi form mutualistic associations and produce arbuscules and that these characters unite them in a monophyletic group. The remaining characters (with the exception of unexplained variations in wall staining with Trypan Blue) are based on spore characteristics that

Vesicular-arbuscular mycorrhizas

20

Glomus

Acaulospora

Entrophospora Gigaspora

Scutellospora

Bilayered flexible inner wall(s) Pregermination shield Knobby auxiliary cells

Spore terminal on sporogenous hypha(e)

Permanent outer layer enclosing laminae of spore wall

Evanescent layer enclosing laminae of spore wall

Sporogenous cell Extraradical auxiliary cells

Intraradical vesicles

Hyphae with knobs and projections, often coiled

Hyphae cylindrical, often with perpendicular branching

(a)

Arbuscules (monophyletic origin?)

I

li

iii

100

rt

Entrophospora sp.

A. rugosa

A. rugosa

95 ^GL 60 97

1971 96

gigantea

l—G/. albida

64

B 100

10 steps

Gi. gigantea

— S. dipapillosa

—G. etunicatum

— G. etunicatum

G. mossae G. intraradices

^ 50

Gi. albida

L - Gi. albida

— S. dipapillosa

- Endogone pisiformis (b)

|— Gi. gigantea \

k

— S. pellucida

99

i

I—Gi. margarita

p^ Gi. margarita

— S . pellucida

100

- £ Columbiana - A. spinosa

9 o L A. spinosa

r^GL margarita

p - A. rugosa

n

Entrophospora sp.

ioo\J

9 o L A. spinosa

75

r

£ Columbiana

£ Columbiana

100

r

G. mossae f. intraradices

L-G.

i

S. dipapillosa S. pellucida

G. etunicatum

m

G. mossae

G. intraradices

Endogone pisiformis

1 substitution/100 nucleotides

1 step

F i g u r e 1.5 Phylogenetic trees f o r V A mycorrhizal fungi, derived f r o m different types o f i n f o r m a t i o n , (a) A cladogram showing t a x o n o m i c and phylogenetic divergence among genera of VA mycorrhizal fungi, based on comparative developmental sequences of t h e spores. Previously unpublished, courtesy of J.B. M o r t o n , (b) Phylogenetic trees of VA mycorrhizal fungi, based on sequences of small subunit r R N A . (i), (ii), (iii) Different trees obtained by using different methods o f analysis. From Simon et al. (1993). Reprinted w i t h permission f r o m Nature, 3 6 3 , 6 7 - 6 9 . Copyright Macmillan Magazines Ltd.

The symbionts forming VA mycorrhizas

21

vary qualitatively and are stable and discrete. Continuously variable characters, such as spore colour and size, are not used. Application of the cladistic approach to determination of evolutionary relationships has yielded the phylogentic tree shown in Figure 1.5a, which can serve as a hypothesis for future investigations. The key features are the separation of the Glomus/Sclerocystis group from Gigaspora/Scutellospora and the existence of Acaulospora/Entrophospora as a line apparently diverging from Glomus. If the assumptions on primitive and advanced characters are correct, Glomus/Sclerocystis represent the ancestral type, as shown in Figure 1.5a, with the other two major lines being more recent in origin. However, the cladistic analysis puts Scutellospora as more highly evolved than Gigaspora, a point which is disputed by Walker (1992) largely on the grounds that Scutellospora has a more restricted present-day range. There is no fossil evidence to help distinguish between these possibilities, but DNA sequence analysis indicates that Gigaspora and Glomus are in a monophyletic group (Bruns, 1992) and also puts Scutellospora ancestral to Gigaspora (Simon et ah, 1993). The molecular information is very important because it provides an independent means of investigating the phylogenetic hypotheses based on cladistics. Sequences of some sections of ribosomal genes have been obtained from 12 species of glomalean fungi and from Endogone pisiformis (Endogonaceae) as an outlier. Three methods of analysing similarities in DNA sequences provided essentially similar phylogenetic trees (Fig. 1.5b) which confirm the glomalean fungi as true fungi of monophyletic origin, divided into the same three families shown by the cladisitic approach. Lipid analysis also supports the existence of three families (Sancholle and Dalpe, 1993), but analysis of the carbohydrates in the fungal walls suggests a less clearcut phylogeny. A large number of investigations have shown that all the walls contain chitin, but the occurrence of p(l,3)glucans in members of the Glomineae and not in the Gigasporineae, suggests that only the latter group (together with Endogone) can be regarded as true Zygomycetes, while Glomus and Acaulospora are more similar to the Entomophthorales in having both chitin and p(l,3)glucan in their walls (Gianinazzi-Pearson et ah, 1994b; Lemoine et ah, 1995). Application of a range of techniques to evaluate the diversity to be found in the Glomales should soon sort out these apparent discrepancies (see van Tuinen et ah, 1994). The approximate dates for the origin of the group and the divergence of the major branches given by the DNA sequence data provide a link with the palaeontological information. The analysis puts the origin of the glomalean fungi via divergence from the group represented by E. pisiformis in the Devonian between 462 and 353 million years BP and the divergence between Glomaceae and the other groups in the late Palaeozoic 250 million years BP. An origin of G/omws-like fungi at the same time as the origin of the land plants (dated at 415 million years BP) is in agreement with the fossil record and provides some support for the theories that colonization of the land by plants such as Aglaophyton {Rhynia), with restricted absorbing axes, may have been dependent on their association with mycorrhizal fungi which increased their capacity for nutrient absorption from poor soils (Pirozynski and Malloch, 1975; Raven et ah, 1978; see Fig. 1.6).

22

Vesicular-arbuscular mycorrhizas Origin of VAMfu ngi •

1

Events A

h

B

D

C

1 II H — i—1 II

11

1 1

II

II

1 Land plants

c CO o

c CO

*s

E (0

"S O

O

c (0 "c

c

i

(75

Q I

?

500

400

Estimated dates of divergence

i-H

H

| Monocots-dicots

V) 13

c

2

CO

o

o

1

"E

o -G

Landmarks

(0 (0

o

-3

»2

Geological 1 epoch

(Q

O

I

I

300

200

O 1 100

Present

Time (million years)

Figure 1.6 Estimated dates of origin and divergence of VA mycorrhizal (VAM) fungi. From Simon et o/. (1993). Reprinted with permission from Nature, 363, 67-69. Copyright Macmillan Magazines Ltd.

Host Plants Systematics The range of potential host plants for VA mycorrhizal fungi is extremely wide and has been responsible for the oft-quoted statement (Gerdemann, 1968) that 'it is so ubiquitous that it is easier to list the plant families in which it is not known to occur than to compile a list of families in which it has been found'. This continues to hold good. Some members of most families of angiosperms and gymnosperms, together with ferns, lycopods and bryophytes, develop VA infections. Trappe (1987) has produced a most valuable compilation of the incidence of all types of mycorrhizas within the angiosperms, taken from published material. Records of VA mycorrhizas are to be found in all the orders from which plants have been examined and are about equally frequent in Dicotyledonae and Monocotyledonae. He stresses that only about 3% of species have actually been examined and our knowledge of the mycorrhizal status of some taxa is very poor indeed. Consequently, it can be said that about 95% of the present-day species of plants belong to families that are characteristically mycorrhizal. But it cannot be said that 95% of the world's species are mycorrhizal: such sweeping and inaccurate statements should be avoided (see Trappe, 1987). Futhermore, only single specimens of some species have been examined, and in these cases generalizations are risky because of variations in the extent of mycorrhizal colonization between sites and at different seasons. Nevertheless, the more we look the greater the number of species that prove to be mycorrhizal. Harley and Harley (1987) have surveyed the literature on the incidence of mycorrhizas at the species level in the very-well-studied British flora. For many families, over 40% and sometimes as high as 80% of the species have been investigated, often more than once. All families listed contained mycorrhizal species, and these frequently constituted a very high proportion of the total.

The symbionts forming VA mycorrhizas

23

Even in families widely thought to be 'non-mycorrhizal', such as the Polygonaceae, Juncaceae, Cruciferae and Caryophyllaceae, mycorrhizas were found, although their presence was not consistent and the colonization often sparse. As in other surveys (e.g. Newman and Reddell, 1987), some species have been recorded as occurring in both mycorrhizal and non-mycorrhizal states and members of some plant families characteristically form mycorrhizas of other types. VA mycorrhizas are found in most herbaceous plants that have been studied (see above for exceptions) but are by no means restricted to herbs. As long ago as 1897 Janse examined 46 species of tree in Java and found them all to have VA mycorrhiza. More recent work, reviewed by Smits (1992) and Janos (1987) confirms this. The Dipterocarpaceae appears to be the only family of tropical trees which are typically ectomycorrhizal. Otherwise, VA mycorrhizas predominate in these taxonomically diverse systems as well as in some temperate forest systems. Thus, Baylis (1961,1962) states that VA mycorrhizas are ecologically the most important type of mycorrhiza in New Zealand forests. Whereas the Pinaceae are ectomycorrhizal, all other conifer families are dominantly VA mycorrhizal, as are most other gymnosperms, all of which are woody. Although VA mycorrhizas have often been ignored by foresters in the past, they are characteristic of such valuable trees as Araucaria, Podocarpus and Agathis as well as all the Cupressaceae, Taxodiaceae, Taxaceae, Cephalotaxaceae and the majority of tropical hardwoods. The importance of considering the appropriate mycorrhizal associates for trees used in reafforestation programmes in temperate and tropical ecosystems is now widely recognized. While most of the experimental work on VA mycorrhiza has been done with herbs, because these are easier to manage under laboratory conditions, some trees have also been used and these include Malus (apple). Citrus, Salix, Populus, Persea (avocado), Coffea, Araucaria, Khaya, Anacardium (cashew) and Liquidambar. It is certainly important to realize that mycorrhizas may be significant in nutrient absorption and in nutrient cycling of arborescent species in forest ecosystems. Work with trees and other perennials is therefore very important both from an ecological point of view and from a need to consider forest and crop production. Indeed, although work with herbs allows greater control of conditions in growth rooms, etc., the propagation of some woody species from cuttings may have great advantages in providing genetically uniform experimental material which may partly offset the long growth periods necessary for the study of long-lived plants. The extensive work on Citrus mycorrhiza by groups in California and Florida is an example of an arborescent species of economic importance being used in experiments designed both to increase crop productivity and to promote an understanding of the development and physiology of the symbiosis. The Fossil History of Mycorrhizal Colonization As in the case of the spores mentioned above, the long fossil history of fungal infections in the absorbing organs of plants is well recognized. The earliest known land plants did not possess true roots, but the protostelic rhizomes of Aglaophyton (Rhynia) and Asteroxylon were clearly infected by fungi (called Palaeomyces) which formed arbuscules, intercellular hyphae and vesicles like modem members of the Glomales (Kidston and Lang, 1921; Remy et ah, 1994; and see Pirozynski and Dalpe, 1989; see Fig. 1.4a).

24

Vesicular-arbuscular mycorrhizas

Many gymnosperm fossils with VA mycorrhizas have been identified in later Carboniferous deposits. The best known and preserved is Amyelon radicans, which again resembled the VA mycorrhizas of living gymnosperms (see Nicolson, 1975). The Triassic flora from Antarctica (250-210 million years BP) has also yielded important evidence for the development of intraradical vegetative structures, including both intercellular hyphae and arbuscules (Stubblefield et al., 1987a,b; see Fig. 1.4b,c,d). Beautifully preserved roots of Antarcticycas, containing both septate and aseptate hyphae and structures strongly resembling mycorrhizal arbuscules, vesicles and spores, have been described by Stubblefield et al., (1987a). Sections and peels of these fossils are virtually indistinguishable from presentday mycorrhizal cycad roots. It is impossible, of course, to be sure about the physiology of these fossil mycorrhizas, but if they functioned in a manner similar to present-day forms their role in colonization of the land and in subsequent plant evolution may have been considerable. The view has been put forward that the soil available to early land plants is likely to have been deficient in available mineral nutrients, so that the intervention of the fungi in their absorption might well have been important to the success of the plants invading the terrestrial environment (Baylis, 1972b; Nicolson 1975; Pirozynski and Malloch, 1975; Raven et al, 1978). In contrast, ericoid and ectomycorrhizas, which are more typical of communities growing on organic soils are envisaged as originating more recently than VA mycorrhizas, as soil organic matter increased. Recent worldwide surveys have greatly increased the known number of plants that form more than one kind of mycorrhiza. Both VA and ectomycorrhizas are most commonly reported in the Rosaceae and Salicaceae; they also occur in at least 10 other angiosperm families, including the Papilionaceae and Rhanmaceae, and occasionally in the Gymnospermae and Pteridophyta (Newman and Reddell, 1987; Trappe, 1987; Brundrett and Abbott 1991; see Table 1.2). In some cases VA mycorrhizal colonization has been reported on young individuals of species usually forming ectomycorrhizas, for example Eucalyptus, Pseudotsuga and Tsuga (Lapeyrie and Chilvers, 1985; Chilvers et al, 1987; Cazares and Smith, 1992,1996). Indeed, it is possible that VA mycorrhizal fungi may have the ability to invade the underground organs of almost all land plants. Such an attribute would explain why the long coevolution of the symbionts has not resulted in the specialization of the fungi to their host range nor in taxonomic specificity of the symbioses (Table 1.3). The form of the root system is important in influencing the extent to which plants respond, in nutrient absorption and growth, to mycorrhizal colonization. Plants bearing magnolioid type roots, characterized by wide axes (up to 1.5 mm in diameter), slow growth and poor root-hair development, are frequently highly responsive, while plants with fine, rapidly growing root systems and long root hairs are not (Baylis, 1975; St John, 1980). The importance of mycorrhizas for growth and nutrient absorption will be discussed in Chapters 4 and 5. Here the important point is that the woody Magnoliales (with magnolioid root systems) which are considered to be the most primitive living angiosperms (Cronquist, 1981), being ancestral to the other dicotyledons, have a huge incidence of species forming VA mycorrhizas and relatively few which are characteristically non-mycorrhizal or bear other mycorrhizal types (see Trappe, 1987). This frequency is greater than that observed in other n\ore advanced dicotyledonous subclasses, as can be seen in Figure 1.7 and Table 1.2.

The symbionts forming VA mycorrhizas

25

Table 1.2 Numbers and percentages of species of subclasses and classes of Angiospermae examined for mycorrhiza formation and percentage of exammed species by type of mycorrhiza Per cent with mycorrhiza types Taxon

Total species

Per cent VAionly Species examined examined

Division Angiospermae Class Dicotyledonae Subclass Magnoliidae Hamamelidae Caryophyllidae Dilleniidae Rosidae Asteridae

223400 173500

6507 5020

3 3

50 50

15 14

5 6

18 17

12000 3400 I I 000 25000 62100 60000

270 265 317 792 1838 1538

2 8 3 3 3 3

66 27 14 33 56 63

3 44 4 29 16 2

4 II 2 7 5 5

17 6 59 20 12 15

Class Monocotyledonae Subclass Alimatidae Arecidae Commelinidae Zingiberidae Lilliidae

49900

1487

3

49

18

2

21

500 5600 15000 3800 25000

26 61 826 28 546

5 1 6 1 2

4 56 55 7! 37

0 3 1 4 48

0 3 2 0 2

88 30 28 II 7

Other

VA plus other

Nonmycorrhizal

Other: mycorrhizas formed by ascomycetes and basidiomycetes Based on Trappe (1987).

Using only those taxa for which the mycorrhizal status is known in at least 10% of the species, and using the phylogenetic classifications of Cronquist (1981), Trappe has prepared dendrograms which allow some preliminary evolutionary conclusions to be drawn. A high incidence of VA mycorrhizas appears to have been retained in the line through the Rosidae to the Asteridae, which show relatively low incidence of other, supposedly advanced, mycorrhizal types. The line to the Caryophyllidae shows a general reduction in the incidence of any type of mycorrhiza while the line through the Hamamelidae to the present day Juglandales and Fagales shows a marked increase in ectomycorrhizas. A more detailed approach is taken within these phylogenetic lines, where data are available, and they provide fascinating hypotheses which can be investigated in future collections. The present tentative conclusions agree with the fossil record in placing VA mycorrhizas as primitive and the other mycorrhizal types as more advanced. Within the Monocotyledonae all lines (considered to be parallel by Cronquist, 1981) are heavily mycorrhizal and VA mycorrhizas predominate except in the Orchidaceae (Liliidae) which have characteristic and probably advanced orchidaceous mycorrhizas formed by Basidiomycetes (see Chapter 13). One of the important conclusions of this work is that the non-mycorrhizal condition has evolved several times in different phylogenetic lines and may therefore have different cellular and physiological bases. Although families containing

26

Vesicular-arbuscular mycorrhizas Juglandales

©A0

Fagales Asteridae

13)/oA [T| 94\ Rosidae

Urticales

©A0

©Ati] -Dilleniidae

©As Hamamelidae-

— Caryophyllidae

©AS Ma£rno///dae

©A[ii] F i g u r e 1.7 Phyologenetic dendrogram for the subclasses of Dicotyledonae, showing the percentage of species w i t h zygomycetous (VA) mycorrhizas (numbers in circles), asco- and basidiomycetous (ericoid and ecto-) mycorrhizas (numbers in triangles), o r no mycorrhizas (numbers in squares). Many species have mycorrhizas in m o r e than one category, so that percentages total m o r e than 100. Reprinted w i t h permission f r o m Trappe (1987) in Ecophysiology of VA. Mycorrhizal Plants. CRC Press, Boca Raton FL.

large numbers of species in which mycorrhizal colonization is characteristically absent are relatively rare, they are worth studying in their own right for mechanisms by which the fungi are excluded, as well as the means by which they acquire nutrients from soil (Lamont, 1981,1982; Pate, 1994; Marschner, 1995; see Chapter 3).

Specificity and Extent of Colonization So far we have confined the discussion to the potential of different taxa of plants to form mycorrhizas in field or experimental conditions, without concerning ourselves greatly with questions about whether or not a species is always mycorrhizal, how extensively the roots are colonized, or how far it may be dependent on the mycorrhizal state for growth or reproductive success. These are complex issues which are important in discussions of cellular interactions and plant-fungus specificity and compatibility, as well as of ecology. Specificity needs to be considered at both taxonomic and ecological levels. Taxonomic specificity or host range indicates whether or not a given species of fungus can form a mycorrhizal relationship with more than one species of host or whether or not a given species of host associates mycorrhizally with more than one species of fungus. This can be extended to lower taxonomic levels, where subspecific genetic strains of fungus may form mycorrhizas attuned in some way to the species or subspecific genetic strains of the host. At a still finer level we need to determine whether or not there is in mycorrhizal symbioses anything resembling the genes for 'resistance' or 'avirulence' that have been recognized in some kinds of

The symbionts forming VA mycorrhizas

27

antagonistic symbioses and which have so profoundly influenced the thinking of plant pathologists. There is no clear evidence that any absolute specificity exists between taxa of VA mycorrhizal fungi and taxa of potential host plants and it is of interest that even before modem methods were available, investigators such as Magrou (1936), Stahl (1949) and Gerdemann (1955) had reached this conclusion. In general (accepting that a few plant families do not form mycorrhizas or usually form another type of mycorrhiza), we might expect, with reasonable confidence, that a VA mycorrhizal fungus isolated from one species of host plant will colonize any other species that has been shown to be capable of forming VA mycorrhizas, thus combining wide host range with permanence of association. It is important to grasp this point clearly, because some authors (e.g. Heslop-Harrison, 1978) have stated specifically that the angiosperm root systems in association with mycorrhizal fungi exhibit a 'high or very high' degree of specificity. This is incorrect and based on extrapolation from work with parasitic fungal biotrophs (e.g. rusts, smuts) where a very high degree of race-cultivar specificity has evolved. Others have recognized clearly that mutualistic and parasitic associations are subject to quite different selection pressures (Brian, 1976; Vanderplank, 1978; Smith and Douglas, 1987) and we can do no better than to quote Vanderplank in this context: 'Opposite selection pressures are clearly involved. In parasitic symbiosis the host plant benefits by mutation to resistance because this ends, for the host, an unwanted symbiosis. In mutualistic symbiosis the host loses by mutation to resistance because this ends the symbiosis. Mutations to resistance in mycorrhizal plants are eliminated by selection because they are disadvantageous; and the elimination also eliminates a major source of specificity' There are, however, considerable gradations in the extent to which species of plants or even genotypes within a species become colonized by mycorrhizal fungi. The converse is also true: different species or isolates of fungi colonize the roots of the same species of plant to different extents and in a few cases the range of potential partners appears so restricted as to constitute specificity. A few examples will illustrate these points. Giovannetti and Hepper (1985) tested the ability of three legume species, Medicago sativa, Hedysarum coronarium and Onobrychis viciaefolia, to be colonized by four Glomus species. Using two soils of different P availability they showed that whereas M. sativa was extensively (though variably) colonized by all four fungi, there were considerable differences in colonization of the other two plant species. Hedysarum coronarium showed the most striking differences, being colonized to the same extent as M. sativa by G. mosseae, but scarcely or not at all by G. caledonium or by one of the isolates of G. fasiculatum. Hedysarum coronarium is certainly not a 'non-mycorrhizal' plant, but there is clearly some degree of specificity in its response to different fungi, and this has not been further investigated. Fungal host range may occasionally also be restricted, for in a survey of 19 species of host plant. Glomus gerdemanni formed mycorrhizas only with Eupatorium odoratum (Graw et ah, 1979). Genotype-dependent effects on the extent of colonization have also been observed in a number of species (see Smith et al, 1992; Peterson and Bradbury, 1995). In one recent example, the extent to which Glomus etunicatum colonized barley differed in different cultivars, and

28

Vesicular-arbuscular mycorrhizas

showed considerable cultivar-dependent response to P application (Baon ei ah, 1993), but care needs to be exercised in these comparisons because environmental conditions can influence the differences in colonization (Azcon and Ocampo, 1981; Vierheilig and Ocampo, 1991). The most extreme examples are the restriction of colonization at different stages in plant mutants or genotypes of otherwise highly mycorrhizal species such as Pisum sativum, Vicia sativa (Due et ah, 1989) and Medicago sativa (Bradbury et ah, 1991). Investigation of these mutants may help to unravel the genetic control and physiological mechanisms determining mycorrhizal colonization. It is already clear that mycorrhizal colonization of roots can be blocked at a number of stages in typical host plants and that a number of different mechanisms are likely to be responsible for failure of colonization in non-mycorrhizal plants. We do not yet know enough about control mechanisms to say whether or not study of mutants will help with understanding those that prevent or restrict the colonization of naturally occurring non-host species by mycorrhizal fungi (see Chapter 3). These interesting but somewhat 'atypical' occurrences must not be allowed to cloud the observation that VA mycorrhizal fungi and their host plants have generally non-specific interactions. Numerous research groups use one or a few species of plant on which to maintain pot cultures of a large number of fungal species. For example, Plantago lanceolata, Trifolium subterraneum and Sorghum sudanense are widely used for the maintenance of pot cultures and they become extensively colonized by a wide variety of glomalean fungi, as shown in Table 1.3. Ecological Groupings and Specificity It is possible to make the broad generalization that ectomycorrhizas and some ericoid mycorrhizas are characteristic of plants growing on soils with relatively large deposits of organic matter, whereas VA mycorrhizas are more frequent on plants growing on mineral soils (see Chapter 15). More significant may be the occurrence of VA mycorrhizas in plant communities with high species diversity, where the large potential host range for the fungi can be used to the full and where the relatively poor means of dispersal is obviated by the large number of potential host plants. This is true of such different plant communities as tropical forest and temperate grassland, both of which are very species-rich. A number of habitats are characterized by a relatively high proportion of consistently non-mycorrhizal species or by species that sometimes occur in the non-colonized state. These include the following situations: very moist or arid; highly disturbed, particularly in the early stages of succession; and very nutrient-rich soils. Tundra and high alpine habitats may also have low incidence of VA mycorrhizas, even in species known to be able to form them (see Vare et al, 1992). Soil conditions may influence VA mycorrhizal colonization in direct ways and the effect of nutrients, particularly PO^ (phosphate), will be discussed later (Chapter 2). In the present discussion the effects of waterlogging are relevant because the generally non-susceptible Cyperaceae and Juncaceae contain many members which are characteristic of wet or waterlogged habitats and even in susceptible plants colonization may be much reduced under wet conditions (Mejstrik, 1972; Read et al., 1976). In contrast, some water plants are typically mycorrhizal. These include Lobelia dortmanna, Littorella uniflora, Cyanotis cristata and Eichhornia crassipes, which

29

The symbionts forming VA mycorrhizas

T a b l e 1.3a Species of glomalean fungi confirmed to form VA mycorrhizas with Plantago lanceolatOy Zea mays and Sorghum sudanense. Plant species

Fungal species Scutellospora

Gigaspora

P. lanceolata

S. calospora S. castanea

G. Candida G. margarita G. rosea

Z mays

S. aurigloba S. scutata

G. Candida G. margarita

S. sudanense S. calospora G. albida S. coralloidea G. descipiens S. dipurpurescens G. gigantea S. erythropa G. margarita S. fulgida G. rosea S. gregaria S. heterogama S. pellucida S. persica S. reticulata S. scutata S. verrucosa

Acaulospora

Entrophospora

A. delicata A. laevis A. longula A. scrobiculata A. spinosa A. troppei

A. delicata A. dilatata A. gerdemannii A. lacunosa A. laevis A. longula A. mellea A. morrowiae A. scrobiculata A. spinosa A. trappei

£ colombiana £ infrequens

Glomus G. G. G. G. G. G. G. G. G. G.

clarum coronatum dimorphicum fasciculatum fistulosum flavisporum geosporum intraradices mosseae occultum

G. G. G. G. G.

albidum fistulosum fragilistratum geosporum mosseae

G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.

aggregatum catedonium claroideum clarum clavispora constrictum deserticola diaphanum etunicatum fasciculatum fistulosum fragilistratum geosporum intraradices invermaium lamellosum leptotjchum manihotis mosseae occultum

The lists are restricted to species of fungi that have been unequivocally identified; numerous other fungal species for wrhich either the nomenclature or identification are uncertain are also associated with these plants. Gaps in the table do not imply that any species of fungus will not form an association with any particular species of plant. Data of C. Walker and J.B. Morton.

30

Vesicular-arbuscular mycorrhizas

T a b l e 1.3b Plant species recorded as f o r m i n g VA mycorrhizas w i t h different species of glomalean fungi Glomus mosseae

Gigaspora margarita

Glomus etunicatum

Glomus occultum

Zea mays

Zea mays

AgrostJs palustris

Sorghum bicolor

Allium cepa

Allium cepa

Sorghum bicolor

Populus eurame

A. porrum*

Trifolium subterraneum*

Allium porrum*

Lycopersicor)

Fragaha vesca

esculer)tum

Glycine max*

Poter)tilla erecta

Plantago lanceolata Schizachrium scopahum Lycopersicon esculentum Trifolium pratense T. repens T. subterrraneum* Data of C. Walker. * Data of S.E. Smith and P. O'Connor.

T a b l e 1.3c N u m b e r s of species of glomalean fungi recorded as f o r m i n g mycorrhizas w i t h different plant species. Plant species

Scutellospora

Gigaspora

Acaulospora

Glomus

Allium cepa

1

3

1

12

A. porrum

1

3

1

12

Asparagus officinalis

NT

NT

NT

3

Nicotiana tabacum

NT

F

1

4

Pisum sativum

1

1

1

2

Platanus acerifolia

NT

NT

NT

3

Vitis vinifera

NT

2

1

3

Lycopersicon esculentum

NT

1

NT

3

Trifolium pratense

5

3

5

9

Vicia faba

NT

NT

NT

2

Glycine max

NT

NT

NT

4

Solanum tuberosum

NT

NT

NT

3

Lotus corniculatus

3

F

1

3

Rubus ideaus

NT

NT

NT

3

In almost all cases the tests resulted in mycorrhiza formation. Data of V. Gianinazzi-Pearson. NT, not tested; F, failure but tests not repeated.

may be highly colonized, and Phragmites communis, Eleocharis palustris, and Salvinia cucullata, which have only low levels of colonization (Sondergaard and Laegard, 1977; Bagyaraj et al., 1979; Khan, 1993). For these species, as for the taxa with low levels of colonization or infections lacking arbuscules, it must not be assumed that the influence of the fungus will be great or even significant under natural conditions. It is also important not to be too dogmatic about the extent of colonization in particular taxa. High levels are sometimes found, even in 'non-mycorrhizal' taxa. In Uncina meridiensis (Cyperaceae) for example, 77% of the root length was colonized in South Georgia and the Falkland Islands (Christie and Nicolson, 1983). In field investigations specificity is more difficult to analyse because of the complexities of potential interactions between different fungi in the same root

The symbionts forming VA mycorrhizas

31

system or effects of the presence of many potential host species. It is important to be aware that the lack of specificity means that a single plant species can be colonized by many different fungi and that individual plants can be linked below ground by common mycorrhizal mycelium. Links between different species of plant have been proved by observation in eight cases (Newman et ah, 1994). A survey of Festuca in western USA (Molina et ah, 1978) found that a single Festuca plant could have two or more species of fungi associated with it and F. idahoensis was associated with five fungal species per site. Nevertheless, there is some evidence that the range of potential partners may be more restricted in the field than under experimental conditions, that is some ecological specificity may be important. By this we imply that although in cultural conditions a symbiotic association between two component organisms can be set up, the kind of mycorrhiza so formed may not occur in natural or ecological conditions. In other words, specificity is closer in competition or in the available natural habitats than in pot culture in glasshouse or growth-room conditions. Furthermore, there are certainly plant species that in the field may sometimes form mycorrhizas and at other times not. These are referred to by some authors as 'facultative mycotrophs' to distinguish them from 'obligate mycotrophs' which are always mycorrhizal in field situations. Although convenient, we avoid the use of these terms, because they have been used in more than one way and, unfortunately, mycotroph has also been used to imply a degree of physiological dependence on the mycorrhizal condition, although most work has not verified this experimentally. There is more evidence for ecological specificity in ectomycorrhizal associations than for VA mycorrhizas, but a few examples will serve to illustrate the phenomenon. Using only the broad distinction between infections caused by the 'fine endophyte' {Glomus tenuis) and coarse infections, McGonigle and Fitter (1990) demonstrated that Holcus lanatus was apparently highly receptive to fine endophyte, which contributed over 60% of the total colonized length, regardless of season. In contrast, in Ranunculus acris and Plantago lanceolata, G. tenuis contributed 10% or less of the colonized length, although the fractions of the root length colonized were the same or higher than Holcus (total colonization for these three species ranged from 15% for Holcus in March to 44% for Plantago in both October and March). In the same pasture, Phleum pratense was only slightly colonized by either group of fungi (3-12%). The precision of identification of fungi colonizing roots is much increased by application of molecular methods. Using a PCR-based method, Clapp et al, (1995) detected three genera {Scutellospora, Acaulospora and Glomus) in roots of the English bluebell (now reasonably well known as Hycinthoides non-scripta), whereas only Scutellospora and Acaulospora would have been predicted from spore collections. The frequency of Glomus was significantly less under Quercus than Acer, emphasizing the complexities to be expected in natural ecosystems. Genetic diversity in the fungal population in an alpine calcarious grassland, investigated by PCR-restriction fragment length polymorphisms (RFLP), has also been shown to be considerable, both between and within populations of morphologically similar Glomus spores (Sanders et ah, 1995). One consequence of the general lack of absolute specificity is the potential for plants of the same and different species to be linked by common mycorrhizal mycelium. Although the quantitative details of nutrient transfer from one plant to another have not been fully worked out (see chapters 4, 5, 14 and 15), one plant

32

Vesicular-arbuscular mycorrhizas

may depend on the mycelium for uptake of mineral nutrients while another plant plays a major part in the C nutrition of that mycelium. This possibility means that even without actual interplant transfer, the fungus has the potential for considerable moderation of the interactions between individual plants and between species. There are certainly quantitative differences in the way that the symbioses function in different plant-fungus combinations and these variations in efficiency or effectiveness may have important consequences for intra- and interspecific interactions.

Conclusions VA mycorrhizas are formed by members of all phyla of land plants. The fungal symbionts appear to be restricted to relatively few genera in the order Glomales (Zygomycetes). These are apparently asexual organisms, with variation dependent on mutation and, possibly, on heterokaryosis. The group is probably of very ancient origin (350-460 million years BP), indicated both by the fossil record and by DNA sequences of living members. The symbiosis is also ancient and may have played an important role in colonization of the land. The number of species of present-day plants forming VA mycorrhizas is very large and their diversity is considerable, not only in taxonomic position but also in life form and geographical distribution. Herbaceous plants, shrubs and trees of temperate and tropical habitats may all form VA mycorrhizas and there is little evidence for specificity between particular fungi and host plants. Only a few families and genera of plants do not generally form VA mycorrhizas and even in these some members are frequently found to be colonized in particular habitats. The lack of specificity in the relationships has important consequences both for the biology of the fungi and for ecological interactions in plant communities.

Plate I. (a) Spores arid subtending hyphae oi Gigaspora margaritOy approximate diameter 400-450 |im. (b) Spore of Acaulospora laevis (arrowed), attached to the neck of the sporiferous saccule (s). Spore diameter approximately 190-210 |im. (c) Spores of Scutellospora n/gro, approximate diameter 300|Lim. Photographs courtesy of V. Gianinazzi-Pearson.

Colonization of roots and anatomy of VA mycorrhizas

Introduction This chapter provides an account of the main characteristics of vesicular-arbuscular (VA) mycorrhizal roots of different types and shows how they develop from sources of inoculum in the soil. The topics covered include the following: (1) the nature of the propagules that initiate colonization, including spores, infected root fragments and hyphae; (2) the development of structures within the root which characterize morphologically different types of VA mycorrhizas; (3) the growth of the extraradical mycelium in soil and the production of spores; (4) the dynamic interactions between fungal colonization and root growth which leads to the development of VA mycorrhizal root systems. Details of the cellular and molecular interactions between plant and fungus before and during colonization, and the importance of these in maintaining the symbiosis, considered only briefly here, will be discussed in greater detail in Chapter 3.

Sources of Inoculum Colonization of roots by VA mycorrhizal fungi can arise from three sources of inoculum: spores, infected root fragments and hyphae - collectively termed propagules. The large spores, with thick, resistant walls and up to several thousand nuclei, appear to be long-term survival structures with some capacity for dispersal by wind and water (Koske and Gemma, 1990; Friese and Allen, 1991; see Gemma and Koske, 1992). Spores and sporocarps can also survive passage through the gut of a number of different invertebrates, birds and mammals and this may be important for localized dispersal, although this has not been directly demonstrated in all cases (Mcllveen and Cole, 1976; Daniels Hetrick, 1984; Reddell and Spain, 1991; McGee and Baczocha, 1994). The distribution of spores and root fragments is altered by the burrowing activities of both large and small animals, and changes in mycorrhiza development associated with ant and gopher mounds have been documented (Koide and Mooney, 1987; Allen and McMahon, 1988; Friese and Allen, 1993).

34

Vesicular-arbuscular mycorrhizas

For many years it was assumed that spores were the most important propagules, possibly the only ones. Using wet-sieving techniques (see below) many fungal species were described and much was learned about their distribution, frequency and role in initiating colonization of roots. In soil, populations are composed of spores of different ages and in different states of dormancy or quiescence (Tommerup, 1983). Consequently, germination may occur rather slowly and variably, providing a reservoir of inoculum which persists for many years but may not always be important in early colonization of root systems (e.g. McGee, 1989; Braunberger et al., 1994). In many habitats the hyphal network in soil, together with root fragments, is probably the main means by which plants become colonized even when significant spore populations are also present (Hepper, 1981; Smith and Smith, 1981; Tommerup and Abbott, 1981; Birch, 1986; Jasper et al, 1992). Consequently, as a seedling grows in an established community it becomes linked into a complex underground network of the mycelium of different fungal species and of roots growing from plants of different ages. Disruption of the network by disturbance or tillage can result in much reduced infectivity of the soil and lower rates of nutrient uptake by the plants (Birch, 1986; Jasper et al, 1989,1991,1992; McGonigle et al, 1990a). It is difficult to distinguish the relative contributions of the different types of propagules to colonization of the root systems of plants growing in any particular field situation, that is to the 'infectivity' of the soil. The density of the spores in soil can be determined, but although this sometimes shows a correlation to the extent of root colonization, this is not always the case, as shown in the examples in Figure 2.1a,b,c. The relationship is complex, because the extent of colonization may be related both to the availability of spores as inoculum and to the capacity of the mycorrhizal root system to produce new spores (see below). Furthermore, the spore population is varied, with respect to species composition, viability, dormancy, etc., and other sources of inoculum may play a significant role in the colonization of roots. The discrete spores and root fragments have been counted or weighed (Thompson, 1987) or collectively assayed by the most probable numbers (MPN) method, using trap plants to determine the presence or absence of infective propagules in the samples. This method involves dilution and mixing of samples, which destroys the hyphal network, but it is useful for enumerating the reserve of infective, robust propagules in soil provided that its constraints and wide confidence limits are appreciated (e.g. Porter, 1979; Wilson and Trinick, 1982; An et al, 1990; Jasper et al, 1992). Assessment of the infectivity of imdisturbed soil, including the contribution of hyphal networks, is much more difficult but can be achieved by bioassays using standard host plants in undisturbed soil cores (Moorman and Reeves, 1979; Gianinazzi-Pearson et al, 1985; Jasper et al, 1989; Braunberger et al, 1994). Both the MPN methods and bioassays have the great advantage that colonization of roots is used to detennine the presence of viable propagules. Unfortunately, they cannot distinguish the relative importance of the different types of propagule. While an extraradical hyphal network associated with a living plant has indeterminate and probably unlimited growth and a long-term capacity to initiate colonization, the growth of mycelium from spores or root fragments is very restricted. There are indications that VA mycorrhizal fungi may have a slight capacity for saprophytic growth in soil (Hepper and Warner, 1983), but all

Colonization of roots

35

attempts to culture mycelium detached from spores have failed and at the moment we must conclude that hyphal growth in soil in the absence of root colonization is of minor importance in ecological situations. VA mycorrhizal fungi are prime examples of the group of perennial fungi which inhabit roots and use the products of current or recent photosynthesis. Although spores are probably widely dispersed by wind, at least in low densities, other propagules are likely to be moved only short distances. Consequently, the fungi depend either on the existence of a perennial mycelium or on dispersal of host species and lack of specificity for particular hosts to avoid spatial or temporal discontinuities in the availability of plants which are their only sources of organic C. Plants which rely on the symbiosis for success are also dependent on the capacity of the fungal propagules to survive in a wide range of soil types and environmental conditions.

Spores Occurrence ar)d lr)fectivity Spores are the best defined source of inoculum and are the only propagules that can be identified to species with any degree of certainty (see Chapter 1). Consequently, they are of central importance in isolating these species, determining their distribution and establishing them in pot cultures for experimental or identification purposes. The most commonly used method is 'wet-sieving and decanting' which was initially developed by Gerdemann and Nicolson (Gerdemann, 1955; Gerdemann and Nicolson, 1963) and details can be found in various handbooks and laboratory manuals (e.g. Schenck, 1982; Norris et al, 1992; Brundrett et al, 1994). The density of spores in soil and their species diversity are very variable. In some habitats spores are not found in all seasons and even the seasonal maxima are quite low (1-5 spores g~^ soil; see Fig. 2.1), but much higher numbers have sometimes been found. For example, Sutton and Barron (1972) surveyed agricultural sites in Ontario and found between 9 and 89 spores g~^, the higher values being from late in the season as plants reached maturity. Similar seasonal changes in density of Gigaspora gigantea were observed in a Rhode Island sand-dune, where spore abundance was at a maximum (about 4 cm~^ sand) in December. As shown in Figure 2.1d, this maximum did not coincide with either maximum germination (December-July) or the infectivity of the soil, which was lowest in September and October (Gemma and Koske, 1988a). In this investigation, dormancy, which was overcome by 5 weeks at a temperature of 5'^C, was important in determining the contribution of spores to infectivity of the soil. A peak in spore density was also observed in late autumn (November) at a woodland site, with nine spore types represented (Clapp et al, 1995). In situations where density of spores is positively correlated with the extent of root colonization, both may increase during the growing season of annual plants. Decreases in density with depth of soil could well be associated with the decline in density of roots and mycorrhizas (Hayman, 1970; Sutton and Barron, 1972; Giovannetti, 1985; Jakobsen and Nielsen, 1983). However, this relationship does not always hold: Louis and Lim (1987) observed an inverse relationship between spore density and colonization in four perennial trees from lowland tropical rainforest

Vesicular-arbuscular mycorrhizas

36

(a)

(i) Aglaonema connadatum

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Month (ii) Clidemia hirta

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Month Ammophila arenaria

MJ

J A S N J M A M J Month

0.1

0.2

0.3 0.4 0.5 Spores per g soil

0.6

0.7

37

Colonization of roots (d) 0.50

100 '

1

0.40

80

i

0.30

60

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o

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0.00

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A S O N D J F M A M J J A S O N

A S 0 N D J F M A M J J A S O N

Month

Month

Figure 2.1 Relationships between spore density and VA mycorrhizal colonization, (a) Seasonal variation in spore density ( • ) and nnycorrhizal colonization (O) associated with two tropical plant species in the Taban Valley, Bukit Timah Nature Reserve. From Louis and Lim (1987), with permission, (b) Seasonal variation In spore density ( • ) and mycorrhizal colonization (O) of Ammophila arenaria. From Giovannetti (1985), with permission, (c) Correlation between spore density and mycorrhizal colonization in a sand-dune system. Regression analysis: y = 3.06 + 0.5x (r = 0.89; P < 0.01). From Giovannetti (1985), with permission, (d) Seasonal variation in spore abundance (i) and percentage germination (ii) of Gigaspora giganteOy collected at monthly intervals. Redrawn from Gemma and Koske (1988a).

and in some investigations no correlation at all has been found between spore populations and infectivity (Powell, 1977). The species composition and abundance of the spore population, as well as the contribution of spores to infectivity, are probably influenced by a whole range of factors related to spore production, dormancy and infectivity. These may differ between fungal species, type of the plant community, as well as with disturbance, seasonality and other environmental variables. These will be discussed later. Spore populations do not necessarily reflect the contributions of component fungi to root colonization and it will require the application of immunological or DNA-based detection methods to provide data on the diversity of fungal populations in roots (see Bachman, 1994; Hahn et al, 1994). Information on the colonization biology of the fungi has usually been gained using spores of individual species, and, consequently, the effects of interactions between species are not well known or understood. Germination and Hyphal Growth

Spores germinate in soil or on agar media and both they and root fragments will produce small amounts of mycelium of the order of 20-30 mm per spore (but see below). An extensive mycelium is not formed unless successful colonization of a root system occurs, and if the spore becomes detached, growth of the hyphae ceases. Germination can take place in three ways: by germination shields, as in Acaulospora and Scutellospora; directly through the spore wall in Gigaspora species and in some Glomus species; and via regrowth through the hyphal attachment, a method which is common in many other Glomus species (see Siqueira et al, 1985).

Vesicular-arbuscular mycorrhizas

38

The spores of VA mycorrhizal fungi are extremely large (see Figure 1.2 and Plate 1) and contain many nuclei and large amounts of stored lipid and carbohydrate. We must assume that this extreme capacity for storage of both genetic information and energy reserve has selective advantages for the organisms, but at present these are obscure, in particular because there is no sustained growth without colonization of roots. The energy reserves occur mainly as membrane-bound lipid droplets, but also include glycogen and protein bodies and small amounts of trehalose (see Sward, 1981; Becard et al, 1991, 1992; Bonfante et al, 1994; Shachar-Hill et al, 1995). The trehalose is rapidly mobilized as the spores germinate and appears to sustain the initial growth of the germ tube, while the lipid disappears more slowly. Estimates of the number of nuclei per spore vary with the species of fungus and with the methods used to count them. Direct counting after staining gives values of between 1000 and 3850 per spore, whereas estimates based on presumed nuclear size and arrangement around the periphery of the spores determined by electron microscopy are approximately an order of magnitude higher (see Table 2.1). The estimates using the second method are very large and it seems likely that the assumptions on which they are based are incorrect because although nuclei are arranged around the periphery of the spores in Gigaspora margarita, and perhaps some other species, in Glomus versiforme they are mixed with the lipid droplets (see also Becard and Pfeffer, 1993). The DNA content per nucleus has also been Table 2.1 Numbers of nuclei per spore of a range of VA mycorrhizal fungi counted directly (a) or estimated from the surface area of the spore and the presumed distribution of the nuclei (b) (see text) (a) Direct counting Species

Diameter (|im)

Stain

Nuclei per spore

Reference

Gigaspora dedpiens

400

DAP!

1000

Gi margarita

250

2000

Gi. gigar)tea

350

2600

Cooke etai, 1987

Scutellospora erythropa

280

Mithromycin Acetoorcein Acetoorcein

Tommerup, in Viera and Glenn, 1990 Becard and Pfeffer. 1993

3850

Cooke eto/., 1987

(b) Estimated from the surface area of the spore and presumed peripheral arrangement of nuclei Burggraaf and Beringer, 1989. Species

Diameter (^im)

Nuclei per spore

Reference

Gigaspora margarita Gi. dedpiens Gi. gigar)tea Scutellospora erythropea Glomus versiforme G. caledonium

300 400 350 280 150 200

20000 35000 27000 17000 5000 9000

Burggraaf and Beringer, 1989 Viera and Glenn, 1990 Viera and Glenn, 1990 Viera and Glenn, 1990 Viera and Glenn, 1990 Burggraaf and Beringer, 1989

Colonization of roots

39

estimated, again with very variable results. Values based on DNA content per spore and estimates of the number of nuclei give values of 1.7 and 3.4 pg for G. versiforme and Scutellospora persica, respectively (Viera and Glenn, 1990). If the values of nuclear number are revised downward then the estimates would be even higher. Fluorimetry of the stained nuclei gives lower values of 0.26 and 0.77 pg for G. versiforme and Gi. margarita, respectively (Bianciotto and Bonfante, 1992). The first set of estimates is extremely high for fungi (0.009 pg in Saccharomyces cerevisiae, 0.02 pg in Neurospora crassa and Aspergillus niger and 1.5 pg in Erysiphe cichoracearum) Cavalier-Smith, 1985) and, if correct, must indicate a high incidence of repeat sequences. Although Hosny and Dulieu (1994) have provided evidence for the presence of repeat sequences in Scutellospora casianea, there is clearly room for more work in this area. There have been many attempts, so far unsuccessful, to grow VA mycorrhizal fungi in pure culture for long periods and to subculture mycelium separated from the subtending spore and in the absence of roots. It is clear that nuclear division does occur during spore germination and early germ-tube extension, despite early reports to the contrary, and that nuclear DNA replication, as well as synthesis of mitochondrial DNA, RNA and proteins can all occur without the intervention of the symbiotic stage (Hepper, 1979; Beilby and Kidby, 1982; Beilby, 1983; Burggraaf and Beringer, 1989; Viera and Glenn, 1990; Becard and Pfeffer, 1993; Bianciotto and Bonfante, 1993). Elevated CO2 concentrations (2%), together with the presence of flavonols and possibly some other flavonoid or isoflavonoid compounds derived from roots or seeds, exert highly stimulatory effects on mycelial extension and branching at least for Gi. margarita (Becard and Piche, 1989a,b; Becard et al., 1992). However, Glomus intraradices appears to be much less sensitive to CO2 (Nantais and Fortin, personal communication) and again there is scope for more comparative studies. It is important to distinguish conditions which influence germination from those which affect growth of germ tubes and mycelium. The majority of studies of germination have used water or nutrient agar, variously modified, but an alternative and ecologically more relevant approach involves burying spores in soil, packaged in such a way that they can be recovered. Erratic germination may be related to dormancy, for as Tommerup (1983) has shown, spores of 'Gigaspora' {Scutellospora) calospora, Acaulospora laevis and two Glomus species are dormant when first formed but, after periods of storage, dormancy is overcome. The spores then become quiescent and capable of germinating rapidly and fairly synchronously, under appropriate conditions of moisture and temperature. This agrees with other investigations showing that periods of storage in dry soil or at low temperature increase the percentage germination, depending on the species (e.g. Sylvia and Schenck, 1983; Tommerup, 1984a,b; Gemma and Koske, 1988a; Louis and Lim, 1988; Safir et al, 1990). Hepper and Mosse (1975) and subsequently Hepper and co-workers (Hepper and Smith, 1976; Hepper, 1979, 1983, 1984a,b; Hepper and Jakobsen, 1983) carried out the most extensive and systematic investigation of germination of a single species. Glomus caledonium. These and other investigations (e.g. Schenck et ah, 1975; Green et al, 1976; Daniels and Duff, 1978; Daniels and Trappe, 1980) allow some very broad generalizations to be made. Species of fungi vary in the optimum pH, soil matric potential and temperature for maximum germination and although the effects of

40

Vesicular-arbuscular mycorrhizas

high concentrations of P and other mineral nutrients are variable, heavy metals (Zn, Mn and Cd), organic acids and a range of sugars are inhibitory. High salinity reduces germination, probably via its effect on water potential (see Juniper and Abbott, 1993). A few investigations suggest that root exudates or extracts from host species stimulate spore germination (e.g. Graham, 1982; Gianinazzi-Pearson et al., 1989), while others report negative effects or none at all (Vierheilig and Ocampo, 1990a; Schreiner and Koide, 1993a,b). Germination is sometimes, but not invariably, increased in the presence of microorganisms or decreased in sterile soil (Azcon-Aguilar et al., 1986a,b; Mayo et aU 1986; Azcon, 1987; Daniels Hetrick and Wilson, 1989; Wilson et al, 1989). Complex interactions between microbial activity and spore germination and mycelial growth are to be expected and possible mechanisms include removal of toxins or germination inhibitors, production of specific stimulatory compounds and the maintenance of elevated CO2 concentrations which increases hyphal growth in Gi. margarita (Azcon-Aguilar and Barea, 1992; Koske and Gemma, 1992), although the effects on germination itself may be minor (Le Tacon et al., 1983). Interactions between mycorrhizal fungi and other members of the soil microflora are of interest, both from the point of view of germination and colonization and with respect to nutrient cycling and control of root-infecting pathogens (e.g. Graham, 1986; Perrin, 1990; Azcon-Aguilar and Barea, 1992; Fitter and Sanders, 1992; Benhamou et al, 1994; Fitter and Garbaye, 1994). Hyphal growth from spores has been studied intensively with the objective of understanding why this is so limited without root colonization, and also in the hope of producing axenic mycelium for use as inoculum. Little understanding has been gained from most of the early work, although it was consistently found that increased P concentration in the medium reduced hyphal growth, that the stimulatory effects of peptone could be traced to the lysine, cysteine and glycine components and that K (rather than Na) salts of sulphite and metabisulphate were more stimulatory than sulphate or thiosulphate. Various metabolic inhibitors, such as actinomycin, cycloheximide and ethidium bromide, also failed to shed a great deal of light, except to indicate that there were no serious limits to DNA or protein synthesis during spore germination (Hepper, 1979, 1983, 1984a,b; Siqueira et al., 1982; Hepper and Jakobsen, 1983; Pons and Gianinazzi-Pearson, 1984; and see Siqueira et al, 1985; Hepper, 1987). The capacity of germ tubes to absorb nutrients from the medium before the initiation of colonization needs to be carefully examined in the light of two interesting but somewhat contradictory pieces of information. Whereas germ tubes of GL margarita apparently actively absorb Pi (Thomson et al, 1990), they do not appear to possess a plasma membrane-boimd H^-ATPase, which would be necessary to establish the proton-motive force required for proton co-transport of the PO^ (phosphate) ion and other solutes (Lei et al, 1991). The work on effects of plant exudates has been much more enlightening. In axenic tests, soluble exudates or extracts from the roots of host species such as Poncirus and Trifolium, as well as from cell cultures, stimulate the growth and branching of mycelium growing from spores (Graham, 1982; Carr et al, 1985; Elias and Safir, 1987; Gianinazzi-Pearson et al, 1989), while exudates from the non-host Lupinus albus had no effect (Gianinazzi-Pearson et al, 1989). In soil, Giovannetti et al (1993a) have shown complex hyphal branching patterns associated with mycelial growth from spores on roots of a number of host species (Fragaria, Helianthus,

Colonization of roots

41

Oncimum, Lycopersicon and Triticum), but not on the non-hosts Brassica, Dianthus, Eruca or Lupinus. At the same time, a number of studies were initiated on the effects of various phenolic compounds produced by roots or seeds and known to influence symbiotic development between Rhizobium and Agrobacterium and their hosts. In summary, flavonoids, in particular the commonly produced flavonol quercetin, have consistent stimulatory effects on the growth and branching of germ tubes of Gi. margarita and some Glomus species (Gianinazzi-Pearson et ah, 1989; Tsai and Phillips, 1991; Becard et al., 1992). There are a few inconsistencies in the results from different groups using related compounds such as hesperetin, apigenin, narengenin, formononetin and biochanin A, but overall the results are most promising and should lead to important advances, in particular when the mechanisms of action have been elucidated. Application of some of the phenolics also leads to increased colonization of roots by the fungi, but the relative contributions of the increased formation of infection units by the more extensive mycelium, and of the more subtle influences, are not yet clear (Nair et al, 1991; Siqueira et ah, 1991) The importance of an elevated CO2 concentration for the development of mycelium from spores of Gi. margarita is now well established, and goes most of the way in explaining the requirement for volatile root products which has occasionally been noted. In the absence of flavonoids CO2 exerts some stin\ulatory effect, but together the effect is much greater (Becard and Piche, 1989a). Starting with pregerminated spores (to avoid confounding effects of germination and growth) and applying 2% CO2 and 10 |LIM quercetin, Becard et al (1992) have achieved mycelial growth of 500 mm per spore of Gi. margarita, which is at least 10-fold higher than growth commonly observed in less well controlled tests. Comparing quercetin with other flavonoids Becard et al. concluded that it is the hydroxyl group on position 3 of the active molecules that is important and they predict that flavonols will be found to be more stimulatory than flavones. The effect of CO2 seems to stem from a need for dark fixation to generate tricarboxylic acid cycle intermediates during the mobilization of the lipid stored in the spores (see Becard and Piche, 1989a; Becard et al., 1992). The effects of CO2 on Gigaspora seem certain, but we know little of the response of other species. Furthermore, Koske and Gemma (1992) and Gemma and Koske (1992) have cautioned that other volatile compounds may play a significant role and that the potassium hydroxide traps used to remove CO2 would also remove some low molecular weight ketones and aldehydes. This point should be followed up, but the work on volatiles is important both for its promise for axenic culture and, more importantly, for understanding the interactions between the symbionts which clearly commence before any physical contact occurs. Spores of some species appear to be adapted to survive situations in which germination is not immediately followed by colonization of roots and establishment of a symbiotic relationship. Tommerup (1984a) showed that infectivity of spores of Acaulospora laevis and Glomus caledonium was retained in moist field soil for at least 4 weeks in the absence of suitable plants, but declined between 4 and 10 weeks. Similarly, spore-based inoculum of G. intraradices retained infectivity up to 38°C in moist soil (Haugen and Smith, 1992). The basis is unknown for these species, but spores of Gi gigantea are capable of producing a number of germ tubes successively if the earlier ones are cut off (Koske, 1981) and it is probable that nuclear division occurs, replacing the nuclei that migrated to the mycelium during the initial stages of growth (Becard and Pfeffer, 1993). Moreover, although

42 Vesicular-arbuscular mycorrhizas

young spores of Gi. margarita germinate with a single germ-tube, old spores frequently produce several (Sward, 1981) and Glomus epigaeus produces secondary sporocarps m long-term storage without any intervening colonization of roots (Darnels and Menge, 1980). It may be that the large spore size and large reserves are important m maintaining infectivity in a large number of species. Root Fragments Root fragments can be an important source of inoculum in many soils, but much less IS known of their biology than that of spores. Regrowth of hyphae from mtected root fragments has been frequently observed (Magrou, 1946; Hall 1976Powell 1976; Hepper, 1984a; Williams, 1990; and many others) and the fragments have often been used to initiate colonization experimentally (Fig. 2.2). We do not know how long vegetative hyphae survive in senescent or dead roots, but the results of Tommerup and Abbott (1981) suggest that they may do so for at least 6 months m dry soil and that infectivity is not related to the presence of vesicles, so that the potential for hyphal regrowth and infectivity was retained in 'Gigasvora' Scutellospora calospora (a non-vesicular species) as it was for Glomus 'fasciculatus'and G. monosporus'. However, vesicles, like spores, store large amounts of lipid and contam many nuclei, which together with their thick walls suggest a function either as propagules or to support the regrowth of intercellular hyphae. The fact that if

Figure 2.2 Growth of mycorrhizal hyphae (h) from a dead root fragment (dr) and mimt^on^f colonization in a growing root of Trifolium subterraneum (arrowed). Photograph

Colonization of roots

43

fragments colonized by Acaulospora laevis fail to retain infectivity has now been partially explained in terms of reduction of the hyphal viability following spore production (Jasper et ah, 1993) and might also be related to the way the intercellular hyphae mature as the roots age. Development of very thick walls by the intercellular hyphae of one mycorrhizal fungus (designated G. fasciculatum) in old Trifolium roots has been studied ultrastructurally by Lim et al (1984). The laminated walls resembled those of sclerotia and surrounded apparently fairly normal cytoplasmic contents. This may represent an adaptation to survival for long periods, but how widespread the phenomenon is, and whether or not the thick-walled hyphae are capable of subsequent germination, are unknown. Within the fragments, regrowth of hyphae often occurs in the lumina of the old hyphae, while outside a cord formed by the intertwining of relatively wide (20 |im diameter), thick-walled hyphae develops. These cords split into distinct hyphal strands when close to a living root and the hyphae form separate appressoria and infection points, so that the contact with the root is similar to the formation of colonization fans by single hyphae (Friese and Allen, 1991; and see Fig. 2.2 ). Thompson (1987) found that with a long fallow period (up to 2 years without plants) numbers of root fragments as well as spores were low and so was infectivity; with short fallow periods the converse was true. The density and distribution of root fragments in pots is important in influencing the rate of colonization of Trifolium subterraneum seedlings and their location on main and lateral roots (Smith and Smith, 1981), and redistribution of root fragments by soil animals has an effect not only on development of mycorrhizal roots, but also on plant distribution. Friese and Allen (1993) showed that harvester ants accumulate very large quantities of clipped root material (and spores) in their underground nests. After the nests were abandoned, Artemisia tridentata and Oryzopsis hymenoides plants adjacent to the nests were colonized very rapidly and extensively by VA mycorrhizal fungi. However, spores of Gi. margarita applied to the soil surface did not move downwards in the soil profile, nor did they initiate significant colonization. In some situations the dead infected roots may be localized in such a way as to provide inoculum exactly where new roots will grow. This seems to be the situation in bluebell woods where the very simple and extensively colonized root system of the bluebell (Hyacinthoides non-scripta) dies off to produce inoculum where the new roots will grow from the bulb the following season (Daft et al, 1980). The capacity of root fragments to maintain infectivity through periods of repeated wetting and drying, even when colonization of roots does not occur, is not yet clear. However, this may have considerable ecological significance in very seasonal habitats where intermittent summer rainfall may wet the soil but not be sufficient to support seedling establishment (Braunberger et al, 1994). The finding that some robust propagules (spores and root fragments) do not germinate well in very warm soils (Braunberger, personal communication) may indicate mechanisms that ensure extensive germination only when soil temperature is cool (during autumn) and soil moisture is therefore likely to be maintained.

Hyphal Networks The importance and extent of the hyphae growing in the soil from VA mycorrhizal roots has been appreciated for a long time. Peyronel (1923) described the extensive development of mycelium and was clearly aware of the importance of hyphae in

44

Vesicular-arbuscular mycorrhizas

linking plants together (see Harley, 1991). Nicolson (1959) was one of the first to make a systematic investigation of the extraradical mycelium associated with grass roots from natural habitats. He described, as others have done since, the striking variation in diameter (2-27 |im) among hyphal filaments, with accompanying variation in wall thickness. He went on to describe and illustrate how the thickwalled hyphae form the permanent bases for the hyphal complexes associated with roots, and the unilateral angular projections, typical of these hyphae, are apparently rermiants of short lived lateral branch complexes. The main hyphae, which normally contained cytoplasm and nuclei, gave rise to these lateral systems of more and more finely branched and septate, lateral hyphae (see Fig. 2.3a). Read and co-workers have consistently emphasized the importance of the living hyphal network in initiating rapid colonization in seedlings (Read et ah, 1985; Read, 1992). Hyphal connections grow from plant to plant, forming hyphal bridges which can be simple or may branch as a root is approached to form multiple colonizing hyphae and appressoria (Friese and Allen, 1991; Fig. 2.3b). Runner hyphae forming external loops along the surface of the root also initiate secondary colonization (Cox and Sanders, 1974; Brundrett et al, 1985; Friese and Allen, 1991; Wilson and Tommerup, 1992; see Fig. 2.3b). Mycelium of Glomus tnosseae has been shown to spread through soil at a rate of 3 mm d~^ to initiate colonization in soybeans (Camel et ah, 1991) and a maximum distance appears to be approximately 20-30 |Lim for a number of species (Warner and Mosse, 1983; Schiiepp et al, 1987), although values up to 90 mm have been recorded. An 'infection front' can spread through a population of plants in sterilized soil at rates between 0.2 and 2.5 mm d~^ depending on the plant and fungal species (Powell, 1979; Scheltema et al, 1985,1987b). The mycelium can be disrupted by the activities of soil organisms, such as grazing coUembolans and burrowing earthworms, although the significance of this for either infectivity of the soil or nutrient absorption has yet to be fully worked out (McGonigle and Fitter, 1988a; Fitter and Sanders, 1992; Pattinson, 1993; Dekkers, 1996). The hyphal network is capable of surviving and retaining infectivity during periods when the vegetation with which it developed is either dormant or actually dead. Work in seasonally very dry and hot (Australia: McGee, 1989; Jasper et ah, 1989,1993; Braunberger et al, 1994) or cold (Canada: Addy et al, 1994) climates has indicated the importance of this survival to rapid colonization when conditions favourable to plant growth return. For some fungi the maintenance of the infectivity of the network may depend on whether or not it has dried before sporulation commenced, so that the significance of spores and/or hyphae and the effects of disturbance will be varied (see Jasper et al, 1993). It will be interesting to determine

Figure 2.3 Variation in the hyphae forming the external mycelium of VA mycorrhizas. (a) Camera lucida drawings showing thick-walled (H) and thin-walled (h) hyphal elements with many angular projections (arrowed) in the mycelium associated with the roots of Dactylis glomerata. Bars, i, iii, iv, 20 |im, li, 10 |im. From Nicolson (1959), with permission, (b) Diagrammatic representation of the types of hyphae and hyphal architecture associated with VA mycorrhizal roots. Hyphae entering roots from root fragments or spores. Hyphae in soil, forming hyphal networks, runner hyphae and bridge hyphae. Intraradical colonization not shown. Redrawn from Friese and Allen (1991).

Colonization of roots

45

(a)

(b)

Hyphae entering root

Root fragment

Germ tubes

Figure 2.3 (Caption opposite)

Hyphae exiting root

46

Vesicular-arbuscular mycorrhizas

if all components of the network are important or whether it is the thick-walled and runner hyphae that are involved in carry-over of infectivity.

Morphology and Anatomy of VA Mycorrhizas Arum- and Paris-type Mycorrhizas It has already been emphasized that there are three important components of any mycorrhizal root system - the root itself and two associated mycelial systems, one within the root apoplast and the other in the soil. The two mycelial systems groW and develop in very different environments: the first is very constant through root homeostasis and the second is highly variable. Descriptions and illustrations of the internal mycelium were made as early as 1897 by Janse, and beautiful drawings illustrating the details of fungal interactions with plant cells and tissues of the root were published by Gallaud in 1905 (Fig. 2.4a,c). Although interpretation of the significance of some of the structures has not stood the test of time, the main features are clearly recognizable. Gallaud's observations indicate that mycorrhizal roots can fall into one of two general anatomical groups depending on the species of plant. The type nowadays regarded as a 'typical VA mycorrhiza' and frequently described in the fast-growing root systems of crop plants, belongs to Gallaud's Arum-type. In these associations the fungus spreads relatively rapidly in the root cortex via intercellular hyphae, which extend along well developed intercellular air spaces. Short side-branches penetrate the cortical cells and branch dichotomously to produce characteristic arbuscules. Hyphal coils may be formed, particularly in the hypodermal (exodermal) cell layers of the root, but they are not usually a major component of the intraradical mycelium. One of Gallaud's illustrations and a photomicrograph of a typical Arumtype infection unit in Allium porrum are illustrated in Figure 2.4a,b and will be described later in greater detail. A single arbuscule is shown in Figure 1.1. In the Pans-type, colonization of the roots is characterized by extensive development of intracellular coiled hyphae which spread directly from cell to cell within the cortex (see Fig. 2.4c,d,e). Arbuscules grow from these coils and there is very little, if any, intercellular growth. In consequence, the rate of growth of the infection units within the root is much slower than for the Arum-type. We do not know how common the Pflrzs-type of colonization pattern really is. Gallaud (1905) described it in the European woodland plants Paris, Parnassia and Colchicum and it has been more recently depicted in members of the Gentianaceae (Jacquelinet-Jeanmougin and Gianinazzi-Pearson, 1983; McGee, 1985) and in a number of woodland species, including Erythronium, Trillium, Asarum (Brundrett and Kendrick, 1990a), Acer saccharum, (Yawney and Schultz, 1990; Cooke et al, 1993), Liriodendron (Gerdemann, 1965), Taxus (StruUu, 1978) and Ginkgo (Bonfante-Fasolo and Fontana, 1985). Smith and Smith (personal communication) have surveyed the literature for information on this type of mycorrhiza and found reports of its occurrence in many families of pteridophytes, gymnosperms and angiosperms. In a semiarid environment of southern Australia, McGee (1986) noted VA mycorrhizas with coils in only two species, Centaurium and Wurmbea. In some cases inoculation has

Colonization of roots

Heme genemlc de Itotanhjue.

Figure 2.4 (Caption on p. 49.)

47

48

Vesicular-arbuscular mycorrhizas Recue genirale de Bolanique.

Tome 17, Planch' S.

Imp.

I.c

/liifnt.

W'

Figure 2.4 (Caption opposite)

Colonization of roots

49

confirmed the glomalean relationships of the fungi, but this type of association may also occur widely in achlorophyllous plants in which aseptate coils without arbuscules predominate and which are accepted as VA mycorrhizas by Leake (1994), even though formal proof of the glomalean identity of the symbionts is lacking. Apart from their development, studied with the light microscope, a few details of the ultrastructure of Gentiana, Ginkgo and Liriodendron mycorrhizas (Kinden and Brown, 1975a,b,c; Bonfante-Fasolo and Fontana, 1985; Jacquelinet-Jeanmougin and Gianinazzi-Pearson, 1987) and a very small amount of experimental work on growth responses in Centaurium, Gentiana and Liriodendron, we are relatively ignorant of the Parzs-type of VA mycorrhiza. We know that the last three plant genera listed are very dependent on symbiosis for satisfactory growth, but we do not know much of the detail of nutrient acquisition by the plants or the roles played by the hyphal coils in the transfer of either mineral nutrients or C between the symbionts (Smith and Smith, 1995, 1996a). Coils, rather than arbuscules, also predominate in heterotrophic plants such as the gametophytes of Psilotum (Peterson et ah, 1981), the achlorophyllous bryophyte Cryptothallus mirabilis (Schmidt and Oberwinkler, 1993) and the roots of some achlorophyllous members of the Gentianaceae and Burmanniaceae (see Leake, 1994). The Paris-type of mycorrhiza in photosynthetic plants will not be considered further here, except for passing references, but for those interested in the diversity of symbiotic interactions, the study of their occurrence, distribution and function is sure to be rewarding.

Establishment of Colonization Precolonization Events Colonization of roots can be initiated from hyphae growing from any of the three sources of inoculum described earlier. Details of the colonization process have been studied chiefly using spores or infected segments of root as inoculum, either in axenic culture in agar (Mosse and Phillips, 1971; Mosse and Hepper, 1975; Hepper, 1981) or on slides buried in soil (Powell, 1976). More recently, direct, nondestructive observations of colonization from natural inoculum in soil have been made in glass-sided boxes (Friese and Allen, 1991) or from pregerminated spores in Figure 2.4 Variations in intraradical VA nnycorrhizal colonization, (a) Drawings of Arumtype mycorrhizas showing intracellular arbuscules originating from intercellular hyphae in a range of host plants (41, 45, 46, 47). Stages in the disintegration of arbuscules also shown (43, 44, 47). From Gallaud (1905). (b) A single. Arum-type infection unit of Glomus versiforme in a lO-day-old root oi Allium porrum. Note the intercellular hyphae (arrowed) growing longitudinally between the root cortical cells. Branches may develop into arbuscules (*) or remain as short projections on the hyphae. From Brundrett et al. (1985), with permission, (c) Drawing of Paris-type mycorrhizas, showing the prolific development of intracellular coils and relatively sparse development of arbuscules see also (a) 42. From Gallaud (1905). (d) Mycorrhizal development in Acer saccharum, showing highly developed coils (d) in cortical cells. Bar, 40 jim. From Cooke et al. (1993), with permission, (e) Mycorrhizal development in Acer saccharum, showing hyphae passing directly from one cortical cell to another (arrowed). Bar, 40 |im. From Cooke et al. (1993), with permission.

Vesicular-arbuscular mycorrhizas

50

an axenic dual culture system employing transformed roots (Becard and Fortin, 1988; Becard and Piche, 1989). Colonization from mycelium has been followed in 'nurse pots' containing mycorrhizal plants, into which the plants of interest are transplanted once the hyphal network has developed (Brundrett et al, 1985; Rosewame, 1993; see Fig. 2.5). In this latter system, very rapid, dense and almost synchronous colonization can occur, permitting studies of the early stages of contact with the root. Primary colonization of roots can be initiated from as far away as 13 mm, as shown by calculations of the effective width of the rhizosphere of Trifolium subterraneum at 12 days. The width of the rhizosphere increased linearly at 0.5 mm d"^ up to this time, suggesting that hyphae grew towards the root at this rate (Smith et a/., 1986a) and confirming values for interplant spread of colonization (see above). Despite the increased mycelial growth in the presence of roots, hyphae do not always appear to make directional growth towards the roots until they are very close to them and in some investigations there was no evidence at all of this occurring (Mosse and Hepper, 1975; Powell, 1976; Becard and Fortin, 1988; Gemma and Koske, 1988b; see Koske and Gemma, 1992). However, once contact occurs, branching on the root surface takes place. In a number of investigations in both soil

100 H

80 H

r^ 60H

o o

0

2

4

6

8

10

Harvest (days) F i g u r e 2.5 Rapid colonization using 'nurse pots'. Total internal colonization ( • )

and

arbuscular colonization of Lyco/>ersfcon esculentum transplanted into *nurse pots' containing 6-week old mycorrhizal plants of Allium porrum

g r o w n in l o w P soil. N o t e that the

development of arbuscules ( • ) , occurs synchronously w i t h a time-span of between 6 and 16 days (see t e x t ) . Means and standard deviations of the means are shown. Unpublished data of G. Rosewarne.

Colonization of roots

51

and axenic systems, the main hypha (diameter 20-30 |Lim) approaching a root gives rise to a characteristic fan-shaped complex of lateral branches (diameter 2-7 |im), which may be septate, and colonization of the root usually occurs from these narrow lateral hyphae. Giovannetti et al (1993a,b) have used an elegant and simple system of millipore membrane sandwiches to study the differential morphogenesis of hyphae in response to the presence of host and non-host roots, while preventing actual contact between them. They showed, as can be seen in Figure 2.6 the development of a densely branched hyphal network on the surface of the membrane immediately over the roots of host, but not non-host, plants. Prevention of actual contact with the roots did, however, result in swelling and septation of the hyphae, which may indicate a stress response and this is being further investigated (Giovannetti, personal communication). These results indicate the existence of prepenetration stimuli which may include the flavonoids already discussed. However, as Friese and Allen (1991) emphasize, the formation of precolonization fans or obvious changes in hyphal morphogenesis does not always occur and sometimes a relatively undifferentiated, thick-walled hypha infects the root directly, confirming the much earlier observations of Nicolson (1959). Contaa and Penetration

Hyphal contact with the root is followed by adhesion and, after about 2-3 days, the formation of swollen appressoria (Becard and Fortin, 1988; Giovannetti et al, 1993b; and see Peterson and Bonfante, 1994). No appressoria are formed on dead roots or on various artificial fibres and a thigmotropic stimulus does not appear to be involved (Giovannetti et al. 1993b). These morphogenetic changes on the surface of the root indicate that the fungus has recognized the presence of a potential host plant (see Figure 2.7). Early stages of colonization at the ultrastructural level have been studied in Allium porrum-Glomus versiforme (Garriock et al, 1989). The elongated and elliptical appressoria are aligned with their long axes parallel to the long axes of the epidermal cells (Fig. 2.7c). There was little variation in either shape or position of the appressoria, but the length ranged from 16.8 to 79.8 \xxa. Large-diameter colonizing hyphae bearing small projections always develop from the appressoria and in a high proportion of cases penetrate both the adjacent epidermal cells (Fig. 2.7d). Colonization by other fungi is less well documented, but it is clear that single hyphae originating from the appressorium are more common for some. At this stage there may also be evidence for recognition of fungal attachment by the plant. Garriock et al (1989) observed the regular occurrence of slight wall thickening on the epidermal cell adjacent to the penetrating hyphae and even in the absence of thickening the walls fluoresced stongly after staining with acriflavine-HCl for polysaccharides with vicinal hydroxyl groups. As there was no fluorescence with aniline blue or berberine sulphate these thickenings probably did not contain either callose or lignin, a point confirmed by Harrison and Dixon (1994) for G. versiforme associated with Medicago. However, G. mosseae did not induce a response in Pisum sativum (GoUotte et al, 1993) and G. versiforme caused no changes in synthesis of phenols in either Allium or Ginkgo (Codignola et al, 1989). It is therefore unclear whether the response was related to plant or fungal

52

Vesicular-arbuscular mycorrhizas

species but, in any event, the thickenings did not prevent the penetration of fungal hyphae through the walls. Penetration of plant cell walls is always associated with narrowing of the hyphal diameter to form a peg, followed by expansion as the hypha enters the lumen of the cell. The cell wall may bulge as the hypha penetrates, indicating that pressure may play a part in the penetration process (Cox and Sanders, 1974; HoUey and Peterson, 1979). However, changes in the middle lamella structure when intercellular spaces are colonized by hyphae indicate the involvement of fungal enzymes such as pectinases (Kinden and Brown, 1975b; Gianinazzi-Pearson et ah, 1981b), a suggestion now supported by biochemical evidence of their production by spores and external mycelium (Garcia Romera et ah, 1990, 1991). The penetration of the outer cell layers of the root is influenced by the development of a hypodermis. In many plant species, including the very well-studied Allium, the layer of cells immediately beneath the epidermis is characterized by the presence of a casparian band on the tangential walls and, as the root matures, by increasing depositions of suberin on both tangential and radial walls (Shishkoff, 1987; Peterson, 1988). In some species this hypodermal layer is dimorphic and whereas the numerous long cells become rapidly suberized, this is delayed in the short 'passage' cells. The casparian band on the tangential walls offers no barrier to the entry of hyphae of mycorrhizal fungi but the suberization on the radial walls appears to do so. Consequently, the fungus enters by the passage cells and always coils within them (Gallaud, 1905; Kinden and Brown, 1975a; Brundrett et ah, 1985; Smith et al, 1989; Brundrett and Kendrick, 1990a,b). The interaction of the fungi with the hypodermal layer may be important for a number of reasons. It may affect the timing of susceptibility of the root (see below) and may also influence the control of the composition of the solution in the cortical apoplast, with consequences for nutrient transfer between the symbionts (see Chapter 14). Development of Infection Units Following the formation of an appressorium and penetration of the epidermis and exodermal cells, hyphal branches pass into the middle and inner cortex of the root and, in Arwm-type mycorrhizas, grow longitudinally in the intercellular spaces. Thus the fungal hyphae develop in a fan-shaped way across the outer cells of the cortex (Fig. 2.7a,b), with a growing colony subtended by one or a few closely associated entry points on the epidermis. These independent colonies were called 'infection units' by Cox and Sanders (1974), a term which is retained, despite the fact that 'colonization' rather than 'infection' is more appropriate for describing mutualistic plant-fungus associations. Each infection unit develops longitudinally and to some extent radially in the cortex of the root. Branches from the longitudinal

F i g u r e 2.6 Differential hyphal morphogenesis elicited in Glomus mosseae by roots of host plants growing underneath a millipore membrane, (a) Dense hyphae showing morphogenetic response (mr) above the roots of Oncimum basilicum. Non-elicited hyphae at some distance f r o m the roots (arrowed). From Giovannetti et al. (1993a), w i t h permission, (b) Detail of a similar response, showing the dense hyphal branching associated w i t h the presence of a root. Photograph courtesy of M. Giovannetti.

53 ^^"^^ft^t??-:^?-^i9'l|^^^®^<^^^^f:;;/C-^5f 'f'f^x:^}ii i:>^t3W^^W'^^^^^'^'':'?'''^^y'^^'^" " "'

Figure 2.6 (Caption opposite)

54

Vesicular-arbuscular mycorrhizas

hyphae give rise to arbuscules in the cells. Hence, the oldest arbuscules are closest to the site of penetration and the young and immature ones are progressively further away (Fig. 2.4b). The rates of growth of the intercellular hyphae have been estimated and, depending on the method, the species of plant and the environmental conditions, these range from 0.13 to 1.22 mm d~^ for Arum-type mycorrhizas (Smith and Walker, 1981; van Nuffelen and Schenck, 1984; Walker and Smith, 1984; Brundrett et al, 1985; Tester et al, 1986; Brundrett and Kendrick, 1990a; Bruce et ah, 1994). The maximum longitudinal extent of the infection imits appears to be about 5-10 mm in each direction from a simple or complex entry point. Intercellular hyphae may branch, anastomose and form multihyphal cords in some particularly large intercellular spaces. With the electron microscope or appropriate staining they can be seen to be multinucleate and they contain dense cytoplasm with numerous organelles, bacteria-like organisms (BLOs) and vacuoles, as well as glycogen and lipid reserves (see Scannerini and BonfanteFasolo, 1983; Bonfante-Fasolo, 1984; Peterson and Bonfante, 1994). The distribution of nuclei is fairly uniform (Bonfante-Fasolo et al, 1987; Cooke et al, 1987; Becard and Pfeffer, 1993; Bianciotto and Bonfante, 1993) and the hyphae are long-lived compared with the arbuscules, at least in fast-growing crop plants (HoUey and Peterson, 1979; Smith and Dickson, 1991; and see below). Thus in Arum-type mycorrhizas the hyphae appear to provide the living and persistent 'skeleton' of a mycorrhizal infection unit, the fimctions of which must include translocation of nutrients to and from the extraradical mycelium and possibly also transfer between the symbionts (Chapter 14). The characteristics of the infection units vary in different fungal species, so that experienced observers can recognize particular fimgi colonizing roots and study aspects of their rates of growth, colonization and competition, together with the extent of development of external hyphae and spore production (Abbott and Robson, 1979; Gazey et al, 1992; Jasper et al, 1992; Pearson et al, 1993, 1994). This approach has yielded some detailed information about the biology of the fungi and this is extremely useful in selecting suitable fungi for comparison and for understanding their interactions. Turnover of Arbuscules Arbuscules are usually relatively short-lived (at least in Arum-type mycorrhizas) and their development, maturation and collapse have been investigated at both the

Figure 2.7 Early stages of VA mycorrhizal colonization of roots, (a), (b) Precolonization branching of external hyphae (EH) and formation of appressoria by Glomus monosporus on the roots of Trifolium subterraneum. From Abbott (1982), with permission, (c) A single appressorium (*) of Glomus versiforme formed as a swollen branch hypha on the surface of a root of Allium porrum. External hypha arrowed. From Garriock et al. (1989), with permission, (d) Infection branches (arrowed) developing from an appressorium (*) of Glomus versiforme on the surface of a root of Allium porrum. From Garriock et al. (1989), with permission, (e) Section of resin-embedded tissue oi Allium porrum showing an appressorium (*) of Glomus versiforme in contact with the epidermal cell (c) and the development of infection branches. From Garriock et al. (1989), with permission.

Colonization of roots

55

F i g u r e 2.7 (Caption opposite)

56

Vesicular-arbuscular mycorrhizas

light and electron microscope levels in many plant-fungus combinations, so that it is possible to generalize about the changes that occur in cells of both symbionts (see Scannerini and Bonfante-Fasolo, 1983; Bonfante-Fasolo, 1984; Peterson and Bonfante, 1994). Detailed studies of arbuscule development and degeneration in several plant species have been made, using morphometric techniques (e.g. Toth and Toth, 1982; Alexander et al, 1989; Toth et al, 1990b, 1991), and will be discussed later. When a hyphal branch penetrates the plant cell wall to form the main trunk of the arbuscule, the plasma membrane is not breached but grows so that the invading hypha and all its branches remain surrounded by it. Hyphal branches dichotomize repeatedly (see Fig. 2.8a,b,c) and the fungus is always located outside the plant cell cytoplasm within an apoplastic compartment. The plant membrane surrounding the arbuscules (periarbuscular membrane, or PAM) is clearly modified functionally, although it retains staining properties and some activities similar to the peripheral plasma membrane of the cell from which it is derived (Dexheimer et al., 1979,1985). Specialization of the PAM is indicated by its reactions with two monoclonal antibodies, originally raised to study root nodules of legumes. MAC 64, which binds to glycoprotein epitopes in the peripheral plasma membrane of the cell and to the peribacteroid membrane (PBM), does not recognize the PAM. However, MAC 26 recognized the same oligosaccharide epitopes on the PAM, as well as the other two membranes (Gianinazzi-Pearson et al., 1990). Together with the fungal plasma membrane in the arbuscule branches the PAM delimits an interfacial zone (the interfacial matrix or apoplast), which appears highly specialized with respect to the molecules deposited within it and which has an important part to play in nutrient transfer between the symbionts (Smith and Smith, 1990; Bonfante-Fasolo et al, 1992; and see Chapter 14 ). At the base of the trunk of the arbuscule a layer of material is laid down (Fig. 2.8d,e) and this has similar chemical composition to the primary wall of the plant cell. It is thick at the base where it is continuous with that wall, but higher up the trunk hypha it becomes gradually thinner and appears to be absent from the finest branches of the arbuscule. A range of affinity probes, such as antibodies, lectins and enzymes. Figure 2.8 Development of arbuscules. (a)-(c) Scanning electron nnicroscopy of stages in the development of arbuscules of Glomus mosseae within cells of Liriodendron tulipifera. (a) Young arbuscule showing penetration point and dichotomous branching, (b), (c) Later stages in arbuscule development showing how the hyphal branches come to fill the cell volume. From Kinden and Brown (1975), with permission, (d) Section through an arbuscule trunk which is giving rise to a branch and a separate arbuscular branch (E). The interfacial matrix (m) can been seen to be an extension of the host periplasm (pe). The densely stained fungal wall is clearly distinguishable from the surrounding coating of fibrils and also from the host wall (hw) and apposition layer (c). Features of fungal and host (H) cytoplasm can be distinguished: both contain mitochondria and a nucleus (N). Bar, = I |im. From Dexheimer et al. (1979), with permission, (e) Detail of a penetration point of a hypha (E) into a host cell (H). The host plasma membrane (hp) is invaginated by the arbuscular trunk hypha, so that the fungus is surrounded by a continuation of the host periplasm (pe), forming an interfacial matrix (m). Within the matrix an apposition layer (c) of fibrillar material has been laid down; it is continuous with the host wall (hw) and clearly distinguishable from the darkly stained fungal wall. Bar = 0.5 jim. From Dexheimer et al. (1979), with permission.

Colonization of roots

Figure 2.8 (Caption opposite)

57

58

F i g u r e 2.9 (Caption opposite)

Vesicular-arbuscular mycorrhizas

Colonization of roots

59

Figure 2.9 Effects of different nnethods of fixation on transmission electron micrographs (TEMs) of mycorrhizal structures, (a) Chemical fixation of a mycorrhiza formed between Gigaspora margarita and Trifolium. The arbuscule branches (ab) are surrounded by the invaginated host membrane, forming a perlarbuscular membrane (arrowed). The interfacial apoplast is apparently about 0.1 jim wide. Photograph courtesy of P. Bonfante. (b) Freeze substitution of the same material shown in (a). The thin fungal wall is In close contact with the perlarbuscular membrane (arrowed). Photograph courtesy of R Bonfante. (c) Detail of the host-fungus interface after treatment with wheat germ agglutinin-gold, to reveal Nacetylglucosamine residues ( • ) in the amorphous fungal wall. Plant cell, p; fungal hypha, f. Photograph courtesy of R Bonfante.

have been employed to investigate the macromolecular composition of the walls of the symbionts and the interfacial apoplast. Wall deposition by both symbionts is clearly curtailed in the fine arbuscular branches. Structural molecules of plant origin such as (3(l,4)glucans, non-esterified polygalacturonans and hydroxyproline-rich glycoproteins (HRGP) are present, but are not polymerized as they are in a typical wall. Transmission electron microscopy (TEM) following chemical fixation suggests that the space between the PAM and branches of the arbuscules is relatively large (Figs 2.8d,e, and 2.9a). However, when material is prepared by freeze substitution, the PAM and fungal wall are closely adpressed, as shown in Figure 2.9b,c. Chitin in the fine branches of the arbuscules is much reduced and this reflects major changes in thickness and composition of the fungal walls as root colonization proceeds from the spore and extraradical mycelium to the finest arbuscular

60

Vesicular-arbuscular mycorrhizas

branches (Fig. 2.9c). Spores have thick, composite walls which contain a high proportion of chitin laid down in a complex helicoidal arrangement (BonfanteFasolo and Grippiolo, 1984; Bonfante-Fasolo, 1988; Grandmaison et ah, 1988); extraradical, intracellular coils and intercellular hyphae also have relatively thick walls (approximately 500 nm) with laminated chitin fibrils^ which may become even thicker in the intercellular phase as the roots themselves age and undergo secondary thickening. In contrast, the walls of the arbuscular branches show progressive thinning to approximately 50 nm as the hyphae themselves are reduced in diameter to 1-2 |Lim. No fibrillar structure is apparent although chitin molecules have been detected, at least in Glomus versiforme (Bonfante-Fasolo and Perotto, 1992; Bonfante-Fasolo et ah, 1992). The solute composition of the interfacial apoplast is quite unknown. We might predict that it would have a relatively low pH, but concentrations of solutes such as inorganic PO^ (Pi), sugars, amino acids, and mono- and divalent cations have not been determined. Nevertheless, they are of key importance in transport between the symbionts, and information is urgently needed. The fact that arbuscules provide a considerable increase in surface area of contact between fungus and plant has led to the belief that they are involved in nutrient transfer and it certainly seems likely that they are the sites for movement of soilderived nutrients, such as P and Zn, to the plant. It is less clear whether or not they are involved in carbohydrate transfer to the fungus and arguments for the possible role of intercellular hyphae, as well as arbuscules, will be discussed later. Here it is important to point out that the elegant calculations of Cox and Tinker (1976) make it clear that the collapse and presumed 'digestion' of arbuscules is quantitatively totally inadequate to explain the rates at which P passes from the fungus to the plant. At the stage when the arbuscule is growing and reaching maturity in the intracellular apoplast the mycorrhiza can be envisaged as an association between metabolically active fungal structures and living root cells, as Dexheimer et ah (1979) pointed out. They showed that the invaginated PAM was similar to the peripheral plasma membrane of the plant cell and that complex plasma membrane formations developed in the apoplastic region and these further increase the surface area of contact between the PAM and the interfacial apoplast (Dexheimer et al, 1985). Although polysaccharide continued to be produced, it was no longer polymerized. This activity was shown to be associated with changes in neutral phosphatase activity, which although low in uninfected cells and on the peripheral plasma membrane of infected cells, was highly active on the PAM (Jeanmaire et al, 1985). In the fungus, changes in activity of other phosphatases have also been observed, so that acid phosphatase is localized in the immature arbuscule branches, while alkaline phosphatase shows high activity in mature arbuscules and intercellular hyphae (Gianinazzi et al, 1979). Membrane-bound H'^-ATPase activity is seen consistently on the plasma membranes of external and intercellular hyphae, but becomes weak or absent on arbuscule branches, particularly as these age (Marx et al, 1982; Gianinazzi-Pearson et al, 1991a; see Fig. 14.8). There is also good evidence for increased physiological activity of the colonized plant cells. Around the arbuscules the PAM shows increased activity of H"^-ATPase and, again, plasma membrane formations are evident (Marx et al, 1982; Dexheimer et al, 1985; Gianinazzi-Pearson et al, 1991a). The nucleus and nucleoli are increased

Colonization of roots

61

in size, and the nucleus moves from a peripheral position close to the cell wall and takes up a central position, suggesting changes in cytoskeletal activity. The volume of the cytoplasm, which contains a full complement of organelles, is also increased and the vacuoles appear to become fragmented. The increased size of the plant nuclei does not seem to involve a change in ploidy, but is associated with an increase in the amount of decondensed chromatin which may indicate greater activity and delayed senescence (Berta et al, 1986, 1990, 1991, 1996; Blair et al, 1988). The longevity of the arbuscules also differs quite markedly from the intercellular hyphae. Again, it was the early workers, Gallaud (1905) among them, who first noted the way in which arbuscules grew in the cells and subsequently collapsed. After a relatively short period the intracellular arbuscules progressively degenerate to form clumps, whilst the plant cell remains alive. During this phase the fungal chromatin condenses and nuclear degeneration takes place (Balestrini et ah, 1992). Rarely, a cell may become reinfected by the fungus and come to enclose several digestion clumps. Following the work of Cox and Tinker (1976), a number of elegant studies of arbuscular turnover have been carried out in different plant species using morphometric techniques applied to electron micrographs of arbuscules in different stages of growth and collapse. Important points to note are that the surface:volume ratio of the plant cells is at first increased considerably, representing a two- to four-fold increase in plasma membrane area as a result of invagination by the arbuscules. Figure 2.10 shows that the species of host plant has a considerable effect on the percentage volume change and surface:volume ratio of plant protoplasts during development of arbuscules of Glomus fasciculatum. Arbuscules in grasses were generally larger than in the non-grasses, with consequent effects on the increase in volume of the plant cytoplasm and the surface area of interface between the symbionts (see Table 2.2). When calculating fluxes of nutrients between symbionts it is necessary to take these differences into account (see Toth et al, 1990b). It appears that the increase in cytoplasmic volume is very closely correlated with the increase in plasma membrane surface area, and may simply reflect the fact that there is always a thin layer of cytoplasm adjacent to this membrane. The duration of the arbuscular cycle also varies. In Triticutn aestivum formation of arbuscules took 2-3 days and the whole arbuscular cycle around 7 days, which agrees well with earlier estimates (Bevege and Bowen, 1975; Brundrett et al, 1985) and appears to be typical of mycorrhizas of rapidly growing crop species (Alexander et al, 1988). However, Brundrett and Kendrick (1990a) have pointed out that in slow-growing woodland plants the arbuscules appear not only to be much longer lived, but may also have wider and more robust branches. These differences may have significance for the functioning of the symbioses, as well as helping to explain how arbuscules could have become fossilized, which is hard to imagine for the fragile and transitory structures normally described. We do not know why arbuscules have such short life-spans in many plants. None of the proposed explanations are well supported by evidence but (apart from the idea of 'digestion') they include a manifestation of a host defence reaction against progressive fungal invasion. However, evidence for host cell involvement is generally lacking (Chapter 3) and a 'dead end' or programmed death for the fungus, which undergoes autolysis in what turns out to be the stressful environment of the cortical cell.

Vesicular-arbuscular mycorrhizas

62

Trunk

Host cytoplasm

Surface:volume ratio (plasma membrane:cytoplasm)

10

20

30

40 50 60 70 Percentage of cycle (%)

80

90

100

Figure 2.10 Arbuscular cycles in Zea mays (M), Triticum aestivum (W), Oryza sativa (O), Phoseo/us vulgaris (B), LycoJ)ersicon escu/entum (T) and Allium cepa (ON). The volume fractions of the different structures are plotted as a percentage of the plant cell volume occupied by each feature and the surface:volume ratio of the cell is given in jim^ |im~~^. From Alexander et al. (1989), with permission.

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•^

1^ CN hv ( N
4-> fd

0 Z Z

S x: ^(d fdN (1)

64

Vesicular-arbuscular mycorrhizas

may be the best explanation (Harley and Smith, 1983; Peterson and Bonfante, 1994; Becard, personal communication). As the individual infection units age, thick-walled vesicles may be formed. Whether or not this occurs depends in the first place on the identity of the fungus, as neither Scutellospora nor Gigaspora ever develop vesicles, but instead produce auxiliary cells on the extraradical mycelium. Members of all the other genera may develop vesicles to varying degrees and in either intercellular or intracellular positions in the cortex (e.g. Abbott, 1982). Environmental conditions strongly affect vesicle development, so that at high P or low irradiance they are reduced in the same way as arbuscules (see below). Vesicles are thick-walled structures of varying shapes, from ovoid, irregularly lobed to box-like, depending on the species of fungus and where the vesicle is formed. They contain abundant lipids and numerous nuclei and it is likely that they are important storage organs and may play a significant role as propagules within root fragments, as already mentioned. Nevertheless, little is known of their biology, in particular with respect to either germination or mobilization of the reserves. The biomass of the fungus associated with roots has been estimated from determinations of chitin content and by calculation, using the volume of fungus and assumptions of its fresh weight:dry weight ratio (see Harris and Paul, 1987; Toth et fl/., 1990b, 1991). Hepper (1977), using chitin content, obtained estimates of the dry weight of the fungus within the roots, which ranged from 4% to 17% of the total dry weight. Bethenfalvay et al. (1982) estimated the chitin content of extraradical and internal hyphae of Glomus fasciculatus grown with Glycine max and found that both the internal mycelium and extraradical mycelium reached a maximum at about 8 weeks, when the fungus contributed 20% of the biomass of the mycorrhizas, approximately one-third of which was external hyphae or spores. Although biomass estimates based on chitin neither distinguish living from dead fungus nor give information on development of arbuscules or vesicles, the values are similar to those obtained using volume, i.e. between 3% and 20% of the root dry weight (Kucey and Paul, 1982; Harris and Paul, 1987; Toth et al 1991).

Growth of External Hyphae and Spore Production External Hyphae As noted above, once the fungus is established in the root and growing vigorously in the soil, the external hyphae form an important source of inoculum for ongoing colonization of the same root system. Infection units developing on the same plant from this mycelium are referred to as secondary. Spores and auxiliary cells are formed in soil, sometimes at very high densities, so that the transfer of C from the roots to soil can be considerable and energy is further expended in both uptake and translocation of nutrients as well as in fungal growth and respiration. Extensive growth of external mycelium does not begin until after the root has been penetrated by the invading fungal hyphae, but it is not clear which stages of colonization are required. Mosse and Hepper (1975) noted considerable growth after the formation of appressoria. Hepper (1981) confirmed this finding and

Colonization of roots

65

observed that growth outside the root could precede the formation of any arbuscules within the cells. This suggests that nutrients could be transferred across the interface from plant cells to intercellular hyphae and that transfer across the arbuscular interface need not be involved, a point supported by the recent observations of extensive intercellular fungal growth in Pisum mutants which have highly reduced arbuscules (Gianinazzi-Pearson et al, 1994a; see Chapter 3). However, Becard and Piche (1989b) came to the conclusion that arbuscules are an absolute requirement for hyphal growth. Outside the root, main hyphae give rise to the characteristic branching systems illustrated in Figure 2.3b. Friese and Allen (1991) measured the branches in glass-sided boxes and showed that up to eight orders of branching were produced, becoming progressively narrower, finest (5th-8th orders) approximately 2 |Lim in diameter. These finely branched fans are clearly well adapted to exploration of soil pores and have also been found consistently associated with organic matter in soil, where they proliferate in localities in which mineralization of nutrients may be occurring (Nicolson, 1959; St John et al, 1983a; Hepper and Warner, 1983). The result is the formation of an absorptive hyphal network which may also be very important in stabilizing soil aggregates (Tisdall and Oades, 1979; Tisdall, 1991,1994; and see Chapter 16). Anastomosis and wound healing among the hyphae, which would be of importance in maintaining the pathways of nutrient translocation from one place to another in the network, have been observed in a number of investigations (Francis and Read, 1984; Becard and Piche, 1989). Because the external mycelium is important in so many ways, considerable effort is being made to overcome the difficulties of studying it. Early work involved picking out the hyphae and weighing them or estimating their extent by the weight of soil adhering to roots (e.g. Sanders et al, 1977; Graham et al, 1982). Immunofluorescence methods have also been tested (Aldwell et al, 1983; Kough et al, 1983; Wright et al, 1987; Wright and Morton, 1989; Sanders et al, 1992) but have not been widely applied. A recent review (Hahn et al, 1994) provides a critical evaluation of work to date and advocates that increased effort should be devoted to this technique. A number of DNA-based methods are now also being developed, in particular to study spores and intraradical mycelium. These include specific PCR primers, cloned probes or RAPD-PCR polymorphisms which have been used to study variation or identify particular fungal species or isolates. When coupled with improved methods of DNA extraction from soil and MPN techniques for satisfactory quantification, these methods certainly have some potential in the study of mycelium in soil, but are only just reaching the stage of being used in ecological studies (Wyss and Bonfante-Fasolo, 1993; Barker et al, 1994; Clapp et al, 1995; Sanders et al, 1995; Sulistyowati, 1995). Most of the published data on hyphal development have been obtained using a membrane filter technique, and the lengths of hyphae estimated after staining using a grid intersect method. Trypan blue and acid fuchsin are the most commonly used non-vital stains, while tetrazolium salts and fluorescein diacetate have been used to determine the proportion of the hyphal length that is active (Schubert et al, 1987; Sylvia, 1987,1990; Hamel et al, 1990; Sukarno and Dickson, 1992). A problem with these essentially destructive approaches is that recovery is certainly incomplete and losses of hyphal viability may arise during sampling. However, some confidence in the methods is derived from the fact that mycorrhizal hyphae within roots do not

Vesicular-arbuscular mycorrhizas

66

lose their ability to reduce tetrazolium salts even when the roots are cut into 100 jim sections (Smith and Dickson, 1991). Nevertheless, alternatives should be sought and so far the two most promising are vital staining of the intact mycelium prior to sampling (Saito et al, 1993) and use of ^^C as a tracer to detect actively translocating hyphae in intact networks (Francis and Read, 1984). However, these methods have a common drawback in that they do not permit repeated sampling of the same pot or field site. Use of phospholipid and neutral lipid profiles also shows promise as a method of quantifying external mycelium (Olsson et ah, 1995) It is difficult to distinguish hyphae of mycorrhizal fungi from those of soil saprophytes and of root pathogens. Absence of septa and of dark coloration, together with characteristic angular branching can be useful diagnostic characters Table 2.3 Hyphal length in soil, expressed as m g ' dry soil or m m ' root length (a) Data from pot experiments Fungus

Plant

Length Reference Length (nn g"' soil) (m m~' colonized root length)

Glomus fasiculatum

Cucumis sativus

27

ND

0.06-1.54 2-15 ND ND 2-25 ND 1-9

ND ND 105.5 7.1 ND 123 ND 79-250 71 71 71 25 142 49

G. clarum Thfolium reperis Acaulospora laevis T. subterraneum A. laevis T. subterraneum Scutellospora calospora Allium cepa S. calospora T. subterrar)eum S. calospora T. subterrar)eum Glomus spp. T. subterraneum Glomus mosseae A. cepa G. mosseae A. cepa G. macrocarpum A. cepa G. microcarpum A. cepa G. fasiculatum T. subterraneum G. ter)ue T. subterraneum Glomus sp. (E3) Lolium perenne

7.8

Jakobsen and Rosendahl, 1990 Schubert et o/., 1987 Jakobsen et o/., 1992a Abbott and Robson, 1985 Sanders et o/., 1977 Sanders et a/., 1977 Abbott and Robson, 1985 Jakobsen et a/., 1992a Sanders 1975 Sanders et a/., 1977 Sanders et o/., 1977 Sanders et o/., 1977 Abbott and Robson. 1985 Abbott and Robson, 1985 Tisdall and Oades, 1979

ND, not determined (b) Data obtained from field soils Soil

Plant

Length Reference Length (m g"* soil) (m m~' colonized root length)

Serengeti

Native grasses

0.03-6.95

ND

Cultivated red-brown earth, Australia

Lolium perenne

13.9

0.96

McNaughton and Oestenheld, 1990 Tisdall and Oades, 1979

Thfolium repens

3.1

46

Tisdall and Oades, 1979

Colonization of roots

67

for the main hyphae, but the fine hyphal branches are very difficult to identify with certainty. Table 2.3 shows estimates of hyphal length per unit of infected root or per unit weight of soil, updated from a compilation by Smith and Gianinazzi-Pearson (1988). The data show that hyphae represent considerable C flow to the soil, which may be distributed to sites well beyond the zone normally designated as the rhizosphere, as can be seen in Figure 2.11a (Jakobsen and Rosendahl, 1990; Jakobsen et al, 1992a; and see Finlay and Soderstrom, 1992). The estimates of hyphal lengths vary considerably (Table 2.3), as different fungi

Glomus

s p . (WUM 10 (1))

^

Scutellospora

0

1 2

3 5 7 9 Distance from roots (cm)

calospora

11

Figure 2.11 Developnnent of extraradical mycelium, (a) Length of external hyphae spreading from mycorrhizal roots of Trifolium subterraneum after 28 days (i) and 47 days (ii). Soil cores were sampled at increasing distances from the Voot compartment', up to I I cm. Bars are standard errors of means. # , Acaulospora laevis; • , Glomus sp.; T , Scutellospora calospora; x, control. From Jakobsen et al. (1992a), with permission, (b) Suggested patterns of development of hyphae within and outside roots for two different VA mycorrhizal fungi. Effects of different densities of hyphae at the root surface are shown. From Abbott et al. (1992a), with permission.

68

Vesicular-arbuscular mycorrhizas

produce different amounts of mycelium, with different densities in relation to distance from the root (Fig. 2.11a; Jakobsen et ah, 1992a). Some, such as Scutellospora calospora, consistently produce extensive external mycelium, which increases as the extent of colonization of the roots increases. With this fungus the hyphal density per gram of soil declines more or less linearly as distance from the root increases. Glomus sp. (WUM 10) has a more limited capacity for mycelial growth, which is related not to internal colonization but to the number of infection units (Abbott et al, 1992b; Jakobsen et fl/., 1992a; see Fig. 2.11b). In another investigation the length of hyphae produced by two species of Acaulospora varied with both inoculum density and time. Length of hyphae of A. laevis per gram of soil was unaffected by the density of the inoculum, whereas that of Acaulospora WUM 18 showed an approximately twofold increase at 42 and 56 days after planting. These investigations did not include information on proportion of living mycelium, which may be much less than the total and may also show differing relationships with the extent of internal colonization (see Sylvia, 1987,1990). A comparison of two isolates of G. etunicatum (at equivalent propagule densities) showed that although there were no differences in total or living length of hyphae per gram of soil, one produced considerably more living mycelium per unit of arbuscular colonization than the other (O'Connor, 1994). There is little information on the effects of soil conditions on development of external hyphae. Abbott et al. (1984) showed that high P levels in soil reduced the length of hyphae, while Schiiepp et al (1987) showed that spread of hyphae occurred at lower rates in calcined clay, peat moss and chopped hay than in various soil-based media. Proliferation of hyphae in organic matter has also been noted (St John et al, 1983b; Warner, 1984). Measurements of mycelial development are much more difficult in field systems than they are in pots. In the Serengeti Park the length of mycelium associated with C4 grasses varied between 0.03 and 6.95 m g~ soil, and in this habitat was negatively correlated with soil organic matter and nutrient status (McNaughton and Oestenheld, 1990). Other measurements of hyphal length in field soils have been related to soil structural stability and varied considerably with species of host plant used in the experiments (see Table 2.3).

Spore Production The external mycelium is important in the production of spores, and must translocate relatively large amounts of carbohydrate into them, adding considerably to the biomass of fungus outside the root in the soil. Sieverding et al. (1989) found a maximum of 28 spores g~^ soil associated with a cassava crop and calculated a biomass of up to 919 kg ha~^ at the highest spore densities which, as they pointed out, was actually greater than some estimates of ectomycorrhizal fruit body production. As previously stated, it is hard to draw inferences from populations of spores in field soils about either their relationships with root colonization or factors influencing spore production, but some experiments with annual plants indicate that spore production increases as plants mature at the end of the growing season (Hayman, 1970; Sutton and Barron, 1972; Koske and Halvorsen, 1981; Giovannetti, 1985). In some cases a general decline in numbers of spores during early growth is followed by an increase as the plants mature (Saif, 1977). Hayman (1970) observed that much

Colonization of roots

69

larger numbers of spores were produced on wheat that had not received high levels of N fertilizer, conditions which also resulted in higher root colonization. In pot experiments spore production, as well as colonization of the root systems, is affected by factors such as plant growth, fertilizer application and light intensity. Factors such as low irradiance or defoliation, which reduce photosynthesis and hence C supply to the plants, have been repeatedly shown to reduce sporulation as well as colonization (e.g. Furlan and Fortin, 1977; Daft and El Giahmi, 1978). Daft and Nicolson (1972) showed that the application of increasing quantities of P to Lycopersicon esculentum plants reduced not only the proportion of the root system colonized by 'Endogone' (Glomus) 'ntacrocarpa' var. Caledonia but also the number of spores associated with each plant after 84 days' growth. Douds and Schenk (1990) found lower spore production in several fungi when complete Hoaglands nutrient solution was applied, compared with the same solution lacking P. Similar effects of P on spores produced in field soils have been found (e.g. Ross, 1971; Hayman et al, 1975; Porter et al, 1978). Low nutrient concentrations are conducive to high colonization so there may be a link between extent of intraradical colonization and spore production. This was certainly shown for two species of Acaulospora, which required different critical lengths of intensely colonized mycorrhizal root before sporulation commenced, and for 'Gigaspora' (Scutellospora) calospora in which numbers of spores formed at 119 days were closely correlated with colonization of the roots of Trifolium subterraneum at 91 days (Scheltema et al, 1987a; Gazey et al, 1992). However, Daniels Hetrick and Bloom (1986) found a poor correlation between spore production and extent of colonization by three Glomus species in five host species, although there were major effects of plant species on numbers of spores produced per plant. Baylis (1969) investigated the possibility that a stimulation of sporing might be provided by drought or intermittent root growth, but found no evidence for either in his experiments with 'honey coloured' spores and the host plant Coprosma robusta. In the tropics, such seasonal influences are unlikely and the results of Louis and Lim (1987; see Fig. 2.1), as already mentioned, showed no clear correlations between environmental factors and changes in spore populations, although these did decline as root colonization increased. The effect of the plant species on spore production has been observed both in pots and in the field. For example, Struble and Skipper (1988) found that four Glomus species and one Gigaspora species always had poor spore production on Glycine and that each of the fungi sporulated to different extents on Zea, Paspalum and Sorghum. Glomus clarum also sporulated better on maize and sorghum than on chickpea and on the first two plant species the process continued until plant growth reached a maximum (Simpson and Daft, 1990). None of the three Glomus species investigated by Daniels Hetrick and Bloom (1986) sporulated well on Asparagus, and spore production was again greater on Sorghum than on the dicotyledons Tagetes, Trifolium or Lycopersicon. Pot experiments on individual fungal species have provided information on factors which result in high rates of sporulation; experimentation is likely to continue, with the aim of producing spore-based inoculum for both research and commercial purposes. Abbott and Gazey (1994) have highlighted the need to extend these investigations to include the effects of interactions between species on their development, including sporulation. This would link experimental work

70

Vesicular-arbuscular mycorrhizas

with field investigations and greatly increase our understanding of the ways in which the populations of fungi and plants interact in natural vegetation systems.

Distribution and Rate of Formation of Infection units Primary colonization readily occurs on young roots but the actual root apices rarely if ever become colonized. Using a 'nurse pot' system with Glomus versiforme and Allium porrum, Brundrett et al. (1985) showed that external hyphae approached the roots after 1 day and their occurrence increased throughout the 8 days of the experiment. The first penetration points were observed at 2 days, arbuscules at 4 days and vesicles between 3 and 6 days. The length of the infection units increased linearly over this period and measurements of the largest infection units gave a rate of growth of 1.2 mm d~^ (combined growth in both directions), as we have already discussed, while roots grew on average 6.0 mm d~^. The first penetration points were found 11 mm from the tip. This distance is similar to that observed for A. porrum growing in pots with evenly distributed fragmented inoculum, and rather longer than distances in roots with slower rates of extension, such as clover (Smith et ah, 1986b, 1992) or slow-growing woodland species (Brundrett and Kendrick, 1990a). There is considerable variation in length of infection units along young roots of both clover and leek, suggesting that there is no particular infectible region behind the root apex (Smith et al, 1986b; Brundrett et al, 1990). A more detailed study showed that the distribution of colonizations behind the root tip in both Trifolium and Allium was no different from that which would be expected if colonizations were random and determined by the length of time a root had been present and available for colonization (Smith et al, 1992). This conclusion is important and contradicts the earlier (and oft quoted) statement by Smith and Walker (1981) that the region immediately behind the apex is ten times more infectible than the root system as a whole. The finding of uniform susceptibility near the tip does not discount the possibility of changes in the susceptibility of roots as they age beyond the 2-3 weeks that are usually used for experiments of this kind. Transplantation of Allium and Trifolium plants of different ages to soil containing mycorrhizal propagules showed that Allium roots retained susceptibility up to 31 days, whether or not they had grown before transplanting, whereas Trifolium showed much reduced susceptibility from 5 days of age. Interestingly, the leeks remained susceptible well after the hypodermis would have become suberized (see Brimdrett et al, 1985; Brundrett and Kendrick, 1990b), suggesting that penetration through the less well suberized passage cells is important. The rate of initiation of primary colonization from propagules in soil is influenced by the availability and density of inoculum, as discussed earlier. Both Smith and Walker (1981) and Carling et al (1979) have shown a linear relationship between numbers of entry points and the density of propagules in soil (determined by MPN or by weight of infective root fragments) at relatively low propagule densities. However at higher densities the number of entry points did not continue to increase, suggesting that availability of susceptible root length may limit the extent of colonization. The propagule density (MPN) was also positively correlated with colonization in Phleum and Agropyron grown in field soil, but not with growth of the plants (Clapperton and Reid, 1992). Temperature, not surprisingly, influences the rate of formation of entry points, as it does other

Colonization of roots

71

aspects of the colonization process (e.g. Smith and Bowen, 1979). Some data indicate that attachment of hyphae and formation of appressoria on Lolium, Trifoliutn and Allium are suppressed at high P concentrations Qasper et al, 1979; Amijee et al, 1989a; Smith and Gianinazzi-Pearson, 1990; Thomson et al, 1990) but the same did not apply to Cucumis, in which the first colonizations were initiated at the same time and at the same density regardless of P supply (Bruce et al, 1994). Reduced irradiance also reduces the rate of formation of entry points, suggesting that there may be links between rates of photosynthesis and rhizosphere or root surface factors influencing the initial stages of colonization (Tester et al, 1986). Percentage Colonization of the Root System Methods of Assessment

Preparation of roots for light microscopy involves clearing the tissue (usually with 10% potassium hydroxide) and, after washing, staining with Trypan blue (Phillips and Hayman, 1970), acid fuchsin (Merryweather and Fitter, 1991) or chlorazole black (Brundrett et al, 1984). Trypan blue was the most commonly used, but is now registered as a carcinogen. Aniline blue (or cotton blue, as it is often called) is reported to be equally effective (Grace and Stribley, 1991) and is non-hazardous, while the other two stains can give excellent results, particularly with epifluorescence or Nomarsky optics (see Merryweather and Fitter, 1991; Brundrett et al, 1994). Vital staining on fresh material to detect the activity of different enzymes, such as succinate dehydrogenase, alkaline phosphatase and hydrolases, has also been used to determine the proportion of the fungus that is active (e.g. Schubert et al, 1987; Sylvia, 1987; Smith and Gianinazzi-Pearson, 1988; Hamel et al, 1990; Smith and Dickson, 1991; Schaffer and Peterson, 1993; Tisserant et al, 1993). Problems with these methods include differential tissue penetration by the chemicals (in particular when fungal walls are thick and impermeable, as they are in vesicles), background staining in plant tissues and poor contrast of stains with soil particles. The extent of colonization is usually expressed as the percentage or fraction of the root length colonized by mycorrhizal fungi. The most widely used method is a modification of a grid intersect method devised to measure root length by Newman (1966). Giovannetti and Mosse (1980) compared this with other methods and concluded that the standard error of estimates of percentage colonization was lower than for any of the methods based on mounting root pieces on slides. The grid intersect method has the additional advantage of permitting relatively large root samples to be scored and, if weighed subsamples of root are used, the length: weight ratio can be obtained, hence providing very important data for the total root length of the plants. Furthermore, the method can be adapted to measure lengths of hyphae on membrane filters. In addition to percentage colonization, it is important to determine its characteristics, that is the intensity of colonization of the cortex and the extent of development of intercellular hyphae, arbuscules and vesicles (sometimes referred to as 'quality of infection'). This can be achieved by applying the grid intersect method at magnifications that are high enough to allow visualization of these structures

72

Vesicular-arbuscular mycorrhizas

(Amijee et ah, 1989a; McGonigle et ah, 1990b) by applying image analysis to appropriately stained, transverse sections of roots (Smith and Dickson, 1991), or by a semisubjective ranking method using root segments observed at high magnification (Trouvelot et ah, 1986). The Progress of Colonization in Root Systems Colonization of a root system by VA mycorrhizal fungi is a dynamic process, in which both root and fungal components grow and develop. The root grows apically by cell division, elongation and differentiation and it initiates lateral roots. At the same time the fungus initiates both primary and secondary infection units, which grow and colonize the root cortex. The rate at which the root system becomes colonized (and hence the percentage colonization) is influenced not only by the rate of formation of the infection units and their rate of growth, but also by the rate of growth of the root system (see Sutton, 1973; Smith and Walker, 1981; Sanders and Sheikh, 1983). It is very difficult to determine direct effects of environmental variables on fungal colonization or growth using percentage colonization of the root system, unless it has been ascertained that there are no environmental effects on root growth itself. Moreover, differences in percentage colonization of different species or different genotypes of the same species of plant may be controlled not by actual susceptibility of the root systems but by differences in the rates of root growth. That said, there is no doubt that percentage colonization is a very convenient parameter to use. It can be measured on appropriately stained, representative subsamples of a root system and large numbers of samples can be processed relatively quickly. A graph of the percentage of the root length colonized against time has a sigmoid form, and examples are illustrated in Figure 2.12a. Similar curves have been obtained for single fungi in pot culture, as well as for mixed populations from

Figure 2.12 Colonization of roots by VA mycorrhizal fungi as affected by a range of different conditions, (a) The progress of colonization in roots of A///um cepa inoculated with four different mycorrhizal fungi, (i) Glomus mosseae; (ii) Glomus macrocarpus; (iii) Gigaspora (Scutellospora) calospora; (iv) Glomus microcarpus. From Sanders et al. (1977), with permission, (b) Progress of colonization (expressed as intensity of colonization, m%, see Trouvelot et o/., 1996) of Glomus intraradices in roots of Allium porrum, revealed by staining with Trypan blue ( • ) , or for activity of succinate dehydrogenase (O) or akaline phosphatase ( • ) . Shoot dry mass of mycorrhizal (A) and non-mycorrhizal (A) plants. Bars are standard errors of means and data points bearing different letters are significantly different at the 95% confidence level at each harvest. From Tisserant et al, (1993), with permission, (c) The effects of differences in propagule density on the progress of colonization in Trifolium subterraneum. Plants were grown in non-sterile soil with a propagule density (determined by the most probable numbers method) of 4.0 g~' ( • ) or in soil diluted with steamed sand to provide a propagule density of 0.4 g~' ( T ) . From Smith and Smith (1981), with permission, (d) The effect of additions of potassium acid phosphate (KH2PO4) to soil on the percentage of the root length of Allium cepa colonized by VA mycorrhizal fungi after 8 weeks' growth. Redrawn from Sanders and Tinker (1983).

73

Colonization of roots

(a)

(b)

i00r

0

2 4 6 8 10 12 Time after transplanting (weeks)

2 4 6 8 10 12 Time after transplanting (weeks) 80 r

(c)

60

•g 40

20

10 15 20 25 30 35 40 Time after planting (days) F i g u r e 2.12 (Caption opposite)

0.1 0.2 0.3 KH2PO4 (g per kg soil)

0.4

74

Vesicular-arbuscular mycorrhizas

field soil. The key elements of the relationship are the lag phase before colonization is detectable, a phase of rapid increase in colonization, during which fungal spread exceeds the rate of root growth, and a plateau phase in which spread of the fungus and growth of the root are constant relative to each other. In some investigations there may also be a late decline, in which the rate of root growth, exceeds the rate of fungal colonization. These well known relationships are based on staining with non-vital stains and consequently take no account of possible death of either fungal hyphae or root cells. A number of studies using different vital stains have demonstrated that while in young plants all hyphae and arbuscules are active, as the plant and fungus age a smaller and smaller percentage of the intraradical mycelium remains alive (Toth et ah, 1991). Smith and Dickson (1991) have used image analysis of sectioned roots to demonstrate that the decline in fungal activity is associated with a reduction in density of arbuscules, while the intercellular hyphae remain alive for longer. Different 'vital stains', such as nitroblue tetrazolium (NBT, for succinate dehydrogenase) or Fast Blue RR in the presence of oc-napthyl acid phosphate (for alkaline phosphatase), do not necessarily give the same picture of fungal activity, so that care in interpretation of data is needed (see Fig. 2.12b and Tisserant et ah, 1993). The progressive loss of activity of the fungus and change in the quality of colonization help to explain why a close relationship between the percentage colonization (determined by a non-vital stain) and a positive growth response of the plant is not always observed (see Fig. 2.12b; see Fitter and Merryweather, 1992). In addition, if colonization is only determined at a final destructive harvest, then a strong correlation is even less likely, because early, rapid colonization (short lag phase and rapid spread) have been shown to be very important in influencing nutrient absorption and growth, rather than the final plateau value of percentage colonization. Influence of Environmental Factors on Percentage Colonization Quite large differences in percentage colonization are frequently observed with different plant-fungus combinations and are often related to differences in rates of root growth and susceptibility of the plants, as well as to different fungal strategies in root colonization. Plants such as cereals, with rapid rates of root growth, tend to have lower plateau values for percentage colonization than those with slower growth, such as clover or leek. Comparisons of fungi are more difficult because it is difficult to standardize inoculum, but where that has been achieved it is clear that major differences can exist (Daniels et ah, 1981; Bowen, 1987; O'Connor, 1994). Sanders et al. (1977) measured the rate of colonization of Allium cepa and found that one fimgus, probably Glomus 'microcarpus', which was very slow to colonize roots, had little or no effect on plant growth or P uptake, whereas Glomus mosseae, Glomus 'macrocarpus var. geosporus' and 'Gigaspora'; (Scutellospora) calospora colonized more quickly and produced increases in P uptake and growth (Fig. 2.12a). This work remains one of the most thorough comparisons of fungi with respect to percentage colonization of the roots as well as P uptake and growth of plants (see Figs 4.1 and 5.2). There are also important environmental influences on percentage colonization: the most extensively investigated are density of inoculum, temperature, light and the availability of nutrients, in particular P. The effects of soil salinity have been

Colonization of roots

75

recently reviewed by Juniper and Abbott (1993) who concluded that although high salinity is often linked with low percentage colonization, it is impossible to pinpoint the mechanism (osmotic or the effects of specific ions) from the currently available data. Many nitrogenous fertilizers have been reported to decrease colonization both in pots and in the field (Lanowska, 1966; Hayman, 1970; Chambers et al,, 1980) and in some cases these acted directly on the fungus, and were not simply the result of changes in root growth. There are interactions between the effects of N and P on plant growth and their effects upon colonization; P exerts a more marked effect in N-sufficient plants than in N-deficient plants (Sylvia and Neal, 1990). However, the mechanisms of effects such as this are not clear. High propagule densities reduce the length of the lag phase in the curve of percentage colonization v. time (Fig. 2.12c) and may also be associated with a rapid spread of the fungus within the roots, which is important in field situations. After primary colonization from propagules has taken place, the growth of the extraradical mycelium gives rise to increased fungal colonization of the soil and also results in the formation of secondary infection units, which increase the number of connections between the internal fungal structures and the external mycelium. Experimentally, it is almost impossible to distinguish between primary and secondary entry points, and data on factors affecting their formation are nearly always combined. Low numbers of propagules in field soils (e.g. in eroded sites) may result in low levels of colonization and the need to evaluate the infectivity or propagule density in soil, in order to predict outcomes in terms of P uptake and growth of crops or in restoration programmes, is very widely recognized (see Reeves et al, 1979; Jasper et a/., 1988; Allen, 1989; Abbott and Robson, 1991; Miller and Jastrow, 1992a,b). The effects of temperature on the rate and extent of colonization are complex, the responses varying with the host plant and the fungus. There is usually an increase in percentage colonization up to about 30°C, but some plant-fungus combinations develop normally up to 35°C or more (see Bowen, 1987). Variations may represent adaptations to different environments - rapid germination and colonization would be an advantage in the humid tropics, whereas more subtle interactions between soil moisture and optimum temperature for germination and colonization may have evolved in more seasonal environments (see above). Experiments have often been carried out above 15°C, but many plants, both wild and cultivated, grow and develop mycorrhizas at lower soil temperatures in temperate regions. Baon (1994) found that barley failed to become colonized by Glomus etunicatum when root temperatures were held at lO^C, although it became colonized at 15°C. However, Allen and Friese (1989) found high (40-60%) colonization oiAgropyron by both field {Glomus spp.) and pot-culture (Gz. margarita) inoculum at \TC, and Daft et al. (1980) commented that in English bluebells colonization increased rapidly in the winter months when soil temperatures were near to 5°C. It is clear that generalizations are dangerous and that there is a need for more work which is specifically directed at understanding the biology of propagule survival, and germination and colonization of roots in particular habitats. It is frequently stated that high P concentrations eliminate mycorrhizal colonization. While it is well known that P availability influences percentage colonization, the magnitude of the effect is strongly influenced by host species and environmental factors, in particular irradiance. A typical response curve of P addition v.

76

Vesicular-arbuscular mycorrhizas

percentage colonization is shown in Figure 2.12d. Very low P availability may in fact inhibit colonization so that small additions result in increased percentage colonization (Tinker, 1975; Bolan et ah, 1984a). Further additions of P always result in reductions and the sensitivity of the response appears to differ in different host plants. Two examples will serve to illustrate this, although many others are to be found in the literature. Baon et al. (1992a) found that whereas wheat, barley and rye grown in soil containing 5 mg kg~^ bicarbonate extractable P had up to 40% of their root length colonized, additions of 5 mg kg~^ markedly reduced colonization and 30 or 60 mg kg~^ effectively eliminated the fungus. Furthermore, different cultivars of barley were not only colonized to different extents by Glomus iniraradices, but the extent of colonization was variably sensitive to P addition (Baon et al., 1993). On the other hand, in Trifolium suhterraneum, application of sufficient P to achieve maximum growth only reduced colonization from 74% to 53% (Oliver et al, 1983; see Table 4.1). Irradiance interacts strongly with P, with marked reductions in percentage colonization following P application to Allium cepa only apparent at low irradiance (Graham et al, 1982a; Son and Smith, 1988; Smith and GianinazziPearson, 1990; Fig. 2.13). The different responses are of practical importance and need to be emphasized because the view that high P levels always eliminate mycorrhizal colonization of roots is still widely held. The effect of increased P supply is partly mediated by increased root growth.

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F i g u r e 2.13 The effects of irradiance and additions of P on VA mycorrhizal colonization of roots of Allium cepa in low-P soil. High light, l o w P, • ; high light, added P, • ; l o w light, l o w P, • ; l o w light, added P, Q. N o t e the combined effect of added P and l o w light in reducing the percentage of the r o o t length colonized. D r a w n f r o m data of C . L Son and S.E. Smith.

Colonization of roots

77

Bruce et al (1994), using the modelling approach of Smith and Walker (1981), have shown that P applied to mycorrhizal Cucumis plants increased rates of initiation and extension of lateral roots, but these effects did not occur early enough to provide a complete explanation for reduced percentage colonization which was first observed at 10 days. Growth of infection units was reduced by added P from as early as day 8 and although the length of the lag phase of colonization was unaffected, the rate of production of entry points was very much lower in the presence of added P from day 20 onwards. This was probably the result of reduced secondary colonization. Effects of P on arbuscule development are somewhat variable. Sanders and Tinker (1973) and many subsequent observers (e.g. Hayman, 1974; Graham et ah, 1982a; Smith and Gianinazzi-Pearson, 1990; Amijee et al, 1989a; Bruce et ah, 1994) reported reductions in the densities of arbuscules in roots, but although Abbott and Robson (1979) observed lower percentage colonization in Trifolium suhterraneum, Erodium botrys and Lolium rigidum they saw no effects on arbuscule development. Given that high P concentrations in the medium reduce the growth of germ tubes, it might be expected that P in the soil might have an effect on hyphal growth from propagules and the initiation of colonization. Alternatively, high P within the roots might influence the rate of spread of the fungus and the growth of the extraradical mycelium. As shown above, the results are variable. Sanders (1975) investigated this problem by injecting P solution into the hollow leaves of onion plants. P was translocated from shoot to root and at the final harvest the percentage colonization was reduced, probably as a result of slower growth of the hyphae along the cortex of roots of high P content, a point which has been confirmed by Bruce et al (1994). The amount of external mycelium produced per centimetre of infected root was reduced from 3.5 to 2 mg, and there were also changes in the anatomy of the infection units. Sanders (1975) therefore concluded that the effects of soil P in reducing colonization were mediated via the root and need not involve any direct effects upon fungal growth in the soil. This early demonstration has been confirmed with other plant species using transplanting experiments and split-root techniques. However, the importance of direct effects via fungal growth in soil are supported by the results of Amijee et al (1993) who showed that the 'odds' on colonization were strongly reduced as soil P was increased. Menge et al. (1978) showed a reduction in numbers of chlamydospores produced by Glomus fasciculatus on both halves of a split root system of sudan grass, even though only one half received high levels (750 mg kg~^ soil) of P. Additional experiments indicated that the reduction in numbers of arbuscules and external hyphae, as well as chlamydospores, was more closely related to the P concentration in the roots than to that in the soil. Jasper et al. (1979) observed that a lower percentage of the root volume was colonized and fewer entry points per unit length of root were produced when Trifolium subterraneum was transplanted from sterilized soil low in P to a soil high in P containing spores of Glomus monosporus. Such an effect was not observed when plants from sterilized high-P soil were transplanted to infective low-P soil. In this experiment high P concentrations were associated with lower soluble carbohydrate concentrations in the roots and the authors suggested that carbohydrate availability to the endophytes might be important in determining fungal establishment, a point which will be elaborated later. Following early work which indicated reductions in colonization at low light and

78

Vesicular-arbuscular mycorrhizas

which provided speculation on the relationships with carbohydrate supply (Peuss, 1958; Schrader, 1958; BouUard, 1959), the effects of light and defoliation have continued to be investigated and it is clear that percentage colonization is almost always reduced in situations where supply of photosynthates might also be expected to be lower. Daft and El Giahmi (1978) found that both defoliation and either shading or short day length reduced percentage colonization and numbers of secondary spores produced by Glomus macrocarpus var. geosporus or G. mosseae in a variety of host plants. Hayman (1974) found that colonization was higher at higher light intensities and that this was correlated to sugar concentrations in the roots. The reduction in colonization at low irradiance is more marked if P supply is high (e.g. Graham et al, 1982a; Son and Smith, 1988; Smith and Gianinazzi-Pearson, 1990; Thomson et al, 1990a) and there have now been a number of investigations of the way light and P supply interact with the pools of soluble carbohydrate in the root and the amount and composition of root exudates, with the aim of correlating this with mycorrhizal colonization. Ratnayake et al. (1978) formed the hypothesis, based on experiments with Citrus, that exudation of substances from the roots of plants growing in low P conditions is increased as a result of the decrease in phospholipids and an increase of permeability of the cell membranes. They found that there was a much greater leakage of amino acids and sugars from the roots and they suggested that these might stimulate the growth of the fungus and the development of mycorrhizal colonization. They followed this up (Graham et al, 1981,1982a; Johnson et al, 1982; Schwab et al, 1982, 1984; and see Schwab et al, 1991) by studying other conditions where increased exudation results in an increase of mycorrhizal colonization, including an example of a non-host plant {Chenopodium quinona) in which extremely slight colonization was induced by the application of the herbicide simazine. In other investigations, the correlation between percentage colonization and concentrations of soluble carbohydrates in the roots systems was closer than that between percentage colonization and concentrations of exudates (Jasper et al, 1979; Thomson et al, 1990b, 1991). The picture is not clear, because negative correlations between P and soluble carbohydrates have also been found (Amijee et al, 1993; Pearson and Schweiger, 1993) and these more recent investigations have highlighted problems in interpretation of the earlier work. Deduction of causal relationships between the size of the soluble carbohydrate pool and extent of colonization is risky, because the pool size is determined by both input and output, i.e. by production and use. Data have been interpreted in two main ways: (1) the higher the pool size the more carbohydrate is available for the fungus (this is the argument used when a positive correlation between colonization and carbohydrate concentration is found) and (2) the higher the pool size the less carbohydrate the fungus has consumed (this is the argument used to explain negative correlations). The possible effects of the fungus itself in inducing changes in carbohydrate status or effects of carbohydrate status on root growth, and hence percentage colonization, have been more or less ignored. Amijee et al, (1993) measured the rate of growth of the fungus within the roots (rather than percentage colonization) and failed to find a close correlation between that and soluble carbohydrate concentration. They emphasized that in Allium the carbohydrate pools were similar in mycorrhizal and non-mycorrhizal plants that contain the same shoot P concentrations. They did observe some effects of mycorrhizal

Colonization of roots

79

colonization on the relative concentrations of monosaccharides (glucose and fructose). Light and P do not necessarily operate in the same way in reducing percentage colonization: Tester et al. (1986) found no effect of reduced irradiance on the rate of growth of infection units in Trifolium, whereas this parameter was influenced by P supply in Allium and Cucumis (Amijee et al., 1989a; Bruce et al., 1994). In summary, while it is clear that both low irradiance and high P reduce colonization, the mechanism(s) by which they do so are unclear and will not be elucidated until we have a better idea of the mechanisms by which the fungus obtains carbohydrate from the roots.

Root Growth The effect of mycorrhizal colonization on root growth has been investigated in Allium and although some work indicates that there are no changes in rates of apical extension or initiation of branches (Buwalda et al., 1984) it is now well established that changes do take place even if the colonization of the root by mycorrhizal fungi is relatively slight. Berta et al. (1990, 1991) have demonstrated that the rate of growth of root apices slows down after colonization by a Glomus species and that this is associated with a decrease in the mitotic index, because of extensions of G l , S and metaphase and marked reductions in the duration of G2. At the same time an increase in initiation of lateral roots is observed, presumably stimulated by the loss of activity of the apices of the main adventitious roots. The mechanisms underlying these changes are obscure and clearly operate at a distance from the actual sites of colonization, but as Koske and Gemma (1992) have pointed out, the increased branching of roots as well as that of hyphae would increase the chances of encounters between roots and infective hyphae. This is not the place to discuss environmental effects on root growth and development, but factors such as nutrient availability, temperature and soil compaction clearly exert marked effects on both apical extension and branching, and these will affect the interpretation of measurements of percentage colonization.

Conclusions The propagules that can initiate VA mycorrhizal colonization have been identified as spores, root fragments and hyphae. The latter form a complex network which links plants of the same and different species and when these are growing the infectivity of the mycelium is very high. The networks appear to be able to survive in both dry and cold conditions, which is probably very important in initiating colonization early in the following season. The processes leading to the colonization of roots and the way in which the fungi develop in the root systems is well understood in a few plant species. The outcome of the colonization process is that the fungus comes to occupy two probably different apoplastic compartments in the root, the intercellular spaces and a more specialized intracellular, arbuscular apoplast. There are thus two interfaces between the symbionts which show different specializations and may well have different functions. Outside the root an extensive mycelium develops and appears to undergo differentiation, so that different types of hyphae perform different functions in colonization, nutrient

80

Vesicular-arbuscular mycorrhizas

acquisition and, possibly, survival. Although variations in colonization patterns were described early in this century, the comparative study of Arum- and Paris-type mycorrhizas has been neglected. Structural, developmental and physiological investigations are required to understand these diverse associations in field situations, as well as for managing mycorrhizas in primary production. The complexities of field situations, where many different species of fungi and plants coexist with other soil organisms, are appreciated but have scarcely been investigated and will provide very significant challenges.

Plate I. (a) Spores arid subtending hyphae oi Gigaspora margaritOy approximate diameter 400-450 |im. (b) Spore of Acaulospora laevis (arrowed), attached to the neck of the sporiferous saccule (s). Spore diameter approximately 190-210 |im. (c) Spores of Scutellospora n/gro, approximate diameter 300|Lim. Photographs courtesy of V. Gianinazzi-Pearson.

Genetic, cellular and molecular Interactions in the establishment of VA mycorrhizas

Introduction The development of vesicular-arbuscular (VA) mycorrhizas involves a well coordinated sequence of events, during which morphogenetic changes to both fungus and plant take place, supporting the maintenance of a compatible, biotrophic symbiosis (see Chapter 2). This chapter will describe the progress of the interactions between plant and fungus as they are influenced by cultivars, mutants and nonhost species, with the aim of highlighting stages in the colonization processes which may act as control points in the different interactions. Research on these topics is relatively recent and although much of the information is preliminary, rapid advances are to be expected in our knowledge of the molecular-genetic regulation of the symbiosis, as new approaches and methods are applied. These methods include specific staining or affinity labelling of different molecules in the walls or interfaces, in situ mRNA hybridization with nucleic acid probes to detect expression of genes likely to be involved in mycorrhizal development and function, and methods specifically designed to determine changes in gene expression during colonization. These last methods include construction and screening of cDNA libraries, differential display and analysis of changes in polypeptide profiles. When linked to detailed studies of mycorrhiza development in host plants with well characterized genomes and in mutants deficient for mycorrhizal colonization, these will be powerful techniques for investigating mycorrhizal symbiosis. In particular, they will help to overcome the difficulties arising from the unculturability of the fungi and they will allow direct investigation of the molecular composition and gene expression in intact mycorrhizal plants. It is important to describe precisely the stages of mycorrhizal colonization, in order to pinpoint differences in the interactions that occur in different plant-fungus combinations. Currently, descriptions are based almost entirely on morphological changes in the development of plant and fungus because there is little supplementary physiological or molecular information (Gianinazzi, 1991; Bonfante-Fasolo and

82

Vesicular-arbuscular mycorrhizas

Perotto, 1992). As outlined in Chapter 2, the main features of the sequence are spore germination, growth of the external mycelium, formation of appressoria and colonization of the root cortex by an intraradical mycelium, development of arbuscules and growth of the extraradical mycelium (see Giovannetti et al., 1994). However, some details are not included in this list and Table 3.1 provides an expanded list of stages at which there may be important developmental changes, together with abbreviations which will be used later in the chapter (Smith, 1995). Schemes of this type are useful if they provide a means of accurate description and comparison of different interactions and consequently help to predict steps which may be under genetic control. However, they must remain flexible and be updated as new information becomes available. The morphological changes that occur in each organism during colonization indicate that a number of developmental switches occur during the establishment of the biotrophic and compatible interaction, and these require the exchange of signals leading to changes in gene expression. In the root it seems very likely that a number of different genes will be expressed in the different cell types as these are progressively colonized by the fungus. We already know that in cells colonized by arbuscules these probably include H^-ATPases, which show increased activity (Gianinazzi-Pearson et al, 1991a; Murphy, 1995; Murphy et ah, 1996) and, in Medicago, phenyl alanine annmonia lyase (PAL) and chalcone synthase (CHS) (Harrison and Dixon, 1994). Conversely, changes in the fungus (including branching pattern and wall characteristics) are probably induced by preinfection signals produced by the plant and by contact with, or penetration of, the different cell types. Unfortunately, there are only a few clues about which genes are involved in either organism or how the spatial and temporal coordination of expression is organized. In VA mycorrhizas the work has centred on the plant responses and genes, rather than on the fungus, for a number of reasons. First, the developmental sequence in Arum-type mycorrhizas is well known and comparisons of different species and cultivars indicate that the plant genotype can affect the extent of colonization and the response (see below and Chapter 4). Second, the plant 'hosts' can be grown with or without mycorrhizal inoculum, so that their development and gene expression can be studied in both mycorrhizal and non-mycorrhizal states. Furthermore, it is now realized that the choice of plant species for molecular genetic work should be based on a number of criteria which will significantly aid the analysis. These are listed in Table 3.2, together with their applicability to four genera currently in use for mycorrhizal research in this area, namely Medicago, Lycopersicon, Pisum and Hordeum. As shown in Chapter 1, there is no evidence in VA mycorrhizas for narrow cultivar-race specificity and the effects of mycorrhizal colonization on plant defence responses are minor. A plant species can probably accept most, if not all of the known glomalean fungi as mycorrhizal partners, while the fungi are similarly undemanding with respect to the identity of plant species that they can colonize. Only a few species of fungi (around 150) can enter into this kind of symbiosis and we know almost nothing of their genetics, except that they may be asexual and may have relatively large genomes compared with other fungi. However, research is becoming increasingly practicable, with the application of methods based on the PCR which permits amplification of specific DNA sequences from very small amounts of target DNA. This is an area of research in which rapid

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progress is expected to occur when molecular biological techniques are applied. In addition, recent advances in axenic culture of several species of fungi in symbiosis with roots will greatly assist this approach.

Variations in Colonization Mediated by the Plant Cultivars The extent of mycorrhizal colonization of root systems and the response of the plants to it certainly varies in different plant-fungus combinations, most notably between the Arum- and P^ns-type mycorrhizas (see Chapter 2). Within the Arumtype, variations in the extent of colonization of the root systems have been observed in different cultivars or lines of single species and may represent differences in susceptibility. Unfortimately, the occurrence of small but potentially significant differences among genotypes in colonization at the cellular level has not been documented and consequently provides no clues to the genetic control of interactions. There are also genotypic differences in the extent to which cultivars respond to colonization with respect to nutrient acquisition and growth (frequently referred to as mycorrhizal dependency; Gerdemann, 1975). This topic will be considered in Chapter 4. Here it is important to note that variations between cultivars have, with very few exceptions (see Krishna et aL, 1985; Smith et al., 1992b), been ignored by plant breeders, even those involved in selecting and breeding for efficiency of nutrient uptake and use; in consequence, useful sources of variability have probably been lost.

Genotypes and Mutants with Altered VA Mycorrhizal Phenotypes Mutants with altered root anatomy, physiology or symbiotic capacity have not been sought to any great extent in any species, largely because of difficulties in screening for root characteristics. Consequently, few have been isolated despite their importance in the study of the control of root development and symbiosis (see Schiefelbein and Benfey, 1991). The first mutant plants to be identified with abnormal mycorrhizal phenotypes were in the legumes Pisum sativum and Vicia faba (Due et al, 1989). In all instances the so-called myc~ plants blocked colonization at appressorium formation and were also nod~, failing to form any nodules with Rhizobium. This mycorrhizal phenotype has been designated myc~^. Subsequent screening of 66 further nodulation mutants in P. sativum has yielded a second mycorrhizal phenotype, in which the fungus is able to penetrate the epidermis (Apr"^, Pen"^) and to grow intercellularly (Ih"^), but in which arbuscule formation is much reduced (Arb~ or Arb^"*^^) and those arbuscules that are formed have very few branches (Gianinazzi-Pearson et al, 1991b). This phenotype has been designated myc~^ and so far has always been found on mutant plants which nodulated normally but did not fix N (nod'^fix"). These two mycorrhizal mutants (see Fig. 3.1a,b) have now been investigated in a number of different ways and considerable information has accumulated on their interactions with mycorrhizal fungi (see below).

Genetic, cellular and molecular interactions

87

Although screening of nod~ soybean genotypes has failed to find any modified mycorrhizal phenotypes (Schenck and Hinson, 1973; Wyss et ah, 1990), an investigation of nod~fix~ and nod"^fix~ Medicago sativa germ plasm has identified mycorrhizal phenotypes which show apparent overproduction of appressoria by Glomus versiforme, G. monosporum, G. fasciculatum and Gigaspora margarita and limited penetration of the tissues by hyphae (Fig. 3.1c; Bradbury et ah, 1991, 1993a). These abnormal phenotypes are stable under growth room conditions (relatively low light), but when grown in a glasshouse with higher light not only were levels of colonization in normal wild-type (myc'^nod'^fix'^) genotypes higher, but the extent of colonization and development of arbuscules on the nod~fix~ and nod^fix" genotypes were also increased, although never to the same extent as on the wild type (Bradbury et ah, 1993b). Bradbury et al (1993b) suggested that the Medicago genotypes may produce lower concentrations of gene product(s) which are required for colonization and that production is influenced by growing conditions, although this remains to be confirmed. Further investigations of the differences in behaviour of these genotypes and the more phenotypically stable mutants of Pisum is certainly required (see Peterson and Bradbury, 1995). In all the mutants or genotypes identified to date, the myc~ alleles are recessive. In P. sativum three genes controlling the myc~^ phenotype have been identified in diallele crosses and in all cases the nodulation phenotype was inherited with the mycorrhizal phenotype (Due et al, 1989; Gianinazzi-Pearson et al., 1991b). This has led to the suggestion that the genes involved in mycorrhiza formation and nodulation are identical and represent symbiosis genes which, having evolved in the more ancient mycorrhizal association, have been taken over by the legume symbiosis (see Gianinazzi-Pearson et al., 1994a). Much more information on gene function is required before this suggestion can be confirmed and it may be advantageous to extend screening of legumes to include more plants which nodulate normally, because preliminary selection for defective nodulation or N fixation introduces a bias in the screening for altered mycorrhizal phenotypes. Not all nod" plants have altered mycorrhizal phenotypes, suggesting that only a few of the nodulation genes are involved in both symbioses. Moreover, none of the genes implicated in the altered phenotypes have been isolated, so that there is no sequence information that can be used to predict their likely functions or the way in which they might be involved in both mycorrhiza and nodule formation. Progress is, however, being made with respect to the mechanism by which mycorrhizal fungi are excluded from the mutant plants (see below). The emphasis on mutants in Pisum and Medicago diverts attention from the fact that common genes must control mycorrhizal colonization in non-legumes as well as legumes and, consequently, mutants in the former are urgently required. Some progress has been made in screening mutagenized populations of Hordeum vulgare and Lycopersicon esculentum, with the identification of several putative mutant plants. In L. esculentum, screening of 209 M2 families (mutagenized by fast-neutron bombardment) has identified abnormal mycorrhizal phenotypes including the formation of complex appressoria rather like the Medicago genotypes (Barker et ah, unpublished). The mutant status of these plants has yet to be confirmed and crosses are being performed to determine the inheritance of the mutated alleles. However, the high frequency with which putative mutants have been found in both legume and non-legume species indicates that many genes are involved in the

88

F i g u r e 3.1 (Caption opposite)

Vesicular-arbuscular mycorrhizas

Genetic, cellular and nnolecular interactions

89

Figure 3.1 Phenotypes of mutants and genotypes with altered patterns of mycorrhizal colonization, (a) myc~' mutant (P6) of Pisum sativum cv. Frisson, showing normal appressorium formation (Apr"^, arrowed), but absence of penetration into the root tissues. Bar, 50 |im. From Due et al. (1989), with permission, (b) myc"^ mutant (DK51) of Pisum sativum cv. Finale, showing an appressorium (Apr"*") and intraradical penetration (Pen ) and growth of intercellular hyphae (Ih"*", arrowed), but poor development of arbuscules. Bar, 50 jim. Courtesy of V. Gianinazzi-Pearson. (c) Hypertrophied appressoria formed on non-mycorrhizal genotypes of Medicago sativa. Bar, 50 jim. From Bradbury et al. (1993a), with permission.

establishment and maintenance of the symbiosis. This is not surprising, because colonization involves fungal interactions with many different cell types in the plant and alterations in both cellular development and physiology. At this stage a mutagenesis approach to analysis of the fungal side of the interaction is not feasible, because the fungus cannot be cultured and does not reproduce unless it is in symbiosis with a plant or axenic root-organ culture.

Interactions of Glomalean Fungi with Non-hosts It is now generally assumed that the non-VA mycorrhizal condition has evolved several times in different taxonomic lines and includes species which do not form any mycorrhizas (e.g. many members of the Cruciferae, Chenopodiaceae, Juncaceae, Caryophyllaceae and Proteaceae), as well as those which associate with fungi other than those in the Glomales, to form ecto-, ericoid, arbutoid and orchid mycorrhizas (see Chapters 1, 6, 11, 12 and 13). There are also species which can

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form more than one type of mycorrhizal association and in which mechanisms supporting VA mycorrhizal colonization coexist with those supporting a second type of mycorrhiza. The evolution of the non-host state is unlikely to have involved simultaneous loss of all genes involved in mycorrhiza development, and is more probably based on presence of one or a few genes conferring 'resistance' or on loss of a single 'switch' or receptor gene (see below). In consequence, it is to be expected that many genes influencing stages of VA mycorrhiza formation and function will be present in non-hosts, as well as hosts and dual hosts, and several different mechanisms resulting in failure of colonization by VA fungi are likely to be found (see Table 3.3). In addition, screening of mutagenized non-host plants should identify myc"^ 'mutants' and lead to the identification of the 'resistance', 'receptor' or 'switch' genes. A systematic comparison of interactions at the cellular level in taxa with different mycorrhizal status is required and currently available information is collated in the next section, with the aim of identifying likely stages at which colonization is blocked or avenues of research that may be productive in elucidating mechanisms of control.

Cellular Interactions in Hosts and Non-hosts The colonization, or lack of it, in various non-host plants has been discussed in many papers and reviews (e.g. Tester et al., 1987; Trappe, 1987). Table 3.3 lists the stages in normal VA mycorrhizal colonization (see Table 3.1 for details) and shows the way in which they are currently thought to be modified a n d / o r controlled in mutants and non-hosts.

Germination Germination of spores (Germ) does not require the presence of a plant and is not affected by exudates from the roots of host species or the non-hosts Spinacea (Chenopodiaceae), Amaranthus (Amaranthaceae) and Lupinus (Leguminosae) (Gianinazzi-Pearson et al, 1989; Vierheilig and Ocampo, 1990a; Schreiner and Koide, 1993a). In contrast, exudates from Brassica species consistently prevent or delay germination of Glomus spores (Tommerup, 1984c; Vierheilig and Ocampo, 1990b; Schreiner and Koide, 1993a,b), and may therefore be important in reducing colonization.

Preinfection Growth and Branching Preinfection growth (Pif) and branching (Pab) of hyphae growing from spores are the first stages of colonization for which morphogenetic effects mediated via the plant have been detected. The processes are influenced by flavonols produced by host plants (see Chapter 2). Phenolics, including flavonols, are known to be important signal molecules in interactions of both Agrobacterium and Rhizobium with host plants, affecting both chemotaxis towards the root and bacterial gene regulation (Peters and Verma, 1990) and it now seems likely that they play similar roles in VA mycorrhizal associations. Much of the work has involved pure compounds applied at relatively high concentrations, and the fungal response to

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identified phenolics produced by plants at physiologically relevant concentrations has been confirmed in only a few cases. These include exudates from roots of carrot seedlings containing the flavonols quercetin and kaempferol, which stimulate hyphal growth of Gigaspora margarita (Poulin et ah, 1993), and from seeds of Medicago containing quercetin-3-O-galactoside, which stimulates hyphal growth from spores of Glomus species (Tsai and Phillips, 1991). It is a reasonable assumption that the stimulatory effects of root exudates from other host plants are based on the production of similar compounds (Gianinazzi-Pearson et aL, 1989; Giovannetti et aL, 1993a, 1994). However, a note of caution is needed - Becard et al. (1995) have failed to detect any flavonoids in exudates of Ri T-DNA transformed roots of Daucus carota. As the roots were colonized by Gi. margarita, flavonoids are not absolutely essential for the process. The non-host plants Lupinus albus, Dianthus caryophyllus and Spinacea oleracea as well as Brassica species do not appear to produce compounds with morphogenetic effects because neither the presence of roots nor exudates from them increase growth or stimulate branching of hyphae growing from spores or sporocarps (Gianinazzi-Pearson et aL, 1989; Giovannetti et aL, 1993a,b; 1994). The same is true for plant species which form other types of mycorrhizas - Abies alba, Pinus nigra (ectomycorrhiza only). Arbutus unedo (arbutoid mycorrhiza) and Vaccinium myrtillus (ericoid mycorrhiza) (Giovannetti et aL, 1994) - but the dual host Alnus glutinosa (ecto- and VA mycorrhiza) does stimulate hyphal morphogenesis and branching of VA mycorrhizal fungi, as does the myc~^ mutant of Pisum (Giovannetti et aL, 1993a, 1994).

Appressorium Formation Hyphal contact with roots is followed by formation and adhesion of appressoria (Apr). The morphogenetic stimulus has not been identified, but it does not seem to be thigmotropic even though appressoria are often seen in grooves between cells on the root epidermis. No mechanisms of adhesion, such as the fibrils seen in ericoid systems, or the various mechanisms of adhesion found in fungal plant pathogens, have been detected. Both contact and appressorium formation are reduced or absent on roots of Brassica napus and Lupinus albus (Tommerup, 1984c; Glenn et aL, 1985, 1988; Gianinazzi-Pearson and Gianinazzi, 1992). There is evidence that a shoot factor is involved in the inhibition in Lupinus, because when shoots of Lupinus were used as scions grafted to rootstocks of Glycine max or Pisum sativum, appressorium formation was reduced, just as it was in intact Lupinus plants (Gianinazzi-Pearson and Gianinazzi, 1992; Tommerup, 1984c). When fungi are grown with the non-host plants Brassica, Dianthus caryophyllus, Eruca sativa. Nasturtium officinale and Spinacea oleracea, hyphae attach to the surface and develop swellings at their tips, which are morphologically quite different from normal appressoria on Basilicum (Giovannetti et aL, 1994). The swellings are more frequent on plants which act as hosts for other types of mycorrhizas than on species which are generally non-mycorrhizal. In no case did hyphal contact with non-host roots induce fluorescence in the host roots, nor was there deposition of lignin or callose, which would indicate initiation of a defence response. Francis and Read (1994) also investigated interactions between mycorrhizal fungi and the nonhost species Arenaria serpyllifolia (Caryophyllaceae) and Arabis hirsuta (Cruciferae).

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They observed hyphal contact with roots, but there was no penetration and no evidence of lesions or any visually apparent defence responses. These results are quite different from the observations of Allen et at. (1989), who observed penetration of roots of Salsola kali (Crucifereae), associated with marked fluorescence and eventual rejection of the fungi. The myc~^ Pisum mutant (Pen~) does not affect appressorium formation (Apr"^), which is normal both with respect to frequency and morphology (Fig. 3.1a; GoUotte et aL, 1993; Giovannetti et al., 1993b). However, a number of plant genes seem likely to affect this stage because overproduction of abnormally shaped appressoria has been observed in mutants of both Medicago sativa and Lycopersicon esculentum (Fig. 3.1c; Bradbury et al, 1991). The mutants in Pisum myc~^ Medicago and Lycopersicon are all resistant to penetration (Pen~) and it has been suggested that overproduction of appressoria may be a fungal response to failure of tissue colonization (Bradbury et al., 1993a). Alternatively, the swollen structures may not be true appressoria, but may be equivalent to the much-branched hyphae produced by mycorrhizal fungi when actual contact with the root is prevented by artificial membranes, as in the studies by Giovannetti and co-workers. In this case the implication is that the myc~ genotypes produce compounds that stimulate hyphal growth and branching (Pif^ and Pab"^) but do not provide the appropriate stimulus for the formation of appressoria and are therefore actually Apr". This is an area where more research could be very productive, in particular with respect to the stimuli required for appressorium formation and mechanisms of adhesion. Normal appressoria are easily identified and in the Pisum myc~^ mutants they occur in the almost complete absence of other developmental stages. Consequently, fungal genes involved could be isolated by differential screening of cDNA libraries prepared from roots at the early stages of colonization of mutant or non-mutant peas, or a similar screen using a fungal genomic library, as has been done for Magnaporthe grisea (Lee and Dean, 1993). If the abnormal, branched structures on Medicago are true appressoria, increased expression of 'appressorium genes' might be expected in the interaction between mycorrhizal fungi and these plants.

Penetration Penetration (Pen) of the outer tangential walls of the epidermal cells follows appressorium formation and is marked by narrowing of the fungal hypha to form an infection peg. Localized production of wall-degrading hydrolytic enzymes by the fungus, as well as hydrostatic pressure exerted by the hyphal tip are both probably involved at this stage (see Chapter 2). In the plant, thickening of the epidermal cell wall occurs in some plant-fungus combinations (Fig. 3.2a) and indicates recognition of fungal contact. This step is clearly affected in the myc~^ mutant, in which well defined wall thickening, with increased deposition of phenolics and P(l,3)glucans (possibly callose), occurs beneath the appressoria (GoUotte et al, 1993), although the appressoria themselves remain alive and apparently normal (Fig. 3.2b). In contrast, in the hypertrophied 'appressoria' on Medicago nod~myc~ genotypes, electron-dense deposits are soon observed, indicating that both plant and fungal responses are involved in the failure of colonization in these interactions.

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Vesicular-arbuscular mycorrhizas

Intraradical Colonization As penetration and colonization of the root tissues proceed, the plant responds in a number of ways, which probably vary in different plant-fungus combinations, although this has not been systematically investigated. In particular, nuclear size increases, possibly indicating genome reduplication or increased rates of gene transcription. In the myc~^ Pisum mutant this occurs to a lesser extent than in wild-type peas, but information for other mutants and for non-host plants is not available (Berta et al, 1991; Sgorbati et al, 1993). Internal colonization of the root involves the formation of intercellular hyphae (Ih) coils (Cof and Cip) and arbuscules (Arb). The plant-fungus interactions have been studied by following changes in the deposition of molecules in the intercellular and intracellular interfaces, including fungal and plant walls (BonfanteFasolo, 1988; Lemoine et ah, 1995) and by the striking changes in fungal morphology during arbuscule formation. In arbuscule-containing cells, the plant nuclei migrate from the periphery of the cortical cells to their centre. They increase in size and the chromatin decondenses, as shown by antibody labelling (Balestrini et al., 1992). These changes, together with changes in the spatial localization of enzymes such as H^-ATPases and other phosphatases, clearly indicate a remarkable degree of ongoing coordinated development (see Chapter 2). Changes in wall chemistry of the fungus could possibly be important in determining the response of different types of plant cell to colonization. For example, there is no evidence that either intercellular hyphae or intracellular coils (both of which have relatively thick and well developed walls) elicit any defence responses in the host cells. In contrast, development of arbuscules, which have very thin and highly modified walls, does result in localized defence responses. A highly speculative explanation might be that stimulatory or elicitor molecules become exposed in the walls of arbuscules (see below). Formation of arbuscules is blocked (Arb~) or reduced (Arb^^^) both in myc~^ Pisum mutants (Gianinazzi-Pearson et al., 1991b) and in non-host plants such as Brassica, Salsola and Atriplex, where some tissue penetration has been reported. However, in none of these examples is the nature of the block understood. In the fungal interaction with myc~^ mutants, the increase of H^-ATPase activity normally observed on the PAM does not occur (Pat~), and a reasonable prediction would be that P transfer from fungus to plant would be considerably reduced, although this has not yet been investigated. Distribution of activity of plasma membrane H"^ATPase in the fungus is normal: intercellular hyphae have high activity and the reduced arbuscules have none (Gianinazzi-Pearson et al., 1995; GoUotte et al., 1996; see also Gianinazzi-Pearson et al., 1991a). The vigorous growth of the intercellular

Figure 3.2 Cellular reactions during epidermal penetration of roots by mycorrhizal fungi, (a) Acriflavine-positive wall thickenings in the epidermal cells of Allium porrum (arrowed), in response to normal colonization by Glomus versiforme. Bar, 10 jiim. From Garriock et al. (1989), with permission, (b) Callose-containing wall thickenings in the epidermal cells of myc~' mutants of Pisum sativum cv. Frisson, in response to appressorium formation (ap) and colonization. Bar, 10 |im. From Gollotte et al. (1993) Planta, 191, Fig. 2, p. I I 5 , with permission.

Genetic, cellular and molecular interactions

F i g u r e 3.2 (Caption opposite)

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Vesicular-arbuscular mycorrhizas

hyphae in these mutant plants and the distribution of activity of ATPases indicate that arbuscules are not required for the fungus to obtain C from the plant, confirming the suggestion of Gianinazzi-Pearson et al. (1991a; see Smith and Smith, 1995, 1996b). Parallel investigations of nodulation and mycorrhizal interactions at the level of molecules present in the symbiotic interfaces are proceeding with the aim of identifying features which will provide clues to the nature of the common controlling genes in the two symbioses. The expression of several nodulins is increased in mycorrhizal plants, including Nod 26, (Gianinazzi-Pearson and Gianinazzi, 1988; Wyss et al., 1990) which is a membrane protein with sequence homology to a number of genes in plants and animals. The high degree of sequence conservation of Nod 26 in distantly related organisms indicates that it may have arisen in a common and very ancient ancestor and it has been suggested that it is a channel protein in plasma membranes (see Verma et al., 1992). If this is so, then its increased expression in both nodulated and mycorrhizal plants may be a consequence of the increase in membrane production during the development of the PBM and PAM. The PAM shows both similarities to, and differences from the PBM, as indicated by the use of antibodies raised to cell surface components in nodules of Pisum (Gianinazzi-Pearson et al, 1990; Perotto et al, 1994; Gollotte et al, 1995). Similarities between the PBM and PAM are not surprising as they are both at least partly derived from the plasma membrane of the cell and both have important transport functions associated with the symbioses. Apart from this, the results do not yet provide a coherent picture of common functional aspects of the membranes and interfacial compartments of the rhizobial and mycorrhizal symbioses.

Changes in Gene Transcription during VA Mycorrhizal Colonization The hypertrophy of plant nuclei, increased staining with DAPI (4',6-diamidino-2phenylindole) and susceptibility to degradation by DNAase have been accepted as evidence that colonization induces higher rates of gene transcription in mycorrhizal roots, leading to observed increases in accumulation of RNA on a fresh-weight basis (Schellenbaum et al, 1992; Franken and Gnadinger, 1994; Murphy et al, 1996). The relative contributions of transcription of specific symbiosis-related plant genes and up-regulation of genes which are also expressed in uncolonized roots is not yet known, but it seems likely that both are involved. Analysis of soluble protein composition (e.g. Pacovsky, 1989; Schellenbaum et al, 1992; Arines et al, 1993; Dumas-Gaudot et al, 1994b) has shown the production of new polypeptides in mycorrhizal roots; some of these are certainly fungal, but others may be plant proteins related to particular stages of colonization, as shown by their presence in Pisum myc~^ mutants with appressorial colonization by Glomus mosseae. So far, no clues on the functions of the new polypeptides (called mycorrhizins) have been obtained. A second method of detecting changes in gene expression in response to mycorrhizal colonization is the preparation of cDNA libraries, which provide a 'snapshot' of the mRNA species being produced at any one time. Differential screening of such libraries identifies cDNA clones representing mRNAs of particular origin, and can therefore be used to help identify genes expressed by plant or fungus at different

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stages of colonisation. Differential display PCR (Liang and Pardee, 1992; Bauer et al., 1993) also of use in detecting genes with low copy number, including fungal genes. There are several preliminary reports of these approaches being used to investigate VA mycorrhizal associations (Rosewarne, 1993; Barker ei al., 1994; van Buuren and Harrison, 1994; Murphy, 1995; Martin-Laurent et al, 1995; Murphy et al, 1996; Ridgeway et al, 1996). Murphy et al (1996) isolated polyA"" mRNA from barley roots in the early stages of colonization by Glomus intraradices and used it to construct a cDNA library. The resulting clones were screened with ^^P-labelled cDNA, obtained by reverse transcription of RNA from non-colonized and mycorrhizal roots, in order to distinguish clones representing mRNAs which were differentially accumulated in the mycorrhizal interaction. Colonization resulted in both up- and down-regulation of genes. Of those clones selected for further analysis, BM 78 shows close sequence homology with H'^-ATPases from Arabidopsis and Lycopersicon and does not hybridize with DNA from fungal spores. Up-regulation of this gene is particularly interesting because it may be associated with the increased activity of ATPase on the PAM and be involved in symbiotic transport. When used as a probe to investigate expression of the gene in roots treated in different ways, accumulation of the mRNA hybridizing to BM 78 increased as mycorrhizal colonization increased, but was unaffected by P nutrition, infection by the pathogen Gaeumannomyces gratninis or water stress. BM 78 therefore appears to be clearly related to mycorrhizal colonization. Using barley addition lines in the wheat variety Chinese Spring, the gene has been mapped to the short arm of chromosome 2 of barley (Murphy et al, 1995; and see Fig. 3.3). The existence of mapping populations of Lycopersicon will facilitate isolation of genes from this genus (Giovannoni et al, 1991). Other up- and down-regulated mRNA species have also been found and mapped to particular chromosomes, but no information that would give clues to the functions of the corresponding genes is yet available. By screening a cDNA library from mycorrhizal Medicago tuncatula, Harrison (1996) has identified a clone with sequence homology to a membrane-bound sugar transporter, showing a four- to five-fold increase in accumulation of transcripts in mycorrhizal roots, but no change in either non-colonized roots or roots of the myc~ M. sativa genotype. Again, the link with an aspect of symbiotic transport is exciting, but in all cases much more work is required. Some of the up-regulated genes may be associated with resistance responses to pathogens (see next section) because small increases in both activity of the enzymes and accumulation of mRNA transcripts of genes involved in resistance have been found. It is for these that a third approach, using cloned probes for genes with known functions, has been applied with some success. This approach could usefully be extended to genes coding for proteins involved in other activities, such as membrane transport (see above), which can be predicted from physiological or biochemical evidence to be important in symbiotic development. The progress in application of molecular methods is promising and, when coupled with immunolocalization of the proteins a n d / o r in situ mRNA hybridization, has the potential to expand enormously our knowledge of the details of symbiotic interactions. So far, gene expression in the fungi has not been investigated to any great extent and the application of DNA technology has been applied only to investigations of relationships between taxa and to the development of taxon-specific probes for ecological investigations. However, a partial genomic library from Scutellospora

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Vesicular-arbuscular mycorrhizas

CSB123

4 5 6

7

Figure 3.3 Use of barley addition lines of Triticum aesHvum cv. Chinese Spring to determine the location of the up-regulated gene represented by BM 78 in the genome of Hordeum vulgare. DNA from 7! aestivum Chinese Spring (lane CS), H. vulgare cv. Betses (lane B) and Chinese Spring containing chromosomes 1-7 of barley (lanes 1-7) probed with BM 78. The gene is located on chromosome 2, as shown by the major bands (arrowed) common to barley and to the addition line containing barley chromosome 2. From Murphy et al. (1995), with permission.

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99

castanea has now been produced and there is some evidence that three of the clones in this library may represent repeat sequences present at high copy number. As they hybridize to DNA from spores and mycorrhizal roots but not to DNA from noncolonized roots, these could be useful as probes for the occurrence of mycorrhizas, as tools for later mapping (Zeze et al., 1994) and, by comparison of base sequences, for analysis of genome evolution. Fungal genes with potential regulatory functions have recently been identified (Delp et ah, 1996) and from a functional standpoint the differential expression of a fungal PO^ transporter in extra- and intra-radical fungal structures is very important (Harrison and van Buuren, 1996) and will be discussed in Chapter 14.

Effects of VA Mycorrhizal Colonization on Resistance Responses Comparisons of plant defence responses to parasitic and mycorrhizal fungi are beginning to yield coherent information. However, it is important to recognize that although compatible interactions in these two types of symbiosis are superficially similar, there are also important differences. In mycorrhizas the only sign of loss of biotrophic status is the regular degeneration of arbuscules (see Chapter 2); it is the fungus, not the plant cell, which collapses and dies. In contrast, in biotrophic symbioses involving fungal parasites the plant cells die, either rapidly in hypersensitive, resistant responses or more slowly in compatible interactions. VA mycorrhizal associations are consequently analogous to compatible interactions, but direct comparisons between them must be made with care. One of the questions asked frequently is how does a mycorrhizal fungus avoid triggering a plant defence response? As described below, some defence responses are certainly mobilized for a short period but are later suppressed. VA mycorrhizas may represent a state of basic compatibility between plant and fungus which probably underlies all the more taxon-specific resistance mechanisms. Having evolved so long ago, the mycorrhizal recognition and signalling systems may predate evolution of specific mechanisms of resistance to plant disease. It seems likely that there would have been selective advantages in independent operation of systems of defence against pathogens and establishment of mycorrhizal interactions. All the work on deployment of defence responses against pathogens during VA mycorrhizal colonization indicates that they are weak and transitory. In typical mycorrhizal interactions there are no major changes in synthesis of lignin or callose in the plant cells. Metabolism of secondary metabolites, including flavonoids, is altered, but to a much lesser extent than in response to pathogen attack. Enzymes of the phenylpropanoid pathway have been investigated at levels of transcription and activity, particularly in legumes. During mycorrhizal development, early stimulation of both transcription and activity of PAL, CHS and isoflavone reductase (IFR) occurs in Medicago and Phaseolus, at least to a small extent. In Medicago an increase in transcription of chalcone isomerase (CHI) has also been reported (Harrison and Dixon, 1993; Lambais and Mehdy, 1993; Volpin et al, 1994). The increase in activity of PAL and CHI is transitory in M. sativa (Volpin et al, 1994), but in M. truncatula accumulation of PAL and CHS transcripts is maintained at 1.75- and 2.25-fold, respectively, above uninoculated controls for some weeks, while IFR transcripts decline. The increases are small compared with plant-pathogen interactions, but

100

Vesicular-arbuscular mycorrhizas

they are apparently consistent. It must be remembered that the values are averaged over the different cell types in the roots, not all of which show increases in levels of transcripts (see below). The pattern of transcription of IFR is correlated with changes in accumulation of the phytoalexin medicarpin, and it appears to indicate the suppression of a defence response in mycorrhizal plants, which is not observed in the nod~myc~ M. sativa genotype (Harrison and Dixon, 1993). In Glycine, small increases in glyceoUin production have been observed following inoculation with Glomus species, but these are either slow to develop or not significantly greater than the uninoculated controls (Morandi et ah, 1984; Wyss et ah, 1991). In M. truncatula, in situ mRNA hybridization has shown that the increased levels of PAL and CHS mRNAs are confined to cells containing arbuscules, possibly indicating a localized stress response or a mechanism controlling intracellular invasion (Harrison and Dixon, 1994). This may help to explain the short life span of arbuscules. At present there are no data on responses in cells containing intracellular coils (e.g. the hypodermal passage cells), so that it is impossible to determine whether or not they differ from arbuscule-containing cells. No increases associated with intercellular hyphae were observed. Localization of HPRG in the arbuscular interface also suggests mobilization of small and localized defence responses (Bonfante-Fasolo et al, 1992), but although Franken and Gnadinger (1994) observed consistent increases in accumulation of transcripts of a gene coding for HRGP during mycorrhizal colonization of Fetroselenium, preliminary in situ hybridization analysis showed that the tissues involved were the stele and root apex, but not the cells colonized by arbuscules. In this non-legume, which has been used extensively to study plant-pathogen interactions, there were no major changes in the accumulation of phenolic compounds, of transcripts from PAL, CHS or 4coumaratexoA ligase (4CL) genes in mycorrhizal plants, nor of genes encoding other enzymes which respond to treatment with elicitors from Phytophthora megasperma f. sp. glycinea, including peroxidase. Chitinases and P(l,3)glucanases are also implicated in responses of plants to parasites, while peroxidase is important in the final stages of lignin deposition, again as a defence response. Chitinase and peroxidase sometimes show transitory increases in activity in plants infected by mycorrhizal fungi (Spanu and BonfanteFasolo, 1988; Spanu et al, 1989; Lambais and Mehdy, 1993; Dumas-Gaudot et al, 1994a; Vierheilig et al, 1994; Volpin et al, 1994), whereas activities of p(l,3)glucanases are either unaffected or suppressed, compared with uninoculated controls (Lambais and Mehdy, 1993; Vierheilig et al, 1994). Activities of these enzymes do not appear to be related to the control of colonization, because the progress of colonization is normal in transgenic Nicotiana plants expressing different forms of chitinase. Spanu et al (1989), using gold-labelled antibodies to bean chitinase, showed that in Allium porrum these enzymes are localized in plant vacuoles and intercellular spaces of both mycorrhizal and non-mycorrhizal plants and they are never found bound to the walls of G. versiforme. Indeed, the lack of reaction may be because the chitin in the fungal walls is rendered inaccessible to the enzymes by the presence of other wall components. In the non-hosts Brassica, Spinacea and Lupinus, increases in chitinase and P(l,3)glucanase activity, as well as ethylene production, have been observed in response to the presence of glomalean fungi, but the effects were all weak or transitory and did not markedly differ from responses of host plants (Vierheilig et al, 1994). In the myc~^ mutant of Pisum the defence responses appear

Genetic, cellular and molecular interactions

10!

to be stronger, accompanied by phenolics and callose, and exclude the fungus (GoUotte et al, 1993). The occurrence of a pathogenesis related protein (Pbrl) in the wall thickenings of myc~^ mutants indicates that other features of host defence against pathogens are mobilized (GoUotte et aL, 1994) and it will be interesting to discover whether or not the plant cells in this and the myc~^ mutant exhibit other characteristics of a hypersensitive response. In conclusion, it appears that rather than inducing major deployment of defence responses, mycorrhizal colonization may provoke a minor, transitory response which is followed by general suppression in both host and non-host plants. This is consistent with the persistent state of compatibility which mycorrhizas represent and there is no evidence from the species so far tested that failure of colonization by glomalean fungi in non-mycorrhizal plants involves mechanisms similar to those which are mobilized in plants resistant to attack by pathogens.

Possible Control Steps In VA Mycorrhizal Colonization Analysis of the interactions of glomalean fungi with normal host plants, with mutants having abnormal mycorrhizal phenotypes and with non-hosts has shed some light on the mechanisms of control operating in the symbiosis and allows some speculative predictions to be made. Figure 3.4 shows how the interactions vary and provides evidence that the mechanisms conferring non-host status are different in different taxonomic groups. The development of the fungus within the root is clearly influenced by the cell type colonized, so that hyphal coils invariably develop within hypodermal passage cells and sometimes within cortical parenchyma, particularly in P(irzs-type mycorrhizas (see Smith and Smith, 1996a). Intercellular hyphae only develop extensively in roots characterized by extensive intercellular spaces {Arum-type mycorrhizas), while arbuscules are characteristic of the cortical parenchyma and are sometimes localized in the innermost cell layer, in a ring surrounding the endodermis. Coordinated changes in development of both fungus and plant are striking, but we have few ideas on either the identity of the signals or the genes controlling the cellular modifications. It is clear that many genes must be involved in both organisms. The general absence of non-host variants in mycorrhizal species suggests that the significant genes could well be present in multiple copies or be so important in root function that their loss is lethal. Study of mutants has the potential to yield important information about the genes involved in normal mycorrhizal development and on any key control steps. Based on their investigations of the Pisum myc~^ mutant, GoUotte et aL, (1993) have proposed a scheme to explain the way in which the defence responses are suppressed in normal host plants. The hypothesis would explain a number of observations. It requires that a mycorrhizal (host) plant carries a dominant gene which codes for a receptor. This receptor would recognize a signal molecule from an appropriate mycorrhizal fungus and, if this occurs, the host defence responses would not be mobilized and colonization would proceed normally. Different receptors could be involved for each mycorrhizal type, and species with more than one receptor would detect signals from more than one type of mycorrhizal fungus. Mutants in which penetration does not occur could have evolved by loss of

Vesicular-arbuscular mycorrhizas

102

Non host

Host/Mutants

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^ PLANT ROOT EXUDATES

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F i g u r e 3.4 Diagrammatic representation of the sequence of events during colonization of hosts, non-hosts and mutants by glomalean fungi. Modified from Giovannetti et al. (1994).

the receptor, explaining their recessive status as well as the mobilization of some defence responses. This picture contrasts with models for gene-for-gene resistance to plant pathogens, in which the plant-fungus interaction is always highly specific and resistance is dominant. In these interactions susceptibility (recessive) has evolved by the loss of gene function. At present we must emphasize that no signal compounds or receptors have been identified in mycorrhizal plants or fungi. However, molecules of this type are known for the interaction between Alternaria alternata and a range of host species (see Smith and Smith, 1996b). In the plants, resistance is recessive, as in mycorrhizal mutants, and it is controlled by a single gene which is thought to code for a receptor. Lack of the receptor prevents colonization of the resistant plants carrying

Genetic, cellular and molecular interactions

103

the recessive allele. The fungus produces a signal molecule (toxin) which binds to the receptor and confers compatibility on the association. In the Alternaria system the signal molecules produced are specific for particular host varieties (host-specific toxins) and confer specificity as well as compatibility. However, this feature would not be required in mycorrhizas and could very well have been selected against, because having a wide host range would confer considerable advantages for a mutualistic, root-colonizing fungus. There are some problems with the hypothesis. The defence responses potentially mobilized against mycorrhizas and pathogens must be different, otherwise mycorrhizal plants, having switched off their defences, would be much more susceptible to pathogens; in fact, the reverse is normally observed. The mechanisms which exclude mycorrhizal fungi from non-host species are quite varied, and involve toxin production by brassicas and apparent failure to produce morphogenetic signals in a number of other taxa. It is necessary to propose that the different mechanisms are the result of the action of different genes. The simplest in concept would be those which actually block colonization at an early stage; these might be expected to be dominant. Others, representing the loss of one or more key signals or receptors, would be recessive. There is as yet no systematic evidence for variation in susceptibility within non-host species, and no mutant screens have yet been undertaken to identify mycorrhizal phenotypes. Such mutants could give important leads in understanding the genes and mechanisms by which different taxonomic groups of plants achieve non-host status.

Independent Fungal Growth Perhaps the most striking result of the normal plant-fungus interaction is the change in growth rate and reproductive capacity of the fungus following colonization of the root. Possible blocks in fungal metabolism in the absence of the plant have been sought (e.g. availability of particular nutrients, absence of key enzymes or metabolic processes and nuclear division; see Chapter 2), but none has been identified. However, there are indications that membrane transport processes may not be fully operational in germ-tubes growing from spores, and that activity of the plasma membrane H"^-ATPase on the fungus, which is important in nutrient uptake and transfer, depends on colonization (Thomson et al., 1990a; Lei et ah, 1991). There are inconsistencies in the results, possibly because of differences in the age of the experimental material (F.A. Smith, personal communication), so that more experiments are needed. It has also been suggested that the fungus lacks some essential genetic information (see Hepper, 1984a). In symbiosis this might mean that the fungus is dependent on the plant at the metabolic level, with key and as yet unidentified processes coded for by plant genes. Alternatively, genetic material might actually be transferred from plant to fungus, before continuing fungal growth can occur. This suggestion is highly speculative and has not been investigated. Conclusions Much of the material presented in this chapter is preliminary and it is most important to appreciate that many of the ideas are still speculative and unconfirmed by

104

Vesicular-arbuscular mycorrhizas

experiment or observation. The aim has been to provide an overview of variations in mycorrhizal colonization at the cellular level which may help to indicate the directions of research which may be fruitful. We know that the development of mycorrhizas must be under the control of plant and fungal genes, which act in a coordinated manner to produce the characteristic, biotrophic and compatible interaction in mycorrhizal host species. However, our understanding of the details of the process at the genetic level is rudimentary. Several research groups are now addressing the problems and have identified plant-fungus combinations and methodologies which will facilitate molecular genetic investigations. It is possible to describe the colonization process in precise terms, so that key regulatory steps can be identified and consequently investigated. At this stage the mechanisms which prevent mycorrhizal colonization in non-host species are still not completely clear, but there are a number of useful leads and a clear recognition that different taxa may exclude the fungi by different mechanisms. As the mycorrhizal condition is probably primitive in land plants, non-host species are likely to have retained many of the genes controlling colonization in host species. When these have been identified we will have powerful tools with which to investigate non-host plants and also to compare the compatible biotrophic symbioses involving mycorrhizal fungi and fungal plant pathogens. Understanding the genetic control of mycorrhizal symbiosis will provide insights into the evolution of the symbiosis and hence the present-day interactions in natural ecosystems. It will also be important in the biology of crop plants, particularly in the development of cultivars which can be designed to fit particular production systems. For example, in low-input agriculture where high efficiency of nutrient uptake is required, a cultivar that is both highly susceptible and highly responsive to mycorrhizas would be appropriate. Conversely, in highly fertilized situations a non-susceptible and non-responsive cultivar might be more suitable, always assuming that mycorrhizal effects on interactions with pathogens or soil structural stability had been adequately considered (see Chapter 16).

Growth and carbon economy of VA mycorrhizal plants

Introduction This is the first of two chapters on the physiological interactions between the symbionts in vesicular-arbuscular (VA) mycorrhizal plants. It will cover the basis of the symbiotic relationship in terms of transfer of organic C and soil-derived nutrients between the symbionts and it will describe the C economy of the symbioses and the growth responses of the plants to colonization by the fungi. The importance of mycelial links between plants will be considered, in so far as they affect C allocation among members of a group of plants. Interactions between glomalean fungi and non-photosynthetic (heterotrophic) plants will be discussed briefly, because of their unusual method of organic C nutrition. The symbiosis between VA mycorrhizal fungi and autotrophic plants is generally regarded as mutualistic, with the basis of mutualism assumed to be the bidirectional transfer of nutrients. With the exception of a few achlorophyllous species, VA mycorrhizal plants are autotrophic and, although normally colonized by mycorrhizal fungi in the field, they are usually capable of satisfactory growth in the absence of colonization, provided that mineral nutrient supplies are adequate. In contrast, the fungi are ecologically obligate symbionts. There is no good evidence that any glomalean fungi have significant saprophytic ability and the limited capacity of their propagules to produce mycelium in the absence of colonization of a plant is based on mobilization of reserves (see Chapter 2). In consequence, the fungus depends on recent photosynthate supplied by the autotroph and, as will be discussed later, utilizes a considerable proportion of the assimilated C. Conversely, the development of the external mycelium of the fungal symbiont in the soil increases the capacity of the plant to absorb nutrients because both roots and hyphae are involved in uptake. The mycelial habit ensures that the fungal symbiont has access to soil-derived nutrients, some of which are passed to the host plant. This simple view of mutualism takes no account of changes in the quantitative balance of transfer between symbionts at different times as the plants develop, nor of the fact that nutritional interactions within an uneven-aged plant community, composed of different species, may be very complex indeed. Furthermore, it

106

Vesicular-arbuscular mycorrhizas

ignores non-nutritional features of the symbiosis. For example, the more stable physicochemical conditions in the root apoplast (e.g. water potential, ion concentrations, pH), compared with the soil environment, may be advantageous for the fungus. For the plant there is increasing evidence that mycorrhizal colonization may increase resistance to pathogens and insect herbivores, as well as tolerance of water deficit. Consequently, it may be difficult to demonstrate that the symbiosis is mutualistic, especially in natural ecosystems, and the term mutualism should be used cautiously. The questions that will be addressed in this chapter concern particularly the way in which mycorrhizal plants allocate photosynthate to growth of roots and shoots and to the development of the symbiotic fungus, and how this is affected by nutrient supply. Understanding these physiological relationships, including movement of organic C and growth responses, requires some understanding of the role played by the fungal symbiont in mineral nutrition, as well as its requirements for C. This will be covered briefly here and the details discussed in Chapter 5. Fungal translocation to the roots of nutrients absorbed from soil and the cellular bases for nutrient transfer from one partner to the other are included in Chapter 14.

Plant Growth The association between the development of VA mycorrhizas and increased growth of the host was made by Asai (1944) in his studies of mycorrhizal colonization and nodulation in a large number of legumes. He concluded that colonization was important both in plant growth and in the development of nodules. Subsequently, many investigators have carried out a large number of experiments which in general demonstrate that colonization is followed by considerable stimulation of growth. This early work has been extensively reviewed, with particular emphasis on the importance of mycorrhizas in P nutrition (e.g. Gerdemann, 1968, 1975; Mosse, 1973; Tinker, 1975a,b; Smith, 1980; Gianinazzi-Pearson and Gianinazzi, 1983; Harley and Smith, 1983; Hayman, 1983; Smith and Gianinazzi-Pearson, 1988; Koide, 1991a). Pioneering work on the potential significance of mycorrhizas in plant nutrition was carried out by Mosse (1957) on apples, Baylis (1959, 1967, 1972a) on Griselinia and other New Zealand plants, and Gerdemann (1968) on Liquidambar and maize. Subsequently, Daft and Nicolson (1966, 1969a, b, 1972), and Hayman and Mosse (1971, 1972; see also Mosse and Hayman, 1971) independently investigated the basis for this growth response in a number of plant species, in particular with respect to soil conditions and inoculum density. They demonstrated that development of mycorrhizal roots and their effect on plant growth is greater in soils of low or imbalanced nutrient status, in particular if P is in short supply, and they made valuable advances in interpretation of the mechanisms of these effects, which are discussed in Chapter 5. Increased growth has been demonstrated for a very wide variety of plant species including many crop plants and trees (Plate 2); it is manifest as increased growth of roots as well as shoots, reduced root:shoot ratio and increased tissue P concentrations (see Table 4.1 and Fig. 4.1). In a few plant species increased flower production and yield have also been demonstrated. Nodulation and N fixation in mycorrhizal legumes and dually colonized actinorrhizal plants are also increased and have been

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30 40 Time after planting (days)

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Figure 4.1 (a) Dry weight changes and (b) P contents oi Allium cepa, colonized by four VA mycorrhizal fungi, compared with controls (C). YV, Glomus mosseae; LAM, G. macrocarpus van geosporus; MIC, G. microcarpus; BR, Scutellospora (Gigaspora) calospora. From Sanders et al. (1977), with permission.

shown to result in higher tissue N concentrations in some experiments (see Barea and Azcon-Aguilar, 1983). Indeed, so many pot experiments have been carried out with such a wide variety of plant species that many of them cannot be cited. Most of the effects on growth can be attributed, directly or indirectly, to improved mineral nutrition and in many cases similar changes have been shown to take place in response to application of fertilizer in the absence of mycorrhizal colonization. However, it must be stressed here that in discussing C use by the fungi it is essential to distinguish direct mycorrhizal effects from those which would inevitably follow any changes in size, form or nutrient content.

Carbon Use by the Fungal Symbiont Evidence for the dependence of the fungus on plant-derived C is both direct and indirect. The indirect evidence is provided by the greatly increased growth of extraradical mycelium and production of spores which take place once colonization is established, and by the influence of irradiance on both the colonization of the roots and the magnitude of the mycorrhizal growth response (see Chapter 2). Fungal biomass associated with roots has been estimated at between 3% and 20% of root weight (see Harris and Paul, 1987), but the estimates have not usually included external hyphae or spores. These could add to the biomass considerably. In an investigation using 26-day-old Cucumis with 95% of its root length colonized

Growth and carbon economy of VA mycorrhizal plants

109

by Glomus fasciculatum, the dry weight of hyphae was calculated at 2.6% of root dry weight (Jakobsen and Rosendahl, 1990) and spores, as was, noted in Chapter 2, can be produced at very high densities and are likely to represent considerable C allocation to the fungal symbiont. Direct evidence of carbohydrate transfer has been obtained from ^^C02-labelling experiments but quantitative measurement of the amounts transferred is difficult. The close and complex association of the two organisms within the root, and the difficulties of extracting the fragile external mycelium from soil have meant that techniques involving their separation cannot be easily applied (see McGee and Smith, 1992). In VA mycorrhizas the destinations in roots and extraradical hyphae of photosynthetically incorporated ^^C have been traced. Ho and Trappe (1973) performed the earliest experiments which demonstrated that, over a period of weeks, small amounts of labelled photosynthate appeared in extraradical hyphae and spores of VA mycorrhizal fungi. Several groups have subsequently established that there is rapid translocation of ^^C-labelled photosynthate to root systems of mycorrhizal plants and that some of this passes into intracellular fungal structures (demonstrated by autoradiography) and into hyphae outside the root (shown by direct counting of harvested mycelium; Bevege et ah, 1975; Cox et ah, 1975; Hirrel and Gerdemann, 1979; Francis and Read, 1984; see Fig. 4.2). The techniques used in the experiments showed that C transfer certainly takes place, but they did not provide quantitative information on the amounts transferred. More recently, a number of groups have extended the ^^C4abelling approach and in addition to confirming the earlier findings have provided much more complete data on the quantities of material transferred. Balance sheets of the C allocation in mycorrhizal and non-mycorrhizal plants, taking into account their P nutrition and growth (see below), have given a reasonably consistent picture of the proportion of plant photosynthate used by the fungal symbiont both in growth and respiration. By feeding ^^C02 to mycorrhizal and non-mycorrhizal plants matched for shoot P concentration or to a single plant with a split (mycorrhizal and non-mycorrhizal) root system, and determining the distribution of label in different fractions after a chase period, it has been calculated that mycorrhizal roots of a range of herbaceous and woody plants receive about 4 20% more of the total photosynthate than non-mycorrhizal roots (see Koch and Johnson, 1984; Harris and Paul, 1987; Douds et al., 1988; Jakobsen and Rosendahl, 1990; Eissenstat et ah, 1993). C is deployed in growth of the intra- and extra-radical mycelium and in respiration to support both growth and maintenance. At this stage there is little indication of the reasons for the variations in the estimates, but they are likely to include species of plant and fungus, fungal biomass and rate of colonization, as well as the metabolic activity of the fungus. Table 4.2 (from Jakobsen and Rosendahl, 1990) shows data for ^^C incorporation into the mycorrhizal roots of young Cucumis plants and the external mycorrhizal mycelium of Glomus fasciculatum associated with it. The C incorporation (|jLg C plant"^ d~^) into extraradical hyphae in this case was 6% of the incorporation by roots and the specific ^^C incorporation (|jLg C mg~^ dry wt~^ d~^) was 2.4 times that of the roots. The hyphae constituted 26% of the extraradical organic ^^C, emphasizing their importance in transfer of organic matter to soil (Table 4.2a). Using a number of reasonable assumptions about the fungal growth yield, specific incorporation of intraradical and extraradical mycelium and intraradical fungal

MO

Vesicular-arbuscular mycorrhizas

• SP

Figure 4.2(Caption opposite)

Growth and carbon economy of VA mycorrhizal plants

I 11

Table 4.2 C allocation in Cucumis sativuSy colonized by Glomus fasciculatum (a) Uptake of C and distribution of '^C in 26-day-old mycorrhizal Cucumis sativus 80 h after labelling of shoots with '^COi for I6h C uptake Total ( m g C d " ' ) Specific (mg C dm"^ h~') '"*€ distribution (%) Shoot Shoot respiration Root External VA mycorrhizal hyphaef Soil organic C Below-ground respiration Ratio ''^C lost from roots:''^C translocated to roots

37.0 ± 0.5* 1.36 ± 0.03 54.1 2.5 13.2 0.8 2.3 27.0 0.70

± 0.6 ± 0.8 ± 0.1 ± 0.1 ±1.1 ± 0.09

* Mean ± SE of five plants; f hyphal densities assumed to be similar in hyphal compartments (HC) and root compartments (RC).

(b) Length, dry weight and C incorporation of hyphae in hyphal compartment (HC) and length, VA mycorrhizal colonization and C incorporation of roots in root compartment of mycorrhizal Cucumis sativus 80h after labelling of shoots with '"^COi for I6h Hyphae in HC Length (cm g~' dry soil)* Diameter (|im) Dry weight (|Lig g~' dry soil):]: C incorporation Total (|ig C plant"' d~') Specific (|ig C mg~' dry wt d~') Roots Total length (cm g~' dry soil) VA mycorrhizal length (cm g~' dry soil) C incorporation Total ()ig C plant"' d~') Specific (|Lig C mg~' dry wt d~')

2708

± 206t

2.6 ± 0.1 34 ± 3 125 ± 14 41 ± 3

24 ± 1 23 ± 1 4965

± 301

17 ± 1

* Data corrected for hyphal counts in HC of non-mycorrhizal plants; t Means ± SE of five plants; ^ Dry wt = biovolume x 0.23 (Bakken and Olsen, 1983). The compartments contained I50g soil so that absolute lengths of colonized root can be calculated (3450cm per plant). For (a) and (b) the plants were grown in pots divided into compartments by mesh, in order to separate hyphae from roots. Data from Jakobsen and Rosendahl (1991).

Figure 4.2 Mycelial links between plants of Plantago and Festuca. (a) Autoradiograph of roots of a donor plant {Plantago, PLR) and a receiver plant {FestucOy FR) linked by a fungal hypha (AH) and showing transfer of '"^C from donor to receiver. Labelled infection unit in Festuca (*). (b) Distal part of an arterial hypha (AH) showing entry points (EP) into a receiver root (Festuca, FR). Labelling of the hyphal system and of a spore (SP) is apparent, following feeding of ''^C02 to a donor plant of Plantago. From Read et al. (1985), Ecological Interactions in Soil, Blackwell Scientific Publications, with permission.

I 12

Vesicular-arbuscular mycorrhizas

biomass, Jakobsen and Rosendahl (1990) calculated that the fungus could have been using as much as 20% of the total ^^C02 fixed by the plant, equivalent to 7.4 mg ^^C per day. These plants were only 26 days old and were already extensively (95%) colonized, so that it is likely that most of the fungal tissue was alive and active (see Smith and Dickson, 1991), possibly explaining the high C use. Different fungal species appear to use different proportions of the total photosynthate. With Cucumis as the plant symbiont, the proportion of total fixed ^^C respired below ground was 16.3%, 17.3% and 26.2% when the roots were colonized by Glomus caledonium, Glomus species WUM 10(1) and Scutellospora calospora, respectively Uncolonized control roots respired only 9.7% (Pearson and Jakobsen, 1993a). The transfer of ^^P to the plant was studied in the same experiment and the results showed that S. calospora was the least efficient fungus and G. caledonium the most efficient in terms of hyphal ^^C use per unit of ^^P transported. The underlying reasons for these differences have not yet been determined, but could involve different colonization patterns, in which efficiency might be related to a higher proportion of fungal biomass in arbuscules (involved in P transfer), compared with intercellular and extraradical hyphae (involved in C transfer and distribution). Alternatively, there may be variations in symbiotic function at the level of rates of transfer across the interfaces or differences in metabolic activity. In Arum-type mycorrhizas available evidence supports the hypothesis that the site of C transfer is the interface between the intercellular hyphae and the root cortical cells, rather than the arbuscules. The hyphae remain alive throughout the life of an infection unit (Smith and Dickson, 1991; Tisserant et al., 1993) and maintain contact with the external mycelium, even when frequency of arbuscules declines as the plants age. The presence of H^-ATPases on their plasma membranes indicates that these are energized and capable of generating the proton-motive force necessary to support proton co-transport of sugars (or other sources of organic C) from the intercellular apoplast (Gianinazzi-Pearson et al., 1991a). Circumstantial evidence that arbuscules do not play a major role in C acquisition from the host comes from the observations of Mosse and Hepper (1975), who showed that extraradical mycelium began to grow as soon as intercellular hyphae began to colonize the root cortex and before arbuscules were formed, and GianinazziPearson et ah (1995) who observed that intercellular hyphae grow rapidly in the root cortex in Pisum myc~^ mutants which form poorly developed, abortive arbuscules (Fig. 3.1b). Furthermore, the distribution of H"^-ATPase in the arbuscular interface is consistent with the role of the arbuscule in polarized P transfer to the plant, rather than bidirectional transfer of both C and P (see Chapter 14). The site of C transfer in mycorrhizas of the Paris-type, with few or no intercellular hyphae, is unknown at present (see Smith and Smith, 1996a). There are no good data concerning the rate of transfer of organic C across the interface. However, the values of C use by Glomus fasciculatum associated with Cucumis (Jakobsen and Rosendahl, 1990), together with information on mycorrhizal development (G. mosseae) in the same host (Smith and Dickson, 1991) can be used to estimate the C flux (as glucose) across the interface, assuming that hyphae alone, or arbuscules, or both, are involved in the transfer process (Table 4.3). If it is assumed that hyphae are the only sites of transfer, fluxes of C could be in the region of 28 nmol m~^ s~^ and are similar to the highest values for P flux through

Growth and carbon economy of VA mycorrhizal plants

I 13

Table 4.3 Fluxes of C (as glucose equivalents) from Cucumis sativus to Glomus fasciculatum C use by G. fasciculatum (mg C plant ~ ' d ~ ' ) Equivalent glucose transfer (nmol s~') Length of nnycorrhizal root per plant (m) Hyphal interface (m^ m ~ ' ) Hyphal interface per plant (m^) Arbuscular interface (m^ m ~ ' ) Arbuscular interface per plant (m^) Total interface per plant (m^)

7.4 1.16 36 1.2 X 10-^ 41.4 X 10-^ 5.9 X 10-^ 212 X 10-^ 253 X 10-^

Flux (nmol m~^ s~') Hyphae only Arbuscules only W h o l e interface

28 5.5 4.5

Calculated from data for C use by the fungus and percentage colonization of the roots from Jakobsen and Rosendahl (1991). Data for development of intercellular hyphae and arbuscules (by G. mosseae) in the same host from Smith and Dickson (1991).

arbuscules in Allium symbioses (Cox and Tinker, 1976; Smith et al., 1994a,b; and see Table 5.4). If arbuscules are the sole site of transfer then the C flux would be approximately 5.5 nmol m~^ s~^ and if both arbuscules and hyphae are important then the figure would be 4.5 nmol m~^ s~^. It must be emphasized that these estimates are preliminary, but they show that combining measurement of C allocation to the fungus by ^^C labelling with determination of the development of arbuscules and intercellular hyphae might supply accurate data for fluxes in single experiments. The identity of the organic C molecules that are transferred from plant to fungus in VA mycorrhizas is not known with any certainty. We can speculate that sucrose is exported from the cortical cells of the root and that plant or fungal invertases in the apoplast might hydrolyse it to glucose and fructose, which could then be absorbed and used by the fungal hyphae. Unlike other mycorrhizal symbioses, there is apparently no single carbohydrate pool characteristic of the fungus, which rapidly becomes labelled with ^^C and which could consequently be used to follow the incorporation of specifically labelled precursors. For many years both trehalose and polyols eluded detection in VA mycorrhizal fungi. Trehalose has now been found in spores and mycorrhizal roots, and spores also contain significant quantities of glycerol and small amounts of mannitol (Amijee and Stribley, 1987; Becard et al., 1991; Schubert et al, 1992; Shachar-Hill et al, 1995). These, together with large amounts of lipid and small amounts of glycogen, constitute the main carbohydrates and storage compounds in VA mycorrhizal fungi. Their identification has paved the way for studies of the fate of sugars that are potential candidates for transfer. The task is still difficult, but recently the incorporation of ^^C-labelled glucose in mycorrhizas has been followed by nuclear magnetic resonance (NMR) spectroscopy, coupled with gas-chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS) (Shachar-Hill et al, 1995). In roots of leek, colonized by Glomus etunicatum, labelling from ^^Ci-glucose was incorporated mainly into trehalose, with very little transfer of the Ci label into other ring positions. The fractional enrichment of trehalose was over 72%, indicating little dilution by host photosynthate during synthesis. The

I 14

Vesicular-arbuscular mycorrhizas

data are consistent with glucose as a major form of fixed C taken up by the fungus and converted immediately to trehalose. Enrichment of the label in glycogen, but not mannitol, was also observed, contrasting with the situation in germinating spores, where mannitol, rather than trehalose and glycogen, became labelled. At this stage it is not clear whether or not other sugars can be used by the fungus and it would be particularly interesting to compare the patterns of incorporation of label from glucose with those from fructose or sucrose. The last seems most likely to be the ultimate source of carbohydrate for the fungus. Mechanisms which could promote the continuing transfer of carbohydrate from plant to fungus include increasing efflux of 'sucrose' from the cells and switching off plant retrieval mechanisms which would further enhance net efflux. The fungus would require a rapid rate of absorption and incorporation, so that the concentration gradient between the plant cell and the apoplast was maintained (see Patrick, 1989; Smith and Smith, 1990; Smith et «/., 1994b). Incorporation into trehalose, together with very rapid glucose catabolism and synthesis of lipid, would also maintain such a concentration gradient, suggesting the operation of a 'biochemical valve', similar to that proposed in other biotrophic symbioses (Smith et al,, 1969). Total lipid levels in mycorrhizas may be considerably higher than in colonized roots, although the lipid fractions do not appear to differ qualitatively in Allium, Trifolium or Lolium (Cooper and Losel, 1978). However, in Citrus, definite qualitative differences have been detected between the lipids of mycorrhizal and non-mycorrhizal roots. Three unidentified fatty acids, which made up 31-41% of the total lipids in mycorrhizal roots, were not present in non-colonized roots. In view of this, it is surprising that increased incorporation of ^^C into the lipid fraction of mycorrhizal roots has not been detected in all investigations. However, this may have been related to the stage of fungal development, because spores and vesicles which contain most of the stored lipid are not formed in the early stages of colonization. In any event, a recent and extremely thorough analysis of C allocation in a highly mycorrhiza-responsive Citrus cultivar has shown, surprisingly, that the development of large numbers of lipid-rich vesicles during colonization by Glomus intraradices increased the C allocation to the fungus by only about 10%, even when there was a negative growth response with high soil P (Peng et al., 1993). This finding emphasizes that the growth of the fungus both within and outside the root, together with respiration necessary to support growth and maintenance, may be a significant process driving carbohydrate transfer to the fungus.

Nutrient and Carbon Acquisition: An Overview The C economy of mycorrhizal plants needs to be considered in the context of the effects of mycorrhizal colonization on mineral nutrition. It is well known that mycorrhizal roots are more efficient in nutrient acquisition, per unit length, than non-colonized roots. Most of the evidence for this comes from experiments conducted in pots, in the controlled conditions of glasshouse or growth room, but some field studies have given similar results (e.g. Jakobsen, 1986a,b, 1987; Dunne and Fitter, 1989). Mycorrhizas have the largest effect on P nutrition. Not only is P required by both symbionts in relatively large amounts, but it is poorly mobile in soil and occurs in very low concentrations in the soil solution, being rapidly fixed as

Growth and carbon economy of VA mycorrhizal plants

I 15

Fe, Al or Ca phosphates. There is also convincing evidence for increased uptake of Zn, which is also poorly mobile and is deficient in some soils, and Cu. Only relatively recently has attention turned to N, which, in addition to its organic forms, occurs either as poorly mobile ammonium (NH4), or as nitrate (NO^), which although highly mobile in moist soil is not so in dry soil. Evidence is accumulating that uptake of both NH4 and NO3" can be increased in mycorrhizal plants (see Chapter 5). The increased efficiency of nutrient acquisition depends on three essential processes: uptake of the nutrients by the fungal mycelium in the soil; translocation for some distance within the hyphae to the intraradical fungal structures (hyphae and arbuscules) within the roots; and transfer to the plant cells across the complex interface between the symbionts. Under conditions of low nutrient availability the hyphae can absorb nutrients from soil beyond the zone depleted through uptake by the roots themselves, so that they increase the effectiveness with which the soil volume is exploited. Furthermore, the soil pores that can be penetrated by hyphae are perhaps an order of magnitude smaller than those available to roots. Consequently, the effects of mycorrhizal colonization on P nutrition are often large and may have indirect effects on other aspects of plant metabolism, so that direct effects of the symbiosis on other nutrients are masked. When the availability of P in soil is low, non-mycorrhizal roots may be unable to absorb it effectively and the plants become P deficient and grow poorly. Mycorrhizal colonization and P uptake lead to relief of this nutrient stress and, in consequence, plant growth is increased. This is the well-known mycorrhizal growth response (the 'big and little plant' effect; see Plate 2) which has been demonstrated for an enormous number of species in pot experiments. In addition to increased growth rate, mycorrhizal plants frequently have higher tissue P concentrations than nonmycorrhizal plants grown in soil of the same P status and allocate a lower proportion of total plant weight to roots (see Table 4.1). As soil P is increased the growth response of the plants to mycorrhizal colonization declines, so that if sufficient P is available to support near-maximum growth of non-mycorrhizal plants, the colonized plants may actually grow less than non-mycorrhizal ones. However, although there is a negative growth response to colonization, the fungus continues to have an effect on the physiology of the plants. The extent of this depends on the sensitivity of the symbiosis to P supply but the mycorrhizal roots continue to function as effective nutrient-absorbing organs, the root: shoot ratio is reduced and the plants accumulate P in luxury amounts (see Smith and Gianinazzi-Pearson, 1988). Nutrient uptake by the fungus does not lead to a positive growth response because another factor, probably the rate of C acquisition via photosynthesis, is now limiting the rate of growth. Indeed, negative growth responses in mycorrhizal plants can be induced by low irradiance and they are most commonly observed in experiments carried out in glasshouses in winter or in growth rooms with poor light sources, a further indication that there may be a delicate balance between the benefits to be gained from symbiosis in nutrient acquisition and the costs incurred by supporting the heterotrophic fungal symbiont (Buwalda and Goh, 1982; Bethlenfalvay and Pacovsky, 1983; Bethlenfalvay et al, 1983; Koide, 1985a; Modjo and Hendrix, 1986; Smith et al, 1986; Modjo et al, 1987; Son and Smith, 1988). It is important to appreciate the way in which plants respond to both mycorrhizal colonization and changes in nutrient availability in soil because this affects the design of experiments aimed at determining the C allocation to the fungus and the

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economics of the syinbiosis in tern\s of bidirectional transfer of P and C. Measurements of the rate of photosynthesis and allocation of photosynthate in the growing plants need to take into account the effects of the symbiosis on P nutrition. Mycorrhizal and non-mycorrhizal plants used for comparison should be as similar as possible with respect to the rate of growth and nutrient status. In other words, if the aim is to determine whether or not the fungus has any direct effects on the physiology of the plant it is not useful to compare a small nutrient-deficient nonmycorrhizal plant with a large, nutrient-sufficient mycorrhizal one because the indirect effects of improved P uptake on the mycorrhizal plants will be impossible to distinguish from the direct effects of the fungus itself. This means that mycorrhizal and non-mycorrhizal plants need to be grown in soil with different P concentrations and, ideally, a full P response curve for the non-mycorrhizal plants should be obtained, so that the most appropriate comparisons with mycorrhizal plants grown in either low- or high-P soil can be made (e.g. Stribley et ah, 1980; Snellgrove et al., 1982; Eissenstat et al., 1993; Peng et al., 1993; Graham and Eissenstat, 1994; Tinker et al., 1994). An alternative is to employ plants with split root systems, with and without mycorrhizal colonization or supplied with different amounts of P (Koch and Johnson, 1984; Douds et al, 1988). The responsiveness of plants to mycorrhizal colonization also varies between species and cultivars and is markedly influenced by nutrient supply. This responsiveness is frequently referred to as mycorrhizal dependency and expressed as the percentage difference in dry matter between mycorrhizal and non-mycorrhizal plants grown in the same soil (Gerdemann, 1975; see Table 4.1). Responsiveness is, as Gerdemann (1975) emphasized, strongly affected by the nutrient status of the soil and by the irradiance. In other words, both the capacity of the plant to absorb nutrients independently and its capacity to support a heterotrophic symbiont with 'excess' photosynthate are important and may be genetically and environmentally influenced. Examples of highly dependent (and responsive) species which make little growth unless they are colonized even when soil P is relatively high, include Manihot (cassava: Yost and Fox, 1979), Elais (oil palm: Blal et al., 1990) and Centaurium (McGee, 1986; Grime et al, 1987). At the other extreme, many cereals show little response even when they become extensively colonized (e.g. Baon et al., 1992). The ontogeny of the plants and the rate of colonization of the roots also need to be considered in the design of experiments; ideally, multiple harvests at different stages of plant development are required to gain a thorough understanding of the symbiotic relationships, including relative growth rate and nutrient uptake rates. The biomass of the fungus, as well as its rate of colonization of the root system and the relative frequency of arbuscules, hyphae, vesicles and development of mycelium in soil are all important in making cost-benefit analyses of the symbiosis, but few investigations have provided data for all these parameters (see Harris and Paul, 1987).

Mycorrhizal Responsiveness: Costs and Benefits of the Symbiosis Cost Benefit Analysis C fixed by the plant during vegetative growth is allocated above-ground to photosynthetic tissue and below-ground to nutrient-absorbing roots and mycori'hizas.

Growth and carbon economy of VA mycorrhizal plants

I 17

Both these investments are essential for the continuing growth of the plant. However, it has been frequently suggested that the C used by the fungus represents a considerable cost to the plant, which may or may not be offset by a benefit in terms of nutrient uptake (Stribley et al, 1980; Fitter, 1991). Mycorrhizal plants may sometimes be limited by C rather than nutrients, with direct evidence coming from the negative growth responses under low irradiance (see above) and indirect evidence from increased nutrient concentrations in tissues a n d / o r increased fresh weightidry weight ratios (Stribley et ah, 1980; Snellgrove et al., 1982; Smith et al., 1986; Son and Smith, 1988). However, cost or benefit of the symbiosis, determined as dry weight difference (or responsiveness), is an estimate of the net effect and does not provide information on the gross effect, including the actual consumption of C by the fungal symbiont which, as shown above, can be up to 20% of the total fixed C. Koide and Elliott (1989) were among the first to advocate the use of a common currency (C) for the 'economic' analysis of the efficiency of mycorrhizal symbiosis. They defined the cost of the symbiosis as the C expended to support the symbiosis, gross benefit as the extra C fixed as a consequence of mycorrhizal colonization and net benefit as gross benefit minus cost. The efficiency of the symbiosis was defined as net benefit divided by gross benefit. Tinker et al. (1994) have extended the approach and provided a useful account of appropriate methods for making cost-benefit analyses under different conditions. Analysis of the C economy is valuable in determining the direct influence of the fungus on the plant, the amount of photosynthate used in fungal growth and respiration, and the relative efficiencies of roots and fungi under particular soil P conditions. It is important to extend the use of this approach to compare the efficiencies of different plant-fungus combinations and in particular to determine the C-use efficiencies of different fungi in slow- and fastgrowing plants. The data will be valuable in identifying model systems for experiments to determine the characteristics of the fungi or plants that lead to efficient or inefficient symbioses; the data might also be valuable in selecting fungi for horticultural and agricultural applications. So far, few systematic investigations of this sort have been made, so that comparisons are mainly based on the less useful measure of mycorrhizal responsiveness (dependency). In simple terms, mycorrhizal colonization of a root system and the involvement of the fungus in energy- and substrate-requiring activities such as nutrient uptake, vegetative growth and spore production would certainly represent a cost to the plant if there were no way of compensating for it. This is the situation in plantpathogen associations. In mycorrhizal plants the involvement of the fungus in nutrient acquisition may directly or indirectly increase the ability of the plants to fix CO2, and, consequently the 'expense' of the fungus is offset (Allen et al., 1981b; Snellgrove et al, 1982; Koch and Johnson, 1984; Brown and Bethlenfalvay, 1988; Fredeen and Terry, 1988; Eissenstat et al., 1993). Increased photosynthesis may be mediated directly via increased availability of inorganic phosphate (Pi) in the leaves (Sivak and Walker, 1986) or by increased specific leaf area (Fredeen and Terry, 1988); it may sometimes be associated with increased hydration of the leaves (Snellgrove et al, 1982). Comparing mycorrhizal Citrus grown in low P conditions with non-mycorrhizal, P-supplemented plants, Eissenstat et al. (1993) showed that the mycorrhizal plants had higher photosynthetic rates than the non-mycorrhizal plants and that a marked

I 18

Vesicular-arbuscular mycorrhizas

increase occurred during the period (7-8 weeks after planting) when fungal colonization was particularly rapid and P concentration in the tissues was increasing. The mycorrhizal plants in low-P soil were highly dependent (in the strict sense of the word) on the symbiosis, so that although the fungus used a considerable proportion of the fixed C this was clearly compensated for by the improved nutrient uptake which resulted in a higher rate of photosynthesis and overall increased growth. If photosynthesis is sink-limited, as some evidence suggests, then increased demand by mycorrhizal roots might also have contributed to the observed increases in rate. When the rate of photosynthesis and growth is limited by nutrient availability, the C cost of producing absorbing organs (roots or mycorrhizas) may be significant. In contrast, the cost of producing a given length of hyphae (approximately 2-20 ixm in diameter) is about two orders of magnitude less than for roots (approximately 200 jjim in diameter), so that under these severely limited conditions a plant may be able to 'afford' hyphae but not roots. However, at high P concentrations the total cost of maintaining a mycorrhiza may be greater than that of maintaining an uncolonized root, as shown by Eissenstat et al. (1993). Mycorrhizal effects on the percentage below-ground recovery of ^^C (fed as ^^CO^) that were independent of P were a reduction in the fibrous root component at early stages of growth, and increases in the soil component (including hyphae) and in below-ground respiration. Overall, these workers concluded that at equivalent P status mycorrhizal plants had a lower efficiency of C production (ratio of change in whole-plant C gain to change in C expended below ground) and were somewhat less efficient than nonmycorrhizal plants in P acquisition. In summary, if P is readily available in soil, then non-mycorrhizal roots can absorb it more efficiently than mycorrhizal roots because of the high C cost of maintaining the fungal symbiont. However, if P is in short supply or only transitorily available, then non-mycorrhizal roots cannot absorb it at rates that will support photosynthesis and growth. Investment of C in rapid root growth to overcome problems of development of depletion zones is not possible (see Chapter 5) and the plant is obligately dependent on the fungus because mycorrhizal roots can obtain a resource that roots alone cannot. Elevated CO2 concentrations in the atmosphere have been shown to lead to higher rates of C acquisition by plants. There are indications that they also result in increased mycorrhizal colonization of roots. It seems likely that the increased availability of photosynthate would also increase the responsiveness of plants to colonization a n d / o r reduce growth depressions at high concentrations of available P.

Variations in Extent of Colonization and Responsiveness The responsiveness of plants to mycorrhizal colonization certainly varies with species and genotype, as well as with environmental conditions. The type of root system has already been mentioned (Chapters 1 and 3) and there is general acceptance that thick, unbranched 'magnolioid' roots, with few root hairs, are frequently associated with high responsiveness, but this is certainly not always the case and a number of other factors have been suggested as being important. These include the extent of colonization of the roots and the rate of root growth, both of which would directly affect uptake of nutrients. The inherent rate of

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growth, together with the plasticity of the rootishoot ratio, may also be important. Koide (1991a) has further argued that another crucial determinant of responsiveness is the P deficit of the species or cultivar. This is essentially the difference between P demand for growth and P uptake, with the latter parameter influenced by P supply in the soil as well as by mechanisms such as rate of root growth and mycorrhiza formation, which influence efficiency of uptake. Koide considered that rapid growth rate and high tissue P concentrations might lead to a high P deficit and high mycorrhizal responsiveness, and that this might particularly apply to crop plants selected for rapid growth rates. It is fairly difficult to separate out all these possible effects (see examples below) and although in some species rapid growth seems to be associated with high dependency, there is an equal number of examples where this is not the case. Certainly, Avena sativa, which has a fast growth rate and lower and less plastic root.shoot ratio, was more responsive to colonization than the wild Avena fatua (Koide et ah, 1988) and cultivars of Lycopersicon esculentum were more responsive than wild accessions (Bryla and Koide, 1990a,b). However, Hall (1978a) found that a variety of Zea mays (cv. PX 610), which had a rapid growth rate, extensive root system and low tissue P concentration, did not respond to inoculation with VA mycorrhizal fungi, whereas Z. mays cv. 415 and Z. mays x robusta cv. Golden Cross Bantam, both of which have slower growth rate and root development but higher tissue P concentrations, were responsive to colonization. Cultivars of barley (Hordeum vulgare) selected for variation in P efficiency responded to mycorrhizal inoculation and to P application when non-mycorrhizal, in similar ways (Baon et ah, 1993). Cultivars that were inefficient with respect to growth on low-P soils (slow growing) when non-mycorrhizal were more responsive to both inoculation and P fertilization than efficient cultivars (see Fig. 4.3a). 'Shannon', the least efficient and most responsive, had the lowest root dry weight (and, presumably, root length), while 'Yagan', the most efficient and least responsive, had the highest. There were also considerable cultivar-dependent differences both in percentage colonization by Glomus etunicatum and in effect of applied P on colonization (Fig. 4.3b). 'Shannon' had high percentage colonization in low-P soil (30%) and 'Yagan' low (15%); several cultivars of intermediate efficiency also showed low colonization. P fertilization markedly reduced colonization in 'Shannon', but not in 'Yagan'. These responses in barley are quite different from those reported for Citrus rootstocks, so that, as yet, there appear to be no consistent mechanisms involved in differences in efficiency. The work on variation in Citrus and related species has included consideration of C use by the colonizing fungus, as well as extent and rate of colonization and mycorrhizal responsiveness in low- and high-P soils. In these plants the rate of spread of the fungus, rather than the maximum extent of colonization, appeared to be important in C use and its effect on responsiveness. Citrus species of low mycorrhizal responsiveness became colonized more slowly than highly responsive species, leading to the suggestion that this character would have been favoured during evolution because of the considerable drain on photosynthate that the fungus exerts during rapid colonization of the roots (Graham et ah, 1991; Graham and Eissenstat, 1994). In highly responsive species, which would be expected to be characteristic of sites low in available P, this expenditure would be offset by increased nutrient acquisition and would not have been

Vesicular-arbuscular mycorrhizas

120

0

1

2

3

Agronomic phosphorus efficiency (g dry wt per pot at no added phosphorus)

Kaniere

Yagan O'ConnorWI 2539 Wl 2767 Galleon

Skiff

Shannon

Cultivars Figure 4.3 Interactions of VA mycorrhizal fungi with cultivars of Hordeum vulgare, differing in phosphorus efficiency. From Baon et o/. (1993), with permission of Kluwer Academic Publishers, (a) The relationship between agronomic P efficiency and response to colonization by Glomus etunicatum or P fertilization with 10 mg P kg~' soil. Cultivars: Sn, Shannon; K, Kaniere; C, O'Connor; Sf. Skiff; W 5 , W l 2539; W 7 , W l 2737; G, Galleon; Y, Yagan. • Mycorrhizal response; O P response. Regressions significant at 0.01 level of probability, (b) Effects of P fertilization on percentage mycorrhizal colonization of cultivars of Hordeum vulgare by Glomus etunicatum. Bars are standard errors of the means of determinations from three replicate plants. P additions (mg P kg~' soil): D zero; B I O ; ! 20.

Growth and carbon economy of VA mycorrhizal plants

121

disadvantageous in evolutionary terms. In these highly responsive plants the rate and extent of colonization are correlated with limited branching of the roots and slow root growth (Graham and Syvertsen, 1985) and, in contrast to the responsive barley cultivar Shannon, is not markedly reduced by high P supply, compared with less responsive species (Eissenstat et ah, 1993; Peng et a/., 1993). Manske (1989) compared 22 land races of wheat {Triticum aestivum) with 22 high yielding varieties and found that the land races were significantly more responsive to colonization in shoot and ear production. In this investigation, growth responses were not related to either percentage colonization or root length, but the picture is confused by the fact that some land races had greater root length than the high yielding varieties and, in consequence, greater colonized root length. P fertilization reduced percentage colonization in both groups and there were no positive growth responses to colonization under these conditions. The extent of colonization was related to P accumulation in a number of inbred lines selected for low or high ear or leaf P content (Toth et ah, 1984). In the field the high-P lines were more highly colonized than the low-P lines and there were no differences in P status when plants were grown in sterile soil without mycorrhizal inoculum. This led to the conclusion that the basis for the differences between the lines was mycorrhizal susceptibility and, consequently, higher P absorption, although this had not been appreciated during the actual selection process. In contrast, Robson and Collins (unpublished; see Smith et ah, 1992) found no relationship between P response and extent of colonization in cultivars of Trifolium subterraneum selected for differences in P efficiency. The picture is far from clear and further work is required, carefully targeting genetic variation in the plants, as well as knowledge of the potential effects of mycorrhizal colonization on growth and nutrition.

The Importance of Variation for Plant Breeding Programmes Not only have breeding programmes ignored potential advantages of mycorrhizas for nutrient acquisition, but, the high fertilizer applications normally used would have ensured that both mycorrhizal colonization and mycorrhizal responsiveness were at their lowest. In consequence, the potential variations in susceptibility and benefits from colonization have been minimized and may have been completely lost in some cultivated species. Furthermore, differences that have been found between highly bred or selected lines with respect to mycorrhizal characters may actually be the result of inadvertent and indirect selection. Toth et al. (1984, 1990a) considered that this was likely both in the inbred wheat lines with different P contents mentioned above and in inbred lines of Zea mays with different resistance to fungal pathogens. In the latter case, resistant inbreds had lower percentage colonization, but they also had longer root systems and matured more slowly than the susceptible varieties, so that the apparent correlation between resistance and mycorrhizal colonization may not be at all related to actual susceptibility at the cellular level. At least three problems arise from ignoring variations in mycorrhizal colonization and response in breeding programmes. First, there is loss of a source of variation which may be useful in developing varieties that are particularly efficient in nutrient acquisition from deficient soils. Second, experiments carried out over a

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Vesicular-arbuscular mycorrhizas

range of soil P levels (e.g. breeding programmes for nutrient efficiency) are confounded by the variations in symbiotic contribution to nutrient uptake and use across treatments, because both colonization and responsiveness are inversely related to fertilizer application (see Fig. 4.2 and Chapters 2 and 5). Third, elimination of mycorrhizal colonization also eliminates potential benefits not directly related to nutrition, such as increased aggregate stability and improvements in soil structural stability, pathogen resistance and drought tolerance (see Chapter 16). The genes involved in the control of mycorrhizal development and function, the activities of which are apparent as cultivar- and species-dependent variation, need to be identified and their function appreciated, so that their role in mycorrhizal responses of wild plants can be understood and they can be used confidently in the development of new cultivars of crop plants.

Responses in Natural Ecosystems In natural ecosystems interpretation of the advantages of mycorrhizal colonization in terms of responsiveness, growth rate and P demand are complicated by the age and species diversity of the plant populations. Many wild plants are relatively slow-growing compared with crops bred for high production of above-ground biomass; they are characteristic of habitats where nutrients may be in short or intermittent supply and have presumably evolved with mycorrhizal fungi. Low growth rate may mean that roots do not extend sufficiently rapidly in soil to overcome problems of depletion of immobile nutrients close to the roots and they might therefore be highly responsive to mycorrhizal colonization. Mycorrhizal dependency may also vary with the stage of growth of the plant or seasonal requirements, in particular with respect to periods of high P demand. Establishment of seedlings of fast-growing crops or ephemerals may well require high rates of P uptake, before the root systems are well developed. Concentrations of P in seeds influence plant establishment and responsiveness to mycorrhizal colonization (AUsopp and Stock, 1992). Rapid mycorrhizal colonization in smallseeded plants may be important in providing adequate P for plant growth, even though the high C demand of the fungus may lead to transitory growth depressions (Smith et al., 1979). P may also be required in large amounts at other stages of growth. For example, Fragaria has a very high P demand when fruiting (Dunne and Fitter, 1989), during which time P uptake occurs at rates that might be strongly influenced by mycorrhiza activity Moreover, Hyacinthoides non-scripta (the English bluebell) has a very limited root system which grows at a season when there is no above-ground tissue. The plant is highly dependent on mycorrhizal colonization for P absorption at this stage (Merryweather and Fitter, 1995a,b, 1996; see Chapter 15). Luxury P accumulation following mycorrhizal colonization in situations where P supply does not (at least temporarily) limit productivity may be a significant advantage for wild plants, providing a stored pool which can be mobilized in periods of high requirement. The possible role of VA mycorrhizas as a mechanism to ensure that transitory peaks of nutrient availability are exploited to the maximum extent has been largely ignored. This stems from the early observations that the fungus itself does not, unlike ectomycorrhizas, store large quantities of P as

Growth and carbon economy of VA mycorrhizal plants

123

polyphosphate. However, the same eventual outcome for the plant could result from luxury accumulation in roots, leaves and seeds. The C available to support the fungal symbiont may also vary depending on the rate of plant growth. Plants with inherently faster growth rates are less likely than those with slow growth rates to produce excess photosynthate, which might be advantageously diverted to the mycorrhizal symbiont, resulting in positive responses to mycorrhizal colonization over a wide range of soil P levels. Slowgrowers might also be less susceptible to C limitation under low irradiance, especially if they are also able to adjust C allocation to roots and shoots (see Chapin, 1980; Lambers and Poorter, 1992; Smith and Smith, 1996a,c). There is increasing recognition that our knowledge of the way in which plants respond to colonization in natural ecosystems is rather scanty (see Chapter 15), and doubts have been expressed concerning the benefits - if any - of colonization in terms of nutrient-C exchange (McGonigle, 1988; Fitter, 1991). Only more research will provide the answers.

Mycelial Links Between Plants: Importance in Carbon Allocation in a Plant Community The potential importance of hyphal links between plants of the same or different species is now widely appreciated (see Read et al., 1985; Newman, 1988). External hyphae are important as sources of inoculum, and probably account, to a very large extent, for the rapid colonization of root systems in many undisturbed habitats, in particular when conditions favour perennial vegetation (see Chapter 2). C allocation amongst plants could be influenced by the hyphal links in a number of ways and it is consequently important to gain as much information as possible about how the links function. There seems to be no doubt that a seedling, growing up in a mycorrhizal community would become very rapidly linked into, and acquire nutrients from, a mycelial network which had developed at the expense of photosynthate from already established individuals. This could represent a considerable saving to the seedling and result in a greater chance of successful establishment, in particular where shade, for example, might limit the photosynthetic capacity beneath the canopy. Growth depressions are observed at the seedling stages of experiments where all plants are of the same age (see Smith, 1980) and are likely to be the result of C utilization as the fungus enters into the stage of rapid root colonization, but before it has a major effect on P uptake. If the hyphae colonizing the roots were supported (at least temporarily) by photosynthate from an established plant, rather than propagules with limited reserves, a considerable benefit might be gained. At later stages of growth, a successful seedling might be expected to become a 'supporter' in terms of C supply, as well as a 'user' of the mycelial network in terms of P uptake. Evidence that the C cost of coloruzation of a 'user' can be borne by a 'supporter' comes from experiments with Centaurium, which are only successfully colonized if they grow with a mycorrhizal companion plant (McGee, 1985), from the very rapid colonization observed in grassland communities (Birch, 1986) and from a number of experimental systems using older 'nurse'

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Vesicular-arbuscular mycorrhizas

or 'supporter' plants to initiate colonization in seedlings (Brundrett et ah, 1984; Rosewame, 1993). Sharing the C cost of maintaining a mycelial network does not necessarily depend on net transfer of fixed C from one plant to the other. All that is required is that the 'supporter' provides the majority of the necessary C to the fungus. Mycorrhizal effects on both frequency and diversity of species in microcosm experiments (see Grime et al., 1987; and see Chapter 15) can be explained if the species vary in the amount of C that they provide to the system and in their response to colonization in terms of nutrient uptake and growth. However, ^^C02 fed to one plant (often termed the 'donor'), certainly does appear in roots and, to lesser extent, in shoots of plants linked to it by the mycelial network (the 'receivers'), as shown in Fig. 4.1 (Hirrel and Gerdemann, 1979; Francis and Read, 1984; Grime et al., 1987). The question is, does this movement of tracer represent a net transfer of C between plants in amounts which could significantly affect growth? Newman (1988) has explained that the movement of ^^C indicates the existence of a pathway for transfer along the hyphae of the network and across the fungusplant interface, in the opposite direction to that normally operating. It does not show that net C transfer between plants occurs, and so far no experiments have shown unequivocally that net transfer of C from fungus to autotrophic plant takes place. A mechanism for bidirectional transfer of C at the membrane transport level exists if sugar is transported from the donor plant to the fungus, while N moves in organic form from the fungus to the receiver (Smith and Smith, 1989, 1990). Operation of these two transport systems in series would explain the low level of ^^C transfer from the shoots of the donor to the shoots of receiver plant. However, as shown in Table 4.4, neither shade nor clipping, which might be expected to increase the driving force for such source-sink transfer of C, increased the labelling in shoots of the receivers, although they did increase it in their roots (Read et al., 1985; Newman, 1988). In consequence, it seems unlikely that plants actually share photosynthate via the hyphal network to any great extent. Rather, support of the network by larger or more photosynthetically active individuals, and sharing of the mineral nutrients acquired by it, probably underlie the effects of mycorrhizal connections observed in communities of photosynthetic plants. If the potential receiver plants are non-photosynthetic (myco-heterotrophic) the situation is quite different. It appears that some achlorophyllous members of the T a b l e 4.4 Effects of shade on C transfer f r o m Plantago lanceolata t o Festuca ovina Shade

Full light Root

Shoot

Non-mycorrhizal

0.08

0.10

VA mycorrhizal

2.8***

0.44**

Root 0.03 36.4***

Shoot 0.04 0.32***

' C supplied as ''*C02 to the leaves of the donor plant. The receiver plants were either fully illuminated or shaded during the transfer period (48 h). Statistical significance of differences between mycorrhizal and non-mycorrhizal: * * * P < 0.001; * * P < 0.01. Figures calculated from raw data in Table 4 of Read et al. (1985); statistical significance by Mann-Whitney L/-test. Data of Read et al. (1985), recalculated by Newmann (1988).

Growth and carbon economy of VA mycorrhizal plants

125

Polygalaceae, Gentianaceae and Burmanniaceae are associated with glomalean fungi, which form mycorrhizas characterized by aseptate hyphae and extensive intracellular coils (see Leake, 1994). It is assumed that these myco-heterotrophs obtain all their organic C via the fungus, and the implication is that this must come from a photosynthetic 'donor' plant, because glomalean fungi have little or no saprophytic capacity. The myco-heterotrophs would thus be epiparasites on the photosynthetic species and net transfer of organic C from fungus to plant must occur. At present there is no experimental evidence to support this idea, but it is clearly an area which is wide open for research.

Conclusions VA mycorrhizal fungi are dependent on an organic C supply from a photosynthetic partner. Between 4% and 20% of net photosynthate is transferred to the fungus and used in production of both vegetative and reproductive structures, and in respiration to support growth and maintenance, including nutrient uptake. There is preliminary evidence that glucose can be absorbed by the fungus and it seems likely that transfer from plant to fungus occurs across the interface between intercellular hyphae and cortical cells of the root, at least in mycorrhizas with distinct intercellular and arbuscular interfaces. Assuming that both these assumptions are correct, the rate of transfer has been calculated in one instance to be as high as 28 nmol of glucose m~^ s~^. Glucose is incorporated into trehalose, with little dilution by other carbohydrate pools, but the fate of sucrose, which is the most likely ultimate source of carbohydrate for the fungus has not yet been determined and more work is required. There appears to be considerable variation in the C expended by different fungi in the transfer of P to the plant and at this stage it is not known whether the differences are due to variations in patterns or rates of colonization or to more subtle differences in membrane transport capacity or respiratory efficiency. More data are needed in all these areas. Expenditure of fixed C by the plant to maintain the fungal symbiont can be regarded as an investment, resulting in greater efficiency of nutrient acquisition when nutrient availability in the soil is low. Attempts to make cost-benefit analyses of the symbiosis have proved difficult, because of the complex ways in which both mycorrhizal colonization and plant growth respond to and interact with niineral nutrition. The C supply to the mycelial network may not be equally contributed by the plants which it links together. The result is that some plants may effectively support others, by reducing the C drain on them, at least temporarily. There is no unequivocal evidence at present that this involves net transfer of C from one plant to another, although experiments with ^^C labelling suggest that there may be a pathway via which this could take place. The way in which myco-heterotrophs associated with glomalean fungi obtain organic C is at present unknown, although it seems likely that epiparasitism via mycelial links may be involved.

Plate 2. Effects of mycorrhizal inoculation of a range of crop plants in fumigate soil. Right-hand block, inoculated with VA mycorrhizal fungi. Left-hand block, not inoculated. Crops (front to back): Allium, Catalpa, Pisum, Vicia, Zea. Non-host plants trimmed. Photograph courtesy of V. Gianinazzi-Pearson.

Plate 3. Lactahus subdulcis mycorrhiza, formed between L subdulcis and Fagus sylvotica. (a) Irregular pyramidally branched mycorrhizal system (X5.5). (b) Irregular pyramidally branchged mycorrhizal system ( X I 0.6). (c) Tip of mycorrhizal axis (X44). (d) Rhizomorph ( X 4 4 ) . From Brand (1987), with permission.

5 Mineral nutrition^ heavy metal accumulation and water relations of VA mycorrhizal plants

Introduction Mineral nutrition of vesicular-arbuscular (VA) mycorrhizal plants has received more emphasis and been the subject of more research than any other aspect of the symbiosis. The effects of increased uptake of P and Zn, as well as Cu and Ni (as NH4 and possibly NO 3) on the growth of plants are the direct result of fimgal colonization and are often related to increased growth when the nutrient in question is limiting. Data have usually been obtained from pot experiments in sterilized or partially sterilized soil, and are available for a very large number of plant species associated with many different mycorrhizal fungi. The main emphasis, as shown in Chapter 4, has been the large influence of mycorrhizal colonization on growth and P nutrition. This major effect has made it difficult to determine the contribution of VA mycorrhizal fungi to uptake of other nutrients or of water. In the 1960s a number of general reviews were written which covered the occurrence of colonized plants and anatomy of VA mycorrhizal roots, and also addressed the problems of experimentation that arise from difficulties in identification of the fungi involved in the symbiosis and production of satisfactory inoculum (Baylis, 1959; Nicolson, 1967; Gerdemann, 1968; Harley, 1969). During that early period, details of the effects of the symbiosis on mineral nutrition were poorly understood, although some clues had begun to appear and key experiments on P nutrition were carried out. In 1957, Mosse published the results of an experiment with apple seedlings which clearly demonstrated increased amounts of K, Fe and Cu per unit weight of tissue in mycorrhizal plants, compared with uninoculated controls. Later, Gerdemann (1964), Daft and Nicolson (1966) and Baylis (1967) established that tissue concentrations of P (which were not measured by Mosse) were higher in mycorrhizal plants. The mechanism(s) underlying this effect had been quickly addressed and the first papers showing increased uptake of P on the basis of root length soon appeared (Sanders and Tinker, 1971, 1973), around the same time as the first demonstrations of differential effects of different fungal species (Hayman and Mosse, 1971; Mosse and Hayman, 1971). Consequently, by 1973 Mosse was able to comment that even though the fungi had not been grown in

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127

pure culture, the methodological problems had been largely overcome and that there had been a change in emphasis of the research towards effects on plant growth and P uptake. Important reviews followed that introduced concepts from soil chemistry to mycorrhizal studies and have influenced the research to the present day (Tinker, 1975a,b, 1978). It is now well established that mycorrhizal roots take up P, Zn and probably Cu and NH4 from soil more efficiently than uncolonized root systems, and that the extraradical hyphae play an essential part in effectively increasing the volume of soil available for acquisition of these nutrients. More recently, research has been extended to cover effects of the symbiosis on many aspects of the physiology of the plants (e.g. Smith, 1980; Smith and Gianinazzi-Pearson, 1988; and see Harley and Smith, 1983; Koide, 1993; Jakobsen, 1995) and a vast array of books and reviews has appeared in the past 15 years, reflecting the interest of researchers in many basic and applied fields (e.g. Powell and Bagyaraj, 1984; Safir, 1987; Smith and Smith, 1990; Read, 1991a,b; Allen, 1992; Gianinazzi and Schiiepp, 1994; Marschner and Dell, 1994; Jakobsen, 1994, 1995). Moreover, the importance of mycorrhizas, rather than roots, as the normal nutrientabsorbing organs of most plant species has been recognized and their effects addressed in more general contexts (e.g. Nye and Tinker, 1977; Clarkson, 1985; Gadd, 1993; Tisdall, 1994). It is the purpose of this chapter to present an overview of the current understanding of the role of mycorrhizas in the mineral nutrition of plants but it is impossible to quote more than a small proportion of the relevant papers. The discussion centres on the mechanisms that have been suggested to account for the mycorrhizal effects and the experimental evidence which supports them. The focus is on P uptake, as well as on the uptake of other nutrients for which there is now unequivocal evidence of mycorrhizal involvement. In addition, the effects of mycorrhizas on the water relations of plants is critically discussed.

Phosphorus Nutrition Effect of Mycorrhizal Colonization on P Uptake Indirect evidence that mycorrhizal roots are more efficient in nutrient uptake than non-colonized roots comes from the fact that mycorrhizal plants are frequently not only larger but also contain higher concentrations of P in their tissues than uncolonized control plants (see Chapter 4). However, increased efficiency of absorption per unit of root tissue is not the only possible explanation for this. Increased total root length in mycorrhizal plants would certainly contribute to increased total uptake, but this would not necessarily lead to increased tissue concentration. If growth kept pace with P uptake, as it would if P supply were the limiting factor, tissue concentrations should remain constant because they are dependent upon the relative rates of uptake and growth. If tissue concentrations rise (as shown in Table 4.1 and Fig. 5.1a,b), some factor other than P must be limiting growth (Pairunan et ah, 1980; Stribley et al, 1980). As shown in Chapter 4, this probably results from increased carbohydrate use in mycorrhizal plants, which means that mycorrhizal plants can be C-limited and accumulate 'luxury' amounts of P. Whatever the

Vesicular-arbuscular mycorrhizas

128

0-5

0-5

• ••

0-4

0 - 3 hr

0-2

(b)

|(a)

•^

[f

-

ft/

0-25

0-1

1

1

1

1

1

i

1

t

200 400 600 800 Shoot dry matter (mg)

1

1

1000

0

1 2 Stioot dry matter (g per pot)

3

Figure 5.1 The relationship between P concentration in the shoots (percentage dry weight) and dry weight of shoots in: (O) non-mycorrhizal plants, and ( • ) plants colonized by G\omus mQ%%^Qt. (a) K\\\un\ porrum (leek) grown on 10 different y-irradiated soils differing in initial P content and receiving five different levels of added P to give the 50 soil treatments. Redrawn from Stribley et o/, (1980). (b) Thfolium subterraneum grown in soil with the addition of superphosphate. Redrawn from Pairunan et al. (1980).

mechanism, the elevated tissue concentrations of P and other nutrients in mycorrhizal plants certainly alerted early investigators to the possible role of mycorrhizas in plant nutrition. More direct evidence about the efficiency of P absorption has been obtained by expressing uptake on the basis of the amount of absorbing tissue. It seems likely that surface area of the root system, either the v^hole or that part involved in uptake, would be the most suitable estimate of absorbing tissue to be related to uptake rates when nutrients are not diffusion limited. In practice, this area is difficult to determine and most results have been expressed on the basis of root length, defined as inflow (mol m~^ s~^) by Brewster and Tinker (1972). Other results have been based upon root weight, to give specific absorption rate in mol g"^ s"^ (Hunt, 1975; Smith and Gianinazzi-Pearson, 1990). Inflow gives a realistic basis for comparison of immobile ions such as PO^, the uptake of which is diffusion limited (see below), because linear extension of the root system into undepleted soil is more important in determining uptake than the surface area presented to a zone of soil in which nutrients are depleted. Sanders and Tinker (1971, 1973) first demonstrated increased inflow of P in mycorrhizal roots in Allium cepa colonized by Glomus sp., and their data are shown in Table 5.1. The inflow into mycorrhizal roots was on average about 3 ^ times greater than into non-mycorrhizal roots. This effect has been shown for a number of other plant-fungus combinations in pot experiments, although the magnitude of the effect varies (see Dunne and Fitter, 1989). Increases in inflow in mycorrhizal plants growing under agricultural field conditions have also been demonstrated for Pisum, Fragaria and Trifolium (Jakobsen, 1986b; McGonigle and Fitter, 1988b; Dunne and Fitter, 1989). However, few data are available for plants in natural ecosystems

Mineral nutrition, heavy metal accumulation and water relations

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T a b l e 5.1 Inflow of P Into mycorrhizal (M) and non-mycorrhizal ( N M ) roots oi Allium cepa and the calculated contribution of hyphae (H) t o inflow in the mycorrhizal roots f o r t w o experiments Experiment

Colonization Inflow (%) (mol P m"* s~' X 10"'^) M

NM

Hyphae

Flux in hyphae* (mol P X 10"^ m"^ s~') Hyphae colonized regions

A B

50 45

13.0 11.5

4.2 3.2

8.8 8.3

17.6 18.5

3.8

The colonized regions of the nnycorrhizal roots had about 600 entry points per metre and entry point hyphae were IS |xm in diameter, with a central lumen of 10 |j,m. The cross-sectional area of hyphae via which P entered the roots was therefore approximately 4.7 X I0~® m^ m~'. These values, together with the percentage root length colonized by the fungus (a Glomus sp.) can be used to calculate the flux of P translocated into the roots via the hyphae. * Mean for two experiments. Data from Sanders and Tinker (1973).

(Fitter et al, 1995; Merryweather and Fitter, 1995a,b, 1996), a point which will be discussed later. Nevertheless, the increased efficiency of uptake requires explanation and this is to be found in the factors limiting uptake of P from soil. P in Soil and its Availability to Plants

The amount and form of P in soil and the factors affecting its availability are important in determining the way in which mycorrhizal fungi influence uptake by plants (see Tinker, 1975a; Nye and Tinker, 1977; Bolan, 1991). P is required in relatively large amounts. It is absorbed as inorganic PO^ ions (H2PO4) from the soil solution where it is present at very low concentrations. Indeed, the proportion of total soil P in solution is commonly less than 1%, controlled mainly by soil chemical reactions and to a lesser extent by biological processes. P supply may also be patchily distributed both in space and in time, affecting its availability to and use by plants and microorganisms (Lodge et al, 1994; Robinson, 1994). P in soil can be broadly categorized as inorganic (Pi) or organic (Po). Pi may be held very firmly in crystal lattices of largely insoluble forms, such as various Ca, Fe and Al PO^s, and may also be chemically bonded to the surface of clay minerals. Some of this P exchanges very slowly with the soil solution and comprises a nonlabile pool, regarded as unavailable to plants. Less tightly bound (or labile) P exchanges relatively rapidly with the soil solution and is viewed as being in isotopic equilibrium with it. It is this labile P which constitutes the pool regarded as being available to plants, although chemical extraction methods to determine its size do not necessarily reflect what the plants actually absorb (see below). P is most readily available at around pH 6.5. At lower pH, the decreasing solubility of Fe and Al phosphates controls the solution concentration, whereas at higher pH decreasing solubility of Ca phosphates becomes important. Localized changes in rhizosphere pH may play a role in altering the availability of these different sources of P

130

Vesicular-arbuscular mycorrhizas

(Grinsted et al, 1982; Hedley et al, 1983; Marschner et al, 1986). Furthermore, the production of chelating compounds, such as organic anions (e.g. citrate and oxalate) could increase the availability of P from some sources (see Marschner, 1995). Po has its origins in the organic P compounds in soil organisms, including plants, microorganisms and animals. The predominant forms that can be extracted from soil are inositol phosphates, phospholipids and nucleic acids, and their conversion to inorganic form and consequent availability to plants depends on hydrolysis either by microorganisms or by enzymes originating in the organisms themselves (autolysis). Organic P does not seem to provide a major pool directly available to plants with VA mycorrhizas, although there is some evidence for the activity of phosphatases on the surface of roots that could effect hydrolysis. Soluble Pi, entering the soil as the result of such hydrolysis (mineralization) or as fertilizer, results in localized and short-term increases in the concentration of phosphate ions in the soil solution. However, much of it is removed from solution by 'fixation', which is rapid at first and continues for a long time without reaching equilibrium. Fixation involves sorption of ions on soil surfaces, precipitation of mineral phosphates and use by organisms, resulting in immobilization of P in the biomass. These fixation processes have important effects on the concentration of phosphate ions in the soil solution and their movement in soil and hence the uptake by roots and microorganisms. It is quite apparent that mass flow of solution in soil is unable to supply phosphate ions to roots at rates that can account for the amount of P absorbed and it is taken as axiomatic that phosphate ions are supplied to absorbing surfaces by diffusion (Tinker, 1975a; Nye and Tinker, 1977). Furthermore, the rate of diffusion of phosphate ions in soil is several orders of magnitude lower in soil (10~^lO"'^^ cm^ s~^) than in free water (10~^ cm^ s~^) and will vary with the P content of soil, the buffering capacity and the tortuosity of the diffusion pathway. Slow diffusion of phosphate ions in the soil solution, contrasted with their rapid absorption by roots and other absorbing organs, results in the development of depletion zones around them (Lewis and Quirk, 1967; Bagshaw et al, 1972; Nye and Tinker, 1977). Uptake is limited by the rate of diffusive movement of phosphate ions into these depletion zones, rather than by the rate of the transport across living membranes into the root or mycorrhiza (i.e. the absorbing capacity). The longer a segment of root remains actively absorbing from soil at a rate greater than that of movement of phosphate ions to it, the wider will be the depletion zone, with consequent reduction in the rate of arrival of P at the absorbing surface and hence on uptake by the plant. It is possible to make approximate calculations of the inflow to be expected once a 'zero concentration sink' has developed roimd the roots. The magnitude of this inflow depends on a number of soil factors including the size of the labile pool and buffering capacity, which affect the solution concentration in the bulk soil, together with characteristics which influence the diffusion coefficient. These include the solution concentration, the pH, redox potential and ionic strength of the soil solution as well as the water content and other factors that affect the tortuosity of the diffusion path. Compared with these soil factors, the effects of root radius and root absorbing capacity on rate of uptake are relatively small. Root hairs effectively extend the diameter of the absorbing surface of the root beyond the depletion zone and it is likely that they have significant effects on P uptake, especially if they are long (see Claassen and Barber, 1976; Clarkson, 1985).

Mineral nutrition, heavy metal accumulation and water relations

131

However, they are short-lived structures and, surprisingly, rather little work has been done on their effects on plants growing in soil (see Tinker, 1990).

Mechanisms Underlying the Mycorrhizal Effect on P Uptake: A Summary Possible explanations for the increased efficiency of P uptake by mycorrhizal roots are outlined briefly below. Some are already well established as playing a significant part, while others are still under investigation. They are not mutually exclusive and the evidence for their operation under different conditions and their relative importance will be discussed in the next section. 1. Extraradical hyphae of mycorrhizal fungi, by growing into soil not colonized by roots, can absorb P from the soil solution and translocate it to the root. These processes, together with subsequent transfer from fungus to plant, are much faster than diffusion through the soil. Consequently, hyphal transfer overcomes the reductions in rate of uptake which result from the development of depletion zones around the roots. The production of hyphae involves a smaller expenditure of C per unit length or per unit absorbing area than the production of roots (see Tinker, 1975a) and their small diameter also allows them to penetrate soil pores of smaller diameter than roots, effectively increasing the volume of accessible soil. The source of soil P is envisaged to be the soil solution and to be the same for both roots and hyphae. As the hyphae absorb P, depletion zones will develop around them also and continuing uptake will depend on hyphal growth into uncolonized soil. The effects of hyphae on increasing the spatial availability of P is well established and good evidence for their operation is available. 2. The hyphae are more effective (as a consequence of their size and spatial distribution) than roots in competing with free-living soil microorganisms for recently mineralized or solubilized Pi. This hypothesis is really an extension of (1), but its operation could result in effective 'short cycling' of Pi, particularly when inputs are as temporal or spatial pulses. Rapid proliferation of hyphae in localized patches of soil with high nutrient concentrations may be important but evidence is not very extensive. 3. The kinetics of P uptake into hyphae may differ from that of roots, with a higher affinity (lower K^) of uptake leading to more effective absorption from low concentrations in the soil solution and possibly lower threshold values below which uptake ceases. The absorbing capacity of either hyphae or roots will be most important in determining the amount of P absorbed when they are growing into undepleted soil. 4. Mycorrhizal roots can use sources of P in soil that are not available to roots. This could involve increased rates of solubilization of insoluble Pi or hydrolysis of Po and would depend on localized alterations in pH, production of organic anions as chelating agents and production of surface or soluble phosphatases. Results are conflicting, despite much effort.

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Vesicular-arbuscular mycorrhizas

Evidence for the Operation of Different Mechanisms of Phosphate Acquisition by Mycorrhizal Roots Extraradical Hyphae and the Spatial Availability of P If extraradical hyphae contribute to uptake by mycorrhizal roots they must be involved in three processes: (a) absorption of phosphate (and of course some other nutrients) from soil; (b) translocation for considerable distances in the soil and into the fungal structures within the root; and (c) transfer from fungus to plant across the symbiotic interface(s). All three processes must occur at rates adequate to support the measured increases in P uptake. The apparent contribution of the fungal hyphae to the increased uptake by mycorrhizal roots can be calculated from inflows determined for mycorrhizal and non-mycorrhizal roots as shown in Table 5.1 (Sanders and Tinker, 1973). The value for maximum inflow from a zero sink was calculated to be 3.5 X 10~^^ mol m~^ s ~ \ and the conclusion was that the non-mycorrhizal roots were absorbing P at the maximum rate possible from the soil used. This value for inflow from a zero sink only applies to the particular soil used for the experiment. As shown above, the value depends on many soil characteristics, and to a small extent on the radius of the root. It is not likely that the uptake by the roots themselves would differ between mycorrhizal and non-mycorrhizal plants, because both would be diffusion limited and uptake characteristics (which might differ with P nutrition) would not have a large effect. Consequently, it is possible to calculate that the fungi contributed about 70% of the P absorbed by the mycorrhizal roots. Other workers have confirmed this important effect and shown that when root growth is restricted, the fungi can contribute up to 80% of the P absorbed (Li et al., 1991a). Sanders and Tinker (1973) calculated that about 50 m of hyphae per metre of root length would be required to supply this additional P, if the hyphae had developed depletion zones themselves and were absorbing from a zero sink in the same soil. They separated hyphae from the soil by sieving and calculated the length from the fresh weight and mean hyphal radius, assuming a specific gravity of 1. They found about 80 m of hyphae per metre of colonized root, so that allowing both for losses during extraction and for the fact that not all the hyphae were alive, the length appeared to be adequate to account for the measured increase in uptake. Data from this experiment can be used to calculate the translocational flux of P within entry point hyphae and the uptake of P by the hyphae on a surface area basis, which were found to be 3.8 X 10"^ mol m~^ s"^ and 7.2-13.0 X 10"^ mol m"^ s " \ respectively. The latter value is of the same order as uptake by free-living fungi or plants absorbing P from a well-stirred solution. The absolute magnitudes of the inflows measured in different experiments vary quite considerably, presumably as a function of soil, plant species and mycorrhizal fungus. Figure 5.2a shows the mean inflows of P to whole root systems of Allium cepa either uninoculated or colonized by four different mycorrhizal fungi (Sanders et aL, 1977). One of the fungi {Glomus 'microcarpus') colonized poorly and had no effect on inflow, compared with the controls. The others increased inflow above the control values and it was possible to relate the hyphal inflow to the colonized root length (Fig. 5.2b) and to show that this was linear above about 10% colonization.

133

Mineral nutrition, heavy metal accumulation and water relations

The effective fungi produced a fairly constant amount of external mycelium per metre of colonized root (360 jig m~^), so that it appeared possible to conclude that, once the fungus has become established, the rate of P uptake by hyphae was related to the length of the external mycelium (Sanders et ah, 1977; Graham et aL, 1982b). A correlation between calculated inflow via the fungus and percentage of the root length colonized has been observed in some investigations, but certainly does not always occur, possibly because of progressive death of the fungus within the root, reduction in the contribution of arbuscules to the colonized length (if these are indeed the sites of P transfer to the plant) a n d / o r death or destruction of the extraradical hyphae. The consequence would be reduction in the ability of mycorrhizal roots to absorb P (Fitter and Merryweather, 1992). Some agricultural and horticultural plants in the field appear to depend on increased inflows due to a mycorrhizal contribution and it has been suggested that any mycorrhizal effect may be underestimated because techniques frequently used to eliminate colonization also change the availability of P to the plants (Jakobsen, 1984; and see Stanley et al., 1993). In natural ecosystems the involvement of mycorrhizas in mineral nutrition is not always clear (McGonigle, 1988). A good correlation between P uptake and colonization has been found in Hyacinthoides non-scripta (Merryweather and Fitter, 1995a,b, 1996) and in Ranunculus adoneus (Mullen and Schmidt, 1993), but this (a:

10

vt

--

10

(b)

E o

6r

8

Q.

I ? 2h "29

»

'

Ii

I

30 40" Doys offer pkinfing

50

_L

10

JL

_L

20 30 40 50 60 Root length colonized (%)

70

Figure 5.2 The effects of mycorrhizal colonization on inflow of P into roots of Allium cepa. (a) Inflows of P into whole root systenns connputed for each harvest date. C, Nonmycorrhizal control; LAM, Glomus macrocarpus var. geosporus; BR, Scutellospora {Gigaspora) calospora; MIC, Glomus microcarpus; YV, Glomus mosseae. (b) Inflow attributable to the hyphae of: A , Glomus mosseae; A , Glomus macrocarpus var. geosporus; O, Gigaspora {Scutellospora) calospora; # , Glomus microcarpus. Values calculated by subtracting inflow into non-colonized roots from inflow into mycorrhizal roots. Note that the ineffective fungus Glomus microcarpuSy which colonized roots very slowly, did not increase the P inflow over that of controls. From Sanders et al. (1977), with permission.

134

Vesicular-arbuscular mycorrhizas

was not the case for the grass Vulpia ciliata (West et al., 1993a), and a number of grassland species (McGonigle and Fitter, 1988b; Sanders and Fitter, 1992). Although mycorrhizas have been implicated in many aspects of plant interactions in natural ecosystems (see Chapter 15), their importance in increasing P inflow has been questioned on the grounds that inflows are often very low (below the zero sink value for inflow by non-mycorrhizal plants given by Sanders and Tinker, 1973) and not related to the extent of colonization (Fitter and Merryweather, 1992). However, the zero sink value can only be applied to well-mixed soil and will vary considerably between soils (see above). Patchiness of both nutrients and inoculum in natural ecosystems will have a very important influence on uptake and on the extent to which mycorrhizas will affect it. There is a need to assess what 'benefits' besides nutrition might be conferred by mycorrhizal colonization and a pragmatic approach to determining the potential significance of mycorrhizal colonization to P nutrition would be to determine whether P supply limits growth, following elimination of colonization using fumigants or fungicides, such as benlate (Plenchette et A/., 1983a,b; West et al, 1993b; Koide et al, 1994; Merryweather and Fitter, 1996). The assumption that non-colonized regions of mycorrhizal roots and roots of non-mycorrhizal plants have the same absorbing capacity needs consideration because it is fimdamental to the calculations of hyphal inflow. It is known that Km and Vmax of uptake systems in roots are influenced by internal P concentration, so that absorbing power is reduced in roots of high P status, at least from wellstirred solutions (e.g. Lefebvre and Glass, 1982; Elliott et al, 1984; Jimgk et al, 1990). This means that the roots of mycorrhizal plants growing in low-P soil and absorbing P via the hyphae may, in fact, have lower uptake capacities than non-mycorrhizal plants of the same species, growing in the same soil. The extent to which these differences actually influence the uptake of P by the plants will depend on the extent to which diffusion (rather than uptake capacity) controls the rate of uptake and on the difference in P status of the mycorrhizal and non-mycorrhizal plants. If these difference are important then the hyphal uptake may be underestimated. Direct methods have been applied to study the way in which the hyphae colonize soil and use P from it (Hattingh et al, 1973; Hattingh, 1975; Rhodes and Gerdemann, 1975; Li et al, 1991a,b,c; Jakobsen et al, 1992a,b; Johansen et al, 1992, 1993a,b; and see Chapter 2). An important development in methodology has been the use of pots separated into root and hyphal compartments using mesh of different dimensions (Fig. 5.3a,b). The hyphal distribution and contribution to uptake by plants or depletion of nutrients in soil can be studied in the absence of the roots themselves. The depletion of bicarbonate- or water-extractable P, hyphal distribution (Fig. 5.4a,b) and the uptake of ^^P from hyphal compartments (Fig. 5.5a,b) have all been measured in different experiments and the results demonstrate the importance of hyphal uptake of P from soil and translocation through the hyphae to the mycorrhizal plants. Although Rhodes and Gerdemann (1975) demonstrated the ability of hyphae of mycorrhizal fungi to absorb "^^P from some distance away from the roots (7 cm), they provided no information on actual depletion of soil P. Owusu-Bennoah and Wild (1979) used ^^P-labelling and autoradiography to study depletion and concluded that mycorrhizal colonization of Allium cepa increased the diameter of the depleted zone from 1 mm in non-mycorrhizal roots to 2 mm, which was calculated

135

Mineral nutrition, heavy metal accumulation and water relations (a)

CQ^

(b) y

PVC tube

[

1 - '"''•^-5''^]

1

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yit

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/

- 37 /fm mesh 8 cm / • '

„^/ y

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\

JC^\^'

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/ \ / /Hyphae/ /^x^ r<> "Pdish

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J~ > v l lOO/tm ) | mesh ."^

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^

-

Bulk Bulk soil Roots soil 3 i^2i--2i--.2.^ 3 — Hyphae Hyphae

V ^ 165 mm

Figure 5.3 Compartnnented pots used for investigation of the effects of extraradical myceliunn on nutrient uptake by plants. The pots are divided into compartments using mesh, so that roots but not hyphae are excluded, (a) System devised by Jakobsen et al. (1992b), in which roots of the plant (in this case Trifolium subterraneum) are excluded from the hyphal compartment by 37 jim mesh. Reprinted with permission, (b) System devised by Li et al. (1991b), in which the plant compartment is separated from the hyphal compartment by 30 |im mesh. The outermost compartments (representing bulk soil) are further separated by 0.45 jim membranes which exclude both roots and hyphae. Reprinted with permission.

to be equivalent to a cylinder of root hairs 1 mm long - a surprisingly small effect. The data in Figure 5.4a demonstrate the effect of hyphal colonization on the depletion of P from soil with two levels of P applied in the hyphal compartment. The percentage colonization of the roots and the hyphal length density in the hyphal compartment differed little between the P treatments (76-79% and around 5.0 m cm~^ soil, respectively; see Fig. 5.4b) and, assuming all the hyphae were alive, values of P inflow to the hyphae of 3.3 and 4.3 X 10"^^ mol P m"^ s"^ for the two P treatments were calculated and are very close to the value of 2.25 X 10~^^ mol P m~^ s"^ that can be derived from the data of Sanders and Tinker (1973) making the same assumptions (Table 5.2). Hyphal uptake extended at least 12 cm into the hyphal compartment (the maximum possible in the experimental system) and had a significant effect on the mycorrhizal plants, which had both higher tissue P concentrations and higher dry weights than the non-colonized controls. Furthermore, the proportional contribution of the hyphae to total uptake was slightly higher with the higher P supply, emphasizing that localized patches of high P in soil (in this case the soil in the hyphal compartment) can be effectively exploited by the mycorrhizal mycelium. This is an important point, because

136

Vesicular-arbuscular mycorrhizas

2

4

6

8

Distance from the roots (cm)

4

6

8

10

Distance from the roots (cm)

Figure 5.4 Phosphorus depletion and hyphal length density in soil in compartmented pots planted with either mycorrhizal or non-mycorrhizal Thfolium repens. (a) Depletion profiles of NaHCOa-extractable (Olsen method) P in the hyphal compartments of mycorrhizal and non-mycorrhizal 7! repens, grown at two P levels. In both cases the mycorrhizal treatments ( I or # ) have depleted the soil to a greater extent than the non-mycorrhizal treatments ( D or O)- (b) Hyphal length density at different distances from the boundary of the root compartment, at the same two P levels used in (a). Note that there were small effects of P on hyphal development, so that the different depletion profiles cannot be attributed to different hyphal densities. From Li et al. (1991a), by permission of Kluwer Academic Publishers.

although the effects of mycorrhizal colonization on inflow are frequently greatest when soil P supply is low, significant effects may be apparent even when levels are adequate for near-maximal plant growth, particularly during later stages of plant development when depletion zones would be maximal and much of the P initially available in the pots had been absorbed (e.g. Smith, 1982; Smith et al, 1986a; Son and Smith, 1988; Dunne and Fitter, 1989). Use of the compartmented pot system to study the effects of an established mycelium on P uptake from temporally discontinuous P supplies might provide further insights into the roles of the external mycelium, particularly in habitats (such as wet tropical forests) where competition between plants and microorganisms for nutrients mineralized from large flushes of organic matter are important in 'tight cycling' (Janos, 1987; Lodge et al, 1994). The importance of hyphal translocation in soil was demonstrated directly as early as 1973 (Hattingh et al, 1973; Hattingh, 1975; Rhodes and Gerdemann, 1975, 1978a,b; and see Rhodes and Gerdemann, 1980). Using compartmented perspex chambers, ^^P, ^^S and ^^Ca were injected at different distances from mycorrhizal onion plants and the appearance of the tracer followed. ^^P was translocated by hyphae of Glomus mosseae and G. Jasciculatus' up to 7 cm through soil to roots of onion. When hyphae between the source of "^^P and the root were cut, no translocation to the root was observed. Extension of this approach using larger volumes of soil and measurements of hyphal length densities in the hyphal compartments (HC) has provided quantitative data for several different fungi (Jakobsen et al, 1992b; see Figs 5.3 and 5.5). Hyphal uptake and translocation of ^^P from a source localized in the HC at different distances (0, 1.0, 2.5, 4.0 and

Mineral nutrition, heavy metal accumulation and water relations

137

5 I

^

10

0-1

1-2

2-3

3-4

4-5

S-7

Distance from root compartment (cm)

Figure 5.5 a-d The time course of appearance of radioactivity in young leaflets of Trifolium subterraneum in association with: • , Acaulospora laevis; • , Glomus sp.; • , Scutellospora calospora; or x, non-mycorrhizal. Distances (cnn) between the ^^P-labelled soil and the root compartment were: (a) 0; (b) 1.0; (c) 2.5; and (d) 4.5. Bars are standard errors of means, (e) Length of hyphae in soil sections from the hyphal compartments of the experimental units with T. subterraneum in association with: • A laevis; M Glomus sp.; or H S. calospora. Values were corrected for background hyphae. From Jakobsen et al. (1992b), with permission. 7.0 cm) from the root plus hyphal compartment (RHC) into Trifolium subterraneum was followed for u p to 37 days. Hyphae of a Glomus sp. (WUM 10(1)) were relatively dense close to the roots and this fungus transferred most ^^P to the plants when the P source was similarly close. In contrast, Acaulospora laevis had a higher hyphal length density between 2 and 5 cm from the root than closei; to it and absorbed ^^P effectively from more distant placements. Scutellospora calospora

138

Vesicular-arbuscular mycorrhizas

T a b l e 5.2 Inflow of P t o hyphae f r o m soil Plant/fungus

Nutrient/system

Inflow (mol m~' s " ' )

Reference

Allium cepa/Qomus

P/soil

2.25 X 10"'^

Sanders and Tinker (1973); see also Tinker (1975)

Trifolium repens/ G. mosseae

P/soil compartmented

3.3-4.3X10"'^

Li et o/. (1991a)

(WUM 12(2)) had relatively low hyphal length densities and did not transfer much '^^P to the plants from any distance. However, hyphae of this fungus contained around four times more ^^P than the others and the conclusion was that although the S. calospora was able to absorb and translocate ^^P, transfer to the plants occurred at a low rate and consequently P accumulated in the hyphae. Only a relatively small total amount of P need be absorbed and retained by the hyphae for this effect to be apparent. The reasons for the low rate of transfer have not been determined, but might relate to low density of active arbuscules (Smith and Dickson, 1991) or to inherently low transfer fluxes across the symbiotic interface(s). The relative effectiveness of the same Glomus and S. calospora has been confirmed in dual labelling experiments in which ^^P was supplied to roots plus hyphae in an RHC and ^^P to hyphae alone in an HC (Pearson and Jakobsen, 1993b). However, hyphal uptake from HC of a third fungus. Glomus caledonium, was as great as uptake from RHC, leading to the suggestion that root uptake was completely inhibited by the presence of the fungus, via an unknown mechanism (Pearson and Jakobsen, 1993b; Jakobsen, 1995). Subsequent re-evaluation of the data indicates that G. caledonium was extremely effective in exploiting the soil and that combined uptake by roots plus hyphae was likely to have depleted the P in the RHC completely. Consequently the apparently low value for root uptake was probably a result of this, rather than more subtle interactions between the symbionts (Jakobsen, personal communication). The rates of translocation of ^^P (as well as ^^Zn and ^^S) in hyphae of G. mosseae to Trifolium repens and Allium cepa plants have been measured in split agar plates by Pearson and Tinker (1975) and Cooper and Tinker (1978, 1981). In these investigations the fluxes were one or two orders of magnitude lower than the theoretical flux calculated for entry point hyphae mentioned above (1.02 X 10~^ to 2.0 X 10"^ mol m~^ s~^; Table 5.3). These measurements involved counting and measuring the hyphae traversing a diffusion barrier in a split agar plate. The discrepancy between the measured fluxes and those calculated from P uptake has been attributed to three factors (Cooper and Tinker, 1978): (a) the strong likelihood of higher fluxes in entry-point hyphae than in those of the general extraradical mycelium; (b) lower growth rates and P uptake of plants growing in agar culture, compared with plants growing in soil and hence lower demand and transfer across the interface; and (c) some of the hyphae used to calculate crosssectional area for flux determinations may have been dead. The second point may be of particular importance, since it has been shown that a mass flow component of the translocation process is increased by rapid transpiration rates in associated host

139

Mineral nutrition, heavy metal accumulation and water relations

T a b l e 5.3 Translocation of nutrients in external hyphae, calculated per unit cross sectional area of hyphae Plant/fungus

System

Translocation flux (molm"^s"')

Reference

Allium cepa/Glomus sp.

Soil/P entry points

3.8 X 10""*

Sanders and Tinker (1973)

Agar/^^P

3-10 X 10"^

Pearson and Tinker (1975)

Agar/^^P

2.0-20 X 10~^

Cooper and Tinker (1978, 1981)

Soil/'^N

7.42 X 10"^

Ames eta/. (1983)

Agar/"Zn

2.1 X I0~®

Cooper and Tinker (1978)

Agar/^^S

16.5 X 10"

Cooper and Tinker (1978)

Thfolium repens/ G. mosseae Thfolium repens/ G. mosseae Apium graveolens/ Glomus sp. Thfolium repens/G. mosseae Thfolium repens/G. mosseae

plants (Cooper and Tinker, 1981). Plants grown in agar may well have lower transpiration rates than plants in pots. Suggested mechanisms of translocation in fungi involve processes based on both mass flow and cytoplasmic streaming (see Chapter 14). In VA mycorrhizal fungi any mechanism must take into account the potential for bidirectional movement of different nutrients (e.g. P and organic C) and the fact that translocation is temperature sensitive and inhibited by cytochalasin b, which also inhibits cytoplasmic streaming. It seems likely that the system of pleiomorphic, motile, vacuolar tubules recently demonstrated in members of the Eumycota, including members of the Glomales (Basidiomycotina, Ascomycotina and Zygomycotina; see Shepherd et al, 1993a,b; Rees et al, 1994; S. Dickson and A.E. Ashford, unpublished data), is involved in fungal translocation and investigations of the factors influencing their behaviour and translocating potential can be expected to shed light on translocation in mycorrhizal systems. Considerable transfer of P from fungus to plant occurs in most of the plantfungus combinations studied. At this point it is essential to emphasize that transfer of P from plant to fungus must involve membrane transport steps at an interface which includes the membranes of both symbionts and an apoplastic region between them (see Chapters 2 and 14). Furthermore, it must be reiterated that arbuscules do not contain sufficient P for their 'digestion' to be a credible mechanism for P transfer (Cox and Tinker, 1976). It is generally assumed that the site of transfer is the arbuscular interface, and although the distribution of membranebound H"^-ATPases does appear to support this (Smith and Smith, 1990; GianinazziPearson et al., 1991a; and see below) there is no definitive evidence that would exclude intercellular hyphae or hyphal coils from involvement in this process. There is no doubt that the interface between the symbionts in cells colonized by arbuscules would provide, as Cox and Tinker (1976) emphasized, a relatively large surface area across which P and other nutrients could also be transferred. They

Vesicular-arbuscular mycorrhizas

140

Table 5.4 Net transfer of P to the plant across the symbiotic interface, assuming the arbuscular interface alone is responsible for transfer Plant/fungus

Nutrient/system

Transfer flux (mol m~^s~')

Reference

Allium cepa/Glomus mosseae

P/soil P/soil P/soil

13 X 10"' 4-29 X 10"' 2.0-3.2 X 10"'

Cox and Tinker (1976) Sukarno et al. (1996) Smith eto/. (1994)

P/soil

5.0-12.8 X 10"'

Smith eto/. (1994a)

A. cepa/Glomus s p . ( W U M 16) A. porrum/G. mosseoe A. porruml Glomus sp.

(WUM 16)

Hyphal contribution to inflow calculated from total P uptake in mycorrhizal and non-mycorrhizal plants. Area of interface deternnined from numbers of arbuscules and invagination of the plant plasma membrane (see Cox and Tinker, 1976).

measured the area of interface in such cells and calculated the flux required to support transfer of P from fungus to plant. P flux via arbuscules of Glomus mosseae to Allium cepa was 13 X 10"^ mol m~^ s~^. Similar calculations have now been made for Glomus sp. (WUM 16) and G. mosseae colonizing Allium porrum (leek) and found to be of the same order (Smith et al, 1994b; and see Table 5.4). The magnitude of these transfer fluxes is of the same order as uptake of P by free living plant and fungal cells, and very much larger than measured rates of efflux (Beever and Bums, 1980; Elliott et al, 1984). As net transfer across a symbiotic interface involves both efflux and uptake operating in series, the two processes must occur at equal rates. The inevitable conclusion is that P transfer from fungus to plant involves special modifications to increase the efflux from the fungus and probably also to suppress reabsorption (uptake) by the fungus, as this would negate the efflux (Smith et al., 1994b,c; and see Chapter 14). The mechanisms underlying the increased efflux are unknown at present. In conclusion, the involvement of hyphae in effectively and economically extending the root system of colonized plants seems very clear and provides a rationale for the negative relationship between development of root hairs (which play a similar role) and the responsiveness of species to mycorrhizal colonization. The idea that mycorrhizas might replace root hairs was first mooted in the nineteenth century and more recently Baylis has discussed the evolution of root systems and their associated mycorrhizas (Baylis, 1972a, 1975). His general thesis has been that plants with thick unbranched roots and few root hairs (e.g. Allium, Coprosma, Citrus) are apparently more responsive to mycorrhizal colonization when growing in low P soils than plants with finely branched roots and long or numerous root hairs, although all may be susceptible to colonization (St John, 1980; see Chapter 1). Competition Between Hyphae of VA Mycorrhizal Fungi and Soil Microorganisms The possibility that extraradical hyphae of mycorrhizal fungi might effectively increase the 'competitive ability' of the mycorrhizal root system, vis a vis soil microorganisms, in acquiring P from the soil solution and thus circumvent the problems of immobilization of P in the soil biomass has been canvassed from time to time (see Linderman, 1992). The dependence of mycorrhizal fungi on recent photosynthate from the plant means that their activity would not be affected by the

Mineral nutrition, heavy metal accumulation and water relations

141

C: P ratio of organic matter in soil, nor by the availability of the C substrates in soil, giving them considerable advantages over saprophytic microorganisms. Barea et al., (1975) were among the first to investigate interactions between P solubilizing bacteria and mycorrhizal inoculation in the mobilization of P from rock phosphate (RP). They observed positive, synergistic effects in growth and P uptake by Zea mays and Lavendula spica, that were significant in some soil-plant combinations. Similarly, the potential of a P-solubilizing fungus Penicillium balaji to increase the availability of RP to Triticum aestivum and Phaseolus vulgaris depended on mycorrhizal activity (Kucey, 1987; Kucey et ah, 1989). Whereas RP alone had no effect on plant growth, the separate positive effects of inoculation with a mycorrhizal fungus and P. halaji were additive. Furthermore, in sterile soil mycorrhizal inoculation was an absolute requirement for the RP-P. halaji system to provide additional P to the plants. Jayachandran et al. (1989) used three soils and showed that if strong iron-chelating agents were added, the P released was available to mycorrhizal but not to non-mycorrhizal plants. They found no evidence for production of chelating agents by the mycorrhizal fungi themselves and concluded that the outcome was the result of effective exploitation of the soil and competition with resident microflora. P mineralized from organic P sources appears to be more readily available to mycorrhizal than to non-mycorrhizal plants, but there is no good evidence that mycorrhizal fungi are actually involved in the mineralization process (Jayachandran et ah, 1992; Joner and Jakobsen, 1994). Joner and Jakobsen (1994) concluded that Glomus sp. (WUM 10) and Glomus caledonium were both capable of intercepting Pi released during mineralization of Po by microorganisms and preventing immobilization in the biomass or sorption on clay minerals. The external hyphae have frequently been seen to proliferate preferentially in organic matter in soil (St John et ah, 1983a,b; Warner, 1984), which would be an appropriate situation for the operation of this competitive effect. Furthermore, the location of living hyphae of VA mycorrhizal fungi within dying roots appears to be important in the redistribution of ^^P to neighbouring plants which are linked into the external mycelium (Ritz and Newman, 1985; Eason and Newman, 1990; Eason et al., 1991; and see Newman, 1988). More work is needed to understand the interactions between the activity of the soil microflora in both mineralizing and immobilizing Pi and the potential capacity of mycorrhizal fungi to short-circuit this aspect of nutrient cycling. The interactions between mycorrhizal colonization and populations of particular functional groups of organisms both in the rhizosphere and further from the root where hyphae may proliferate are complex and as yet their significance is rather poorly understood (Azcon-Aguilar and Barea, 1992; Linderman, 1992; Fitter and Garbaye, 1994). The findings might be significant in studies of the fate of nutrient flushes and of fertilizer P, especially in situations where leaching contributes to losses from the soil and possible accumulation in water supplies. In this context, seasonal development of mycorrhizas in the fine lateral and cluster roots of some dryland species in the Restionaceae and Cyperaceae has been observed to coincide with the first period of winter rains, when nutrient mobilization might be maximal (Meney et ah, 1993). The potential for mycorrhizal interception of nutrient flow through soil deserves investigation.

142

Vesicular-arbuscular mycorrhizas

Differences in Kinetics of Uptake Between Hyphae and Roots Mosse et al. (1973) suggested that mycorrhizal colonization might alter the threshold concentration from which plants were able to absorb P and consequently increase the effectiveness of absorption from the soil solution. This possibility was investigated using tomato {Lycopersicon esculentum) and cassava (Manihot sp.; Cress et al, 1979; Howeler et al, 1979). P uptake from solution by tomato roots was studied at concentrations between 1 and 100 |LIM KH2PO4. The lower part of this range corresponds realistically with the concentration in soil solutions, an important point if we are to use the results to help interpret mycorrhizal effects on plant growth in soil. Both non-sterile and axenically grown non-mycorrhizal roots were used for comparison with mycorrhizal roots and the initial internal P concentra-x tions of the roots used were fairly similar. This was achieved by growing mycorrhizal plants on Ca3(P04)2 and non-mycorrhizal plants on NaH2P04. Over the solution concentration range 1-20 |XM, the Vmax of the uptake system (based on uptake per unit fresh weight of roots) was not very different in mycorrhizal and non-mycorrhizal plants, but the lower X^ (1.6 as opposed to 4.0 |IM) indicated that the affinity of the uptake sites for P was higher in the mycorrhizal roots. Thomson et al. (1990a) determined the kinetics of uptake in germ tubes of Gigaspora margarita grown with different P supplies and concluded that, like roots and other fungi, there were two transport systems which could operate simultaneously. The high affinity System I had a Xm of 1.8-3.1 |LIM and a V^ax of 3.13.6 nmol mg protein"^ h~^. AH that can be concluded from the comparison of the two sets of data is that the affinity of System I in hyphae is similar to that of the colonized roots and it does not seem likely that the hyphae would influence the threshold concentration for absorption by mycorrhizal roots. It is possible to argue that if P uptake is limited by the rate of diffusion of ions through a depleted zone around a root or hypha, then uptake characteristics of the root, mycorrhiza or hypha are irrelevant to considerations of P uptake from the soil, except when they grow into microsites with localized high solution concentrations and before any depletion zones develop. The work with cassava (Yost and Fox, 1979) illustrates two points. This species appears to have a very high P requirement, coupled with a very inefficient P uptake system in the absence of mycorrhizal colonization. Despite this, cassava is well known for its growth on soils of low fertility and its efficiency of uptake is markedly increased when roots are colonized by mycorrhizal fungi. Thus the importance of mycorrhizal colonization to a particular species or variety of host plant (its responsiveness or dependency; see Chapter 4) may depend on the concentration of P in the soil, the relative affinities of the root and fungal systems for P, the characteristics of the root system in extending into the soil and also upon the P requirement of the host (Koide, 1991a).

Use of Sources of P Unavailable to Roots The suggestion has frequently been made that mycorrhizal roots might be able to exploit sources of P in soil not normally available to plants. These sources include relatively insoluble forms of Pi, such as rock phosphate (RP) and Fe and Al

143

Mineral nutrition, heavy metal accumulation and water relations

phosphates, as well as sources of Po such as phytate. It has certainly been shown that growth of mycorrhizal plants does respond to the application of RP or tricalcium phosphate, whereas these fertilizers had little or no effect on the growth of non-mycorrhizal plants at the rates of application used (e.g. Murdoch et ah, 1967; Wibawa et ah, 1995). Similar results have been obtained following application of insoluble phosphate fertilizers for a variety of host plants, usually in soils of low pH. In most investigations, comparisons were made at one or two rates of fertilizer application and Pairunan et al. (1980) have suggested that the conclusion that mycorrhizal plants had increased access to the P supply was invalid. They showed that it is essential to compare growth over complete P response curves for both soluble and insoluble fertilizers. The curves for superphosphate and RP are of similar form (Fig. 5.6), but there are important quantitative differences between them. First, the amount of P as RP which has to be added to soil to achieve maximum growth of Trifolium subterraneum was about 40 times greater than the amount of P from superphosphate that was required both for mycorrhizal and nonmycorrhizal plants. Second, the maximum growth with RP was less than that with superphosphate, irrespective of mycorrhizal colonization. These differences apart, and assuming that the RP contained no traces of soluble P, nor indeed toxic substances, the results do suggest that there is no absolute difference in the availability of RP to mycorrhizal and non-mycorrhizal plants. Nevertheless, at moderate and realistic levels of application, and at any level of P equivalent to the superphosphate range (0-0.8 g P kg soiP^; see Fig. 5.6), mycorrhizal plants were more effective at extracting P from the fertilizer. The mechanisms underlying the increased uptake might depend upon hyphal exploitation of the soil volume. In addition, both synergistic action between mycorrhizas and P-solubilizing microorganisms (see above), and the possible excretion of H"^ or hydroxyacids by hyphae, would increase the availability of RP to mycorrhizal plants (Johnston, 1956; Johnston and Miller, 1959; Smith, 1980). As indicated above, there is no experimental support for production of chelating agents by

1-2 2-4 0 43-2 Phosphorus applied (g per pot)

86-4

Figure 5.6 The effect of phosphorus fertilization and inoculation with Glomus mosseae on dry weight of shoots of Trifolium subterraneum after 7 weeks' growth, (a) Superphosphate fertilizer; (b) C-grade rock phosphate: O, uninoculated control plants; A , plants inoculated with G. mosseae. From Pairunan et al. (1980), with permission.

144

Vesicular-arbuscular mycorrhizas

hyphae of VA mycorrhizal fungi. However, reductions in, soil pH (up to 1.0 unit) in hyphal compartments has been shown concurrently with P depletion in a cambisol, when P was supplied as Ca(H2P04)2 and N as (NH4)2S04 (Li et al, 1991b; Fig. 5.7a,b). The mechanism by which the pH was reduced is likely to have been via the extrusion of H^, following use of ammonium by the hyphae (Raven and Smith, 1976; Smith, 1980; Smith and Smith, 1984), but other mechanisms such as increased CO2 production may also have contributed (Li et ah, 1991c). In addition to studies specifically on the use of insoluble phosphate fertilizers, there has been considerable interest in the ability of mycorrhizal fungi to access P in the non-labile fraction in soil. This question has been addressed by assuming that only the labile P would exchange with added ^^P and that it was the pool from which non-mycorrhizal plants absorbed P. Although mycorrhizal onions, rye grass and soybeans took up more total P from soil with the labile fraction labelled than did non-mycorrhizal plants, there was no difference in specific activity of P in the two groups of plants. These results suggest that mycorrhizal plants had no access to non-labile P sources, which would have diluted the ^^P and lowered the specific activity (Sanders and Tinker, 1971; Hayman and Mosse, 1972; Mosse et al, 1973; Powell, 1975a; Pichot and Binh, 1976; Gianinazzi-Pearson et al, 1981a). In contrast, mycorrhizal potatoes apparently did take up fixed P from the latosols on which they were grown, the evidence being that the specific activity of "^^P in mycorrhizal (a)

Net (30 /im)

Membrane (0-45 ;/m)

(b)

0

5 10 15 20 25 30 35 40 45 Distance from root plane (mm)

50

Figure 5.7 Depletion profiles of: (a) H20-extractable P; and (b) soil pH in the hyphal and bulk soil compartments (refer to Fig. 5.3b) from non-mycorrhizal ( Q or Q ) and mycorrhizal ( # or • ) Trifolium repenSy grown in a cambisol. Bars represent standard errors of means. From Li et al. (1991b), with permission.

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plants was higher than in uncolonized plants when they were absorbing ^^P from the fixed P fraction (Swaminathan, 1979). Furthermore, heating soil to provide a range of concentrations of fixed P gave no evidence of significant difference between mycorrhizal and non-mycorrhizal plants in accessing P in the different fractions (Barrow et ah, 1977). This confused picture probably results from the assumptions made about the labelling of the different P fractions in soil (Bolan et al, 1984b; Bolan, 1991) and a careful re-examination of the problem has shown the need for more work in this area. Bolan et al. (1984b) added iron hydroxide to soil, with the expectation that P 'fixation' as iron phosphates would take place. The P was labelled by the addition of ^^P. Subsequent extraction with 10 mM CaCl2, 0.5 M NaHCOa or acid NH4F was used to determine the amount and specific activities of P in different fractions. Addition of iron hydroxide reduced the amount of P available to non-mycorrhizal plants, but made no difference to mycorrhizal plants of Trifolium subterraneum. However, despite the fact that addition of iron hydroxide reduced the amount of P that could be extracted by CaCl2 or NaHCOs (but not by NH4F) there were no differences in the specific activities of any of the extracts nor between mycorrhizal and non-mycorrhizal plants. The conclusion must be that labelling techniques of this sort are of doubtful usefulness and results cannot eliminate the possibility that mycorrhizal plants obtain P that is unavailable to non-mycorrhizal plants. There has not been much work on the ability of enzymes from roots or VA mycorrhizal fungi to hydrolyse Po. Mosse and Phillips (1971) found that phytates were satisfactory sources of P for plant and fungal growth in agar cultures, and that calcium phytate stimulated fungal growth. It has also been shown that both mycorrhizal and non-mycorrhizal soybeans could hydrolyse calcium phytate in soil, but that there was no differential growth response (Gianinazzi-Pearson ef al, 1981a). Such an effect might be mediated by increased acid phosphatase activity in the rhizosphere, as shown by Dodd et al (1987), although its significance is still doubtful and has not been observed in all investigations (Allen et al., 1981a; Krishna and Bagyaraj, 1982). More recently, '^^P labelling techniques have been applied in pots compartmented by mesh (Joner, 1994; Joner and Jakobsen, 1994) and again there was no evidence for direct effects of mycorrhizal hyphae on solubilization of Po; rather, the hyphae increased the competitive ability of the plants for P mobilized by other soil organisms. Phosphate Metabolism in the Fungus External hyphae of VA mycorrhizal fungi must absorb Pi by active transport after the roots have become colonized. They have an active H^-ATPase on the plasma membrane which would be capable of generating the required proton-motive force (Lei et al., 1991) to drive H'^-phosphate co-transport, and P is certainly accumulated to high concentrations. Rapid absorption of P by mycorrhizal fungi is followed by the synthesis of inorganic polyphosphate in the fungal vacuoles. The granules observed following staining with toluidine blue and in the electron microscope (Cox and Tinker, 1976; Callow et al, 1978) are probably artefacts of the preparation methods, but polyphosphate is certainly important in mycorrhizal P metabolism (see Chapter 14). Recent work using freeze-substitution techniques has shown that in the ectomy-

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Vesicular-arbuscular mycorrhizas

corrhizal fungus Pisolithus tinctorius, and probably also in many other fungi, the polyphosphate is present as soluble, short chain 15-mers, stabilized by K"^ ions (Orlovich and Ashford, 1993; Ashford et ah, 1994). Synthesis of polyphosphate prevents excessive accumulation of orthophosphate in fungal cells, when external supply is plentiful and uptake is rapid. Such a storage role is common in microorganisms and may be important in reducing osmotic stress; it is found in ectomycorrhizas, in rusts and other obligate fungal parasites, in lichens, in many unicellular algae and in some free-living fungi and in bacteria (Harold, 1966; Beever and Bums, 1980). In VA mycorrhizas formation of polyphosphates might account for the increased P concentrations in the root (Sanders, 1975; Smith and Daft, 1977; Smith, 1982), which are associated with mycorrhizal colonization. Polyphosphate accumulation might also constitute an energy store but this is not likely to be large (Beever and Bums, 1980). Polyphosphate is probably also important in translocation of P in the hyphae, although not in granule form, as originally envisaged by Cox and Tinker (1976). The system of pleiomorphic, vacuolar tubules observed in many fungi contains polyphosphate and pulsation of the tubules results in transfer of their contents over short distances. Transfer by this mechanism over the long distances which would be required in external mycelium of VA mycorrhizas is still an open question (Ashford and Orlovich, 1994). Calculations of P content based on numbers of (artefactual) granules and amount of P per granule are probably still valid. Combined with rates of cytoplasmic streaming they indicate that movement of polyphosphate within the hyphae might account for measured fluxes into the root (Cox and Tinker, 1976; Cox et ah, 1980). Enzymes of polyphosphate metabolism have been detected in VA mycorrhizas by Capaccio and Callow (1982). The distribution is consistent with synthesis of polyphosphate in external hyphae and breakdown in hyphae within the root. Furthermore, the absence of polyphosphatases in external hyphae is interesting, as it suggests that polyphosphate turnover and unloading from tubules might be low. This might have consequences for long-distance translocation, but much more work on control of polyphosphate metabolism is required before this could be regarded as certain or even likely (see Chapter 14). Other information on P metabolism by the fungi still does not give a very coherent picture. 'Mycorrhiza-specific' alkaline phosphatases have been detected cytochemically in the vacuoles of the mature arbuscules and intercellular hyphae (Gianinazzi et ah, 1979). There is some circumstantial evidence that development of activity is linked to the presence of arbuscules and transfer of P to the plant (Gianinazzi-Pearson and Gianinazzi, 1978), leading to the suggestion that this enzyme might be a useful marker for efficient P metabolism in the fungi. Furthermore, it has been claimed that activity develops only in structures within the host tissues. Activity in germ tubes was restricted to the tip region and there was apparently a lag in development of activity within the root, which did not occur with succinate dehydrogenase activity (Tisserant et al., 1993). However, these observations are at odds with those of Dodd (1994) who suggested that alkaline phosphatase activity might be an effective vital marker for the external mycelium. Furthermore, Larsen et al. (1996) have shown that while the fungicide benlate inhibits P uptake and transfer to plants via mycorrhizal hyphae, it does not affect alkaline phosphatase activity, so that alkaline phosphatase activity was not a

Mineral nutrition, heavy metal accumulation and water relations

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suitable physiological marker in this instance. The actual role of the alkaline phosphatase in P metabolism of mycorrhizas has yet to be determined. Membrane-bound H'^-ATPases in the plant-fungus interfaces are likely to be important in transfer processes between the symbionts, because these enzymes are essential for generation of proton-motive force, which drives H^ co-transport of solutes across membranes. TTie electrochemical potential gradient for orthophosphate between the soil solution or the mycorrhizal interface and either hyphae or root cells is such that uptake is always active and the distribution of H'^-ATPase on the plant plasma membrane surrounding the arbuscules indicates that this membrane is likely to be energized and capable of the necessary transport of P (or other actively transported ions) to the plant. There is further discussion of the roles of H^ATPases in different interfaces in Chapter 14.

Nitrogen Nutrition Mycorrhizal Effects on Modulation and M Fixation Increased N concentrations have been reported in VA mycorrhizal plants. Of course, where they are also symbiotic with N-fixing bacteria or actinomycetes this can be attributed to increased rates of N fixation induced secondarily, e.g. by increased P uptake, rather than to direct uptake of N compounds from the soil. The assimilation of N fixation of dinitrogen in rhizobial root nodules is certainly increased when plants, growing in low-phosphate soils are also colonized by mycorrhizal fungi. This effect was probably first observed by Asai (1944) who made detailed observations of growth, nodulation and mycorrhizal status of a large number of legumes. More recently, nodulation and N fixation by mycorrhizal and non-mycorrhizal legumes, as well as plants nodulated by Frankia, have been the subject of many experiments (e.g. Rose, 1980; Rose and Youngberg, 1981; Gardner, 1986). In most cases, improved nodulation and N fixation in mycorrhizal plants appears to be the result of relief from P stress and possibly uptake of some essential micronutrients, which result in both a general improvement in growth and indirect effects upon the N-fixing system. The differences between mycorrhizal and non-mycorrhizal plants usually disappear if the latter are supplied with a readily available P source. More detailed information can be obtained from the many reviews of this area (e.g. Bowen and Smith, 1981; Barea and Azcon-Aguilar, 1983; Bethlenfalvay and Newton, 1991; Azcon-Aguilar and Barea, 1992; Barea et ah, 1992; Bethlenfalvay, 1992b). The fact that the effects are not directly attributable to the mycorrhizal fungi themselves should not be allowed to detract from the interest of these tripartite symbioses, which may be very important both in natural ecosystems and in revegetation programmes, where nutrients are in short supply in soil (Lamont, 1984; Pate, 1994). Uptake of Mineral Nitrogen (NH4 and NO3") The N concentration in plants is about ten times greater than the P concentration, emphasizing a much greater requirement for this nutrient. The availability of

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Vesicular-arbuscular mycorrhizas

mineral N in soils depends to a very great extent on the activities of microorganisms in mineralizing N from organic sources, effecting conversions such as nitrification and denitrification, and immobilizing N in the soil biomass. Major inputs derive from organic matter and from biological N fixation. In contrast to the situation with other mycorrhizal types, particularly ericaceous mycorrhizas (see Chapter 12), there is no unequivocal evidence that the extraradical hyphae of VA mycorrhizas are directly involved in mineralization of organic forms of N. However, there is increasing evidence that mycorrhizal fungi are involved in uptake and transfer of inorganic N, although it is not clear whether this always occurs in amounts that are significant for plant nutrition. The two most important sources of inorganic N for plants, and potentially for VA mycorrhizal fungi, are nitrate (NO^) and NH4 (NH4) ions. In agricultural soils NO^ usually predominates, because of the rapid nitrification of NH4. In contrast, in many undisturbed soils, particularly those which are on the acid side of neutrality, ammonium predominates and nitrate may be almost entirely absent (Rice and Pancholy, 1974; Stewart, 1991; Stewart et al, 1993). N O J is not adsorbed on soil colloids and is mobile, at least in moist soil, so that mass flow of the soil solution to roots absorbing both water and nutrients allows uptake to be maintained at rates dependent on the root absorbing power. Consequently, absorbing power and the surface area available for absorption (dependent on root length and radius) are likely to be the factors limiting uptake. A mycorrhizal effect would not be expected for this ion, except in dry soil when the mobility was reduced. In contrast, NH4 is adsorbed and is relatively non-mobile, so that even in moist soils depletion zones develop readily and, as with P, diffusion rather than root absorbing power, limits the rate of uptake and so mycorrhizal fungi might be important in increasing the rate of uptake. Preliminary results on N uptake and transfer were somewhat inconclusive. As early as 1976, Haines and Best reported that loss of NH4, NO3 and nitrite (NO2) from soil by leaching with water was retarded when plants of Liquidambar styraciflua were mycorrhizal with Glomus mosseae. Unfortunately, the root systems of the mycorrhizal plants were considerably larger than the uncolonized systems, so that the results did not indicate unequivocally that the mycorrhizal fungi themselves were involved. Subsequently, Ames et al. (1983) showed that although ^^N supplied to extraradical hyphae in organic form reached mycorrhizal Apium graveolens (celery), transfer required a considerable period of time. They assumed that mineralization by soil microflora was an essential step in making the organic N available and that this caused the delay. Increased inflow of N to roots of Trifolium subterraneum supplied with inorganic N was also observed (Smith et al., 1986d), but the results of different experiments were not consistent (Smith et ah, 1986c) and uptake of N by the hyphae did not seem to play an important role in supplying N to the plants because increased uptake per plant was not observed. The picture is now clearer and again the use of mesh-compartmented systems has played an important part. Ames et al. (1983), supplied (^^NH4)2S04 to the HC of such a system and observed considerable ^^N transfer to the mycorrhizal plants. The amount was correlated with the percentage colonization of the roots of the celery plants {Apium graveolens) by Glomus mosseae, with the hyphal length density in the HC and with the number of hyphal crossings of the mesh. Using the number of crossings, and again assuming that all these hyphae were alive.

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Mineral nutrition, heavy metal accumulation and water relations

Ames et ah (1983) were able to calculate a translocation flux for N of 7.42 X 10~^ mol N m~^ s~^. This is roughly the same as the flux of P through entry-point hyphae calculated by Sanders and Tinker (1973) and, considering the high N requirement of plants, the value seems low (see Table 5.3). A similar approach has now been used with different plant-fungus combinations and the data have confirmed hyphal ^^N transfer to mycorrhizal Cucumis sativus, Trifolium subterraneum and Zea mays when ^^NH4 was supplied in the HC colonized by hyphae of Glomus intraradices (Frey and Schuepp, 1992; Johansen et ah, 1992, 1993a). In two of these investigations the ^^N in the HC was significantly depleted (Fig. 5.8) in the hyphal compartment (Frey and Schuepp, 1992; Johansen et ah, 1992). However, none of these demonstrations of N translocation and transfer have been associated with increased plant N content or growth. Johansen et ah (1992) suggested that the small physical size of the experimental systems might have contributed to this. They proposed that where soil N is present as NH4, or strong competition exists for recently mineralized N, a mycorrhizal mycelium might play a significant part in N acquisition. Differences between species of fungi in accessing ^^N were found to be related to differences in distribution of hyphae in the HC (Frey and Schuepp, 1992). Additional evidence for the competitive effects of mycorrhizas in accessing less available forms of N has been obtained using N dilution techniques (Azcon-Aguilar et ah, 1993; Tobar et ah, 1994b). There has been only one investigation of mycorrhizal effects on ^^N-labelled NO^ (Tobar et ah, 1994a). Again using mesh-compartmented pots, no difference in ^^N enrichment between mycorrhizal {Glomus fasciculatum) and non-mycorrhizal plants of Lactuca sativa supplied with ^^N-labelled NO3 under well-watered conditions was observed. However, in dry soil the enrichment of the mycorrhizal plants was four times higher, probably reflecting the much lower mobility of NO3 in dry soil.

13

5

13

5

1 3 B

13

5

Distance from root compartment (cm) F i g u r e 5.8 Effect of mycorrhizal hyphae of Glomus intraradices on depletion of K C I extractable N H 4 and N O ^ in the hyphal c o m p a r t m e n t of plants of Cucumis sativus fertilized ( H C A ) , o r n o t ( H C B ) , w i t h added N O ^ . G l , mycorrhizal plants; N M , n o n mycorrhizal plants. Bars are standard e r r o r s of the means of f o u r replicates. From Johansen et al. (1992), w i t h permission.

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Vesicular-arbuscular mycorrhizas

Mycorrhizal effects on N nutrition have been studied under field conditions and the potential for increased uptake of N from soil, as well as the P-mediated effects on N fixation demonstrated in Hedysarum coronarium (Barea et al, 1987; Table 5.4). In mixed plantings a twofold increase in ^^N transfer from soybean to maize has been observed in mycorrhizal plots, together with a relative increase in productivity of maize (Hamel and Smith, 1991). This suggests that mycorrhizal fungi may be involved in redistribution of N in a plant community. The type of mycorrhiza formed by plants also appeared to have an influence on their capacity to use NO^ in a Banksia woodland. At recently burned sites N O ^ predominated over NH4 and was stored and used most effectively by non-mycorrhizal and VA mycorrhizal, herbaceous plants. Woody species, including some that could be hosts to both VA and ectomycorrhizal fimgi, appeared to use a wider range of N sources. There is also an indication that 6^^N values may differ in mycorrhizal and non-mycorrhizal plants, due to imknown fractionation processes (Handley et al, 1993), and this might account for the variability in 5^^N values in plants of different mycorrhizal status and life form in the Banksia woodland. It might also have contributed to the problems encoimtered by Hamel et al. (1991) using natural abundance methods to elucidate the mycorrhizal interactions between soybean and maize (see Chapter 15). Both absorption and assimilation of inorganic N by the fungi are prerequisites to translocation and transfer to the plants. As NH4 is frequently present at very low concentrations in soil it is thought that assimilation depends on the activity of glutamine synthetase (GS) and glutamate synthase (GOGAT), rather than glutamate dehydrogenase (GDH), because of the higher affinity of GS for NH4 (Miflin and Lea, 1976). GS activity is increased in mycorrhizal root systems, partly due to a contribution from the fimgi themselves because activity has been detected in fungal tissue separated from VA mycorrhizal roots. Improved P nutrition in the plants resulted in only a small increase in activity, confirming the important contribution of the fungi. In contrast, GDH activity showed no direct relationship with colonization (Smith et al, 1985). This limited evidence suggests that the fungi may have the capacit)^ to assimilate NH4 and in consequence N is likely to be transferred from fungus to plant in organic form. Nitrate reductase activity has been detected, albeit at very low levels, in isolated spores of Glomus mosseae and G. 'macrocarpus' (Ho and Trappe, 1975). Since most fungi that reduce N O ^ have been shown to have an NADP-dependent enzyme, Oliver et al. (1983) assayed NAD- and NADP-dependent activity in mycorrhizal roots of Trifolium subterraneum (80% colonized). They observed that although there was no NADP-dependent activity, NAD-dependent nitrate reductase activity was present. This enzyme, more characteristic of higher plants than of fungi, increased both in colonized plants and in plants receiving additional P. The effect was apparent in shoots as well as roots and the most likely explanation for the mycorrhizal effect is that increases in enzyme activity were the result of improved P nutrition. The results confirm those of Carling et al. (1978) who, however, only assayed nitrate reductase in non-mycorrhizal tissues of colonized and nonmycorrhizal soybeans, i.e. their nodules and shoots. The capacity of the fimgus in mycorrhizal roots to assimilate NO^ must remain in doubt and it seems likely that the main site of nitrate reductase is in the plant. An increase in N uptake in mycorrhizal roots (whether directly mediated by the

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fungus or not) may be important, in order to compensate for the relative loss of absorbing area in mycorrhizal plants with reduced root:shoot ratios. The involvement of VA mycorrhizas in N transformations obviously needs more attention and the interactions between enzyme activity (in both plant and fungus) and P nutrition will need to be addres^sed in further experiments on the apparent i^N/i^N fractionation. Transfer of N between plants via mycorrhizal hyphae has attracted considerable attention and is discussed separately below.

Uptake of Other Nutrients Cu, Zn and Other Micronutrients After some years during which the importance of mycorrhizas in micronutrient uptake received only limited emphasis, there is now consistent evidence that the efficiency of uptake of both Zn and Cu is increased in VA mycorrhizal plants. Some of the earliest work on physiology of VA mycorrhizal plants showed an increase in concentration of Cu in mycorrhizal apple seedlings (Mosse, 1957); subsequently, similar results have been obtained in such diverse species as Zea mays (Daft ei al, 1975), Avena sativa (Gnekow and Marschner, 1989), Phaseolus vulgaris (Kucey and Janzen, 1987), Allium porrum (Gildon and Tinker, 1983) and Trifolium repens (Li et al, 1991c). Using a compartmented mesh system Li et al. (1991c), demonstrated hyphal uptake and translocation of Cu to T. repens. This not only contributed up to 62% of the total Cu uptake, but was also independent of the effects of P nutrition (see below). Increased Cu uptake in mycorrhizal plants has also been confirmed for a number of plant-fungus combinations (e.g. Manjunath and Habte, 1988; Killham and Firestone, 1983) although transfer from the fungus to the plant is often very small (see Manjunath and Habte, 1988) and the mechanisms controlling transfer have not been investigated. As with P, the mobility of Zn in soils is very low and its uptake by organisms is diffusion limited. Consequently, similar effects of mycorrhizal colonization on uptake were expected but proved difficult to demonstrate because of strong PZn interactions. It was shown at an early stage that VA mycorrhizal colonization increased uptake of ^^Zn by Araucaria roots (Bowen et al., 1974) and that ^^Zn was translocated along hyphae of Glomus mosseae into Trifolium repens growing in an agar plate system (Cooper and Tinker, 1978). The rate of ^^Zn translocation was 2.1 X 10~^ mol m~^ s~^, considerably lower than the rate of P translocation (Table 5.3), but probably adequate given the lower requirement of plants for this micronutrient. The tracer studies gave little quantitative information on the transfer of Zn to the plants and effects on Zn nutrition, but Zn deficiency symptoms in peach disappeared as mycorrhizas developed (Gilmore, 1971). Subsequently, a number of studies have shown unequivocally that Zn uptake via mycorrhizas is important and can alleviate Zn deficiency in several species in both pot and field experiments (e.g. Triticum aestivum, Thompson, 1990; Trifolium subterraneum, Burkert and Robson, 1994; Cajanus cajan, Wellings et al., 1991; Zea mays, Evans and Miller, 1988; Lu and Miller, 1989; Leuceana leucocephala, Manjunath and Habte, 1988; Faber et al, 1990). Vesicular-arbuscular mycorrhizal involvement in Zn nutrition

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Vesicular-arbuscular mycorrhizas

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65zn placement (mm from roots) Figure 5.9 Effect of placement of ^^Zn at different distances from the roots of nonmycorrhizal and mycorrhizal clover plants on the ^^Zn activity of the whole plants at (a) 21 days and (b) 35 days. # Non-mycorrhizal plants; / \ Acou/osporo laewis; O Qomus sp.; and • Scutellospora calospora. Bars indicate standard errors of means. From Bukert and Robson (1994), with permission.

has been implicated in the negative effects of both tillage (Evans and Miller, 1988; Fairchild and Miller, 1988) and long fallow (Thompson, 1987, 1990; Wellings et ah, 1991) on crop growth. Differences in the abilities of the same three VA mycorrhizal fungi used by Jakobsen et al. (1992a,b) to absorb and transfer ^^Zn to plants, have been demonstrated. Using a mesh-compartmented system and different distances of placement of ^^Zn source in the hyphal compartment, Acaulospora laevis obtained Zn from distances of up to 40 cm, whereas Glomus sp. (WUM 10(1)) and Scutellospora calospora (WUM 12(2)) were less effective. As with P uptake, the distribution and length density of hyphae in soil were important contributors to the differences between the fungi (Burkert and Robson, 1994; Fig. 5.9). Uptake of other micronutrients via VA mycorrhizal hyphae is not well established (Marschner and Dell, 1994) and the uptake of Mn is most commonly reduced when plants are mycorrhizal. This effect was attributable to lower Mn"^ reducing potential in the rhizosphere mycorrhizal plants, probably because the populations of microorganisms responsible were lower (Kothari et al,, 1991). Interactions between phosphate fertilization and deficiencies (or toxicities, see below) of trace elements, are well known in several species of typically mycorrhizal plants (Wallace et aL, 1978). In general, when the availability of P is increased, P uptake and plant growth also increase. Concentrations of Cu and Zn in the tissues fall, sometimes to levels at which deficiency symptoms become apparent. Mycorrhizal colonization has been shown to affect these interactions (Lambert et al., 1979; Timmer and Ley den, 1980), so that at moderate levels of phosphate fertilization

Mineral nutrition, heavy metal accumulation and water relations

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deficiency symptoms are alleviated as mycorrhizal fungi increase uptake of the trace elements and tissue concentrations rise. At very high levels of P, mycorrhizal colonization itself may be reduced (see Chapter 2) with consequent reductions in uptake and reappearance of the deficiency symptoms. Interactions such as these may be involved in the apparent alleviation of Zn toxicity in polluted sites (e.g. Dueck et ah, 1986). If the sites are P deficient then mycorrhizal P uptake could result in increased growth and dilution of Zn in the tissues.

Potassium Analyses of potassium (K) concentrations in plant tissues have occasionally ir\.dicated increases in K uptake, which might be expected considering the relative immobility of this ion in soil (Mosse, 1957; Holevas, 1966; Possingham and Groot-Obbink, 1971; Powell, 1975b; Huang et al, 1985). However, in the majority of investigations K was found to be at lower concentrations in the tissues of mycorrhizal plants than in those of non-mycorrhizal plants. Extrapolation from tissue concentrations can be dangerous, as we have seen for other nutrients, because of the simultaneous effects of P nutrition on growth. Smith et al, (1981) observed elevated concentrations of K in shoots (but not roots) of mycorrhizal Trifolium subterraneum when plants were grown on P-deficient soils. If sufficient P was supplied to soil to remove any mycorrhizal growth response, then K concentrations in both groups of plants were very similar. This suggests an indirect effect of mycorrhizas on PO^ uptake in P-deficient plants, an effect which has also been indicated with sulphate (Rhodes and Gerdemann, 1978c). However, K depletion in a hyphal compartment colonized by Glomus mosseae, and increased accumulation in associated mycorrhizal Agropyron repens has been observed (George et ah, 1992) so that direct effects are also possible. Accumulation of K is strongly influenced by the form of N available (NOs" or NH4), as well as by other cations, particularly Na"^. It might also be influenced by the synthesis and storage of polyphosphate (see Chapter 14) and carefully designed experiments to investigate the influence of mycorrhizal colonization on K nutrition need to take all these potentially confounding factors into account.

Toxic Elements Some essential elements are required in very small quantities and when accumulated at high concentrations in plants they may become toxic. Consequently, heavy metal toxicity may derive from excessive uptake of Zn, Cu, Fe and Co as well as from other elements and ions which are normally regarded as toxic (e.g. Pb, Cd, Ni, Ti, Ba). General aspects of the interactions between fungi, including mycorrhizal fungi, and these metals have been reviewed by Gadd (1993). Large effects of mycorrhizas in increasing accumulation of Cu, Ni, Pb and Zn in the grass Ehrhartia calycina have been found, especially at low soil p H (Killham and Firestone, 1983). However, El-Kherbawy et al. (1989) observed increased tolerances, at least at some soil p H values. Results are not always consistent between species: Rogers and Williams (1986) found marked increases in ^^^Cs in Melilotus officinalis, in which

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Vesicular-arbuscular mycorrhizas

no additional ^°Co was accumulated, whereas in Sorghum sudanense the converse was true. The possibility that metals may be sequestered in the hyphae and not transferred to the plant has been examined in mycorrhizal roots of Pteridium aquilinum. Electron energy loss spectroscopy showed greater accumulation of Cd, Ti and Ba in the fungal structures than in the root cells themselves. The plants had been growing on a site previously treated with Cd dust and were regarded as tolerant of the toxicity. It was suggested that sequestration of the metals by polyphosphate in the fungus might have been important in minimizing transfer to the plant (Tumau et ah, 1993) but this requires confirmation. The possibility that heavy metal accumulation might result in damage to the fimgi and consequently have significant negative effects on mycorrhizally mediated P or Zn uptake has also been addressed. It appears that prolonged exposure to Cd can result in the development of tolerance in Glomus sp., but again the mechanism is not known, Weissenhom et al. (1994). Arsenate tolerance in plants is most interesting because it appears to depend on modifications to the P transport system (which also transports arsenate) and hence the root absorbing capacity (Meharg, 1994). Tolerant genotypes of Hocus lanatus are more highly colonized by mycorrhizal fungi in the field than non-tolerant genotypes, leading to the suggestion that the tolerant plants are dependent on the fungi for P uptake and consequent success (Meharg et al., 1994). A number of questions still require clarification. If uptake of P and arsenate are diffusion limited it is not clear how modification of transport (root absorbing power) will actually affect the rates of uptake (see discussion on P uptake above). Nor is the mechanism of fungal tolerance to arsenate apparent. If the fungi also have modified P transporters, how can they take up P and compensate for the effects in roots? It is clear that interactions with P nutrition and other aspects of mycorrhizal physiology must be taken into account in studies of accumulation and tolerance of toxic elements. Tissue dilution of a toxic element can occur as a consequence of improved P nutrition and increased plant growth, even in situations where the uptake of the heavy metal per plant is actually greatly increased (El-Kherbawy et al., 1989). Only measurements of inflow or use of matched plants a n d / o r compartmented systems will determine whether the hyphae of VA mycorrhizal fungi are directly involved in uptake of heavy metals by plants.

Interplant Transfer of Nutrients Hyphal links between plants offer potential pathways for the movement of soilderived nutrients, just as they do for plant-derived C, and could play important roles in interplant and interspecies competition and redistribution of nutrients in ecosystems. Consequently, the technical problems in determining whether observed tracer fluxes of these nutrients between plants are likely to be nutritionally or ecologically significant need to be overcome (Newman, 1988; Miller and Allen, 1992; and see Chapter 4). The survival of mycorrhizal hyphae within senescent roots could lead to rapid transfer and redistribution of P released by autolysis, or by the activity of microorganisms, to plants linked by the mycelial network. Transfer of mineral nutrients between living plants is more problematic. There is certainly evidence for the

Mineral nutrition, heavy metal accumulation and water relations

155

transfer of ^^P and ^^N from 'donor' to 'receiver' plants (Whittingham and Read, 1982; Ritz and Newman, 1984; van Kessel et al, 1985; Newman and Ritz, 1986; Haystead et al., 1988; Hamel and Smith, 1991), but in most cases the results did not show net movement of the nutrient in question. Francis et al. (1986) did not use tracers, but instead supplied either nutrient solution or water to one half of the root system of the donor plants (either Plantago lanceolata or Festuca ovina). Receivers were grown with the other half of the root system either inoculated with VA mycorrhizal fungi or uninoculated. Mycorrhizal receivers (P. lanceolata or F. ovina) responded positively in both growth and nutrient content when nutrients were supplied to the donors, but the non-host Arabis hirsuta and non-mycorrhizal P. lanceolata and F. ovina did not, apparently indicating net transfer of nutrients from the larger nutrient-sufficient donors to the receivers via the hyphal network. However, Newman (1988) has suggested that these results could be explained not by direct transfer but by changes in the competitiveness of the donor plants as a result of differences in nutrient supply to them, with large nutrientsufficient donors competing less strongly with the receivers. Newman's suggestion is supported by data of Ocampo (1986) indicating that competition could have been the dominant effect in nutrient redistribution between large and small plants of Sorghum vulgare. Eissenstat (1990) re-examined this question using both nutrient applications and ^^P and ^^N in a split pot system. By measuring the ratio of tracer in the receiver to tracer in the donor, he concluded that P transfer between individuals of Plantago lanceolata was increased following P fertilization, while N transfer was unaffected. In quantitative terms the transfer of P was too small to have any effect on plant growth, whereas the transfer of N was about tenfold higher, as would be expected from the relative requirements of plants for these nutrients, and could have had an effect on the receivers in very nutrientdeficient soils. His overall conclusion was that alteration in the competitive balance, rather than direct transfer, was the most important effect. This is supported by the results of Johansen et al. (1992), who found that although external hyphae of Glomus intraradices effectively depleted total N from soil in a hyphal compartment (HC) and transferred it to plants of Cucumis sativus, very little ^ N was transferred via the plant to a second hyphal compartment. The results do not indicate a major pathway for N transfer from plant to fungus across a symbiotic interface, which is what would be expected if the transfer systems in the interfaces are polarized for transfer of different nutrients in different directions (Smith and Smith, 1990; see Chapter 14). All in all, considerable uncertainty still surrounds the idea that competition between plants may be modified by direct net transfer of nutrients (P and N) from plant to plant via a common mycelium. The lack of convincing data underlines the fact that very carefully designed experiments are required in this area. In addition, short-cycling of nutrients from a dying plant to a linked living plant may be considerable and should not be overlooked in ecological studies. W a t e r Relations Mosse and Hayman (1971) observed that mycorrhizal onions did not wilt when transplanted, but that non-mycorrhizal plants did. Subsequently, several similar observations have been made (Busse and Ellis, 1985; Huang et al., 1985) and there is

156

Vesicular-arbuscular mycorrhizas

no doubt that mycorrhizal colonization does affect the water relations of plants. As with other aspects of the physiology of mycorrhizal plants, it is relevant to distinguish direct effects of fungal colonization from indirect effects resulting from changes in plant size or P status. The subject is complex and there are many inconsistencies in the literature, not all of which can be easily explained (Fitter, 1988; Koide, 1993; and see Nelsen, 1987). The problem was first investigated systematically with soybean by Safir et ah (1971, 1972), who showed that mycorrhizal plants had lower resistances to water transport than uncolonized plants, and in this instance it appeared that most of the difference was attributable to changes in root resistance, since shoot resistances were small and did not differ in the two groups of plants. Safir et al. (1972) concluded that the effect was probably due to improved nutrition, because the differences could be eliminated if nutrients or fungicide were applied. Transpiration rates of mycorrhizal plants are generally higher than those for nonmycorrhizal plants (Allen et al, 1981b; Allen, 1982; Nelsen and Safir, 1982; Huang et al, 1985; Koide, 1985b; Fitter, 1988). As Koide (1993) shows, the discussions and inconsistencies centre on whether this increase is due to increased stomatal conductance or whether decreased resistance to water transport in the below-ground system is also important, as suggested by the data of Safir et al (1971, 1972). Levy and Krikun (1980) used Citrus jambhiri and a fungus similar to Glomus Jasciculatus', with growth and fertilizer conditions that permitted the comparison of mycorrhizal and non-mycorrhizal plants of similar size and growth rate. The major effect of mycorrhizal colonization was an increase in transpirational flux and stomatal conductance, both during stress and recovery. There were apparently no differences between mycorrhizal and non-mycorrhizal plants in terms of resistance of the root to water movement. Similar conclusions were reached with Trifolium pratense (Fitter, 1988), Helianthus annuus (Koide, 1985b), Leucaena leucocephala (Huang et al, 1985) and Bouteloua gracilis (Allen et al, 1981b; Allen, 1982). Again, transpiration rates were increased in mycorrhizal plants, while stomatal resistances were greatly reduced. Both Koide (1985b) and Fitter (1988) came to the conclusion that the high stomatal resistance in P deficient non-mycorrhizal plants was a nutritional effect and Figure 5.10 shows the good correlation between stomatal conductance and leaf P concentration in T. pratense, regardless of whether the differences were induced by P fertilization or mycorrhizal colonization. However, there have been suggestions that stomatal behaviour is influenced by the hormonal changes in the plant that certainly occur as a result of either changes in P nutrition or mycorrhizal colonization per se (e.g. Allen et al, 1980, 1982; Allen, 1982; Dixon et al, 1987; Baas and Kuiper, 1989; Danneberg et al, 1992; Druge and Schonbeck, 1993). In B. gracilis the changes in stomatal conductance could not be explained in terms of differences in gross anatomy or morphology and there were no changes in size of mesophyll cells, bundle sheath cells or stomata, nor was the stomatal density altered following mycorrhizal colonization. Resistance to water transport was not separated into contributions of root and shoot by Allen et al (1981b), but they did comment that increased branching of the roots in mycorrhizal plants could Pb to substantial increases in root surface area without changes in root biomass, and that this might reduce the root resistance to water uptake. However, Koide (1985b) measured the hydraulic resistance to water transport between soil and leaf, and between soil and stem below the leaf (root plus stem resistance) over a range of

Mineral nutrition, heavy metal accumulation and water relations

157

transpiration rates in well watered soils. When mycorrhizal and non-mycorrhizal plants of the same size and root length were compared there were no differences in hydraulic properties and the effects of mycorrhizas appeared to be entirely explicable on the basis of decreased stomatal resistance. Increased stomatal conductivity and increased transpiration rates in Leucaena were accompanied by higher xylem pressure potentials and the stomata responded more rapidly to changes in humidity. The mycorrhizal plants lowered the water potential of the soil in the pots, indicating much higher water uptake than into the non-mycorrhizal plants (Huang et ah, 1985). The conclusion in this case was that higher water use in mycorrhizal plants was offset by increased C gain, because the stomata remained open for a greater part of the day. Large root systems and rapid stomatal response were also important in the overall water economy. The results of a number of investigations have, however, suggested that VA mycorrhizal colonization can reduce the hydraulic resistance to water uptake in the roots (e.g. Hardie and Leyton, 1981; Nelsen and Safir, 1982; Graham and Syvertsen, 1984; Bildusas et al., 1986). On a whole-plant basis this could occur by increases in the size or branching of the root system. However, Hardie and Leyton (1981) could attribute only part of the decrease in hydraulic resistance of the root systems of Trifolium pratense colonized by Glomus mosseae to this effect. They therefore concluded that hyphal growth in the soil was important in reducing root resistance to water flow and this point is discussed further below. However, Koide (1993) has emphasized that because root hydraulic conductivity is not linearly related to root system size (see Ficus and Markhart, 1979), it is not valid to compare values obtained from root systems of different sizes, even if the results 121

^ 10 I w E

£

8

®

o c

B § "D C

o o E

6 o

o

CO

Leaf P concentration (mg g~^)

F i g u r e 5.10 Relationship between stomatal conductance and tissue P concentration, compiled by Fitter (1988). Symbols represent Rosa ( A A ) ' HelianthuSy ( 0 # ) and Trifolium ( D B ) . Mycorrhizal plants, solid symbols and non-mycorrhizal plants, open symbols. Reprinted f r o m the Journal of Experimer)tal Botariy by permission of O x f o r d University Press.

158

Vesicular-arbuscular mycorrhizas

are expressed per unit root length. Consequently, orUy data from matched mycorrhizal and non-mycorrhizal plants can be used for comparison and in these cases either no effects were observed (Graham and Syvertsen, 1984; Koide, 1985b; Graham et ah, 1987) or there was a decrease in hydraulic conductivity (Levy et ah, 1983). Koide (1985b) further noted that apparent effects of mycorrhizal colonization on the root hydraulic conductivity could have been recorded because this parameter varies with transpirational flux, which is normally higher in mycorrhizal plants. The possible roles of mycorrhizal hyphae in water uptake require discussion, despite the fact that there is little imequivocal evidence implicating them in water uptake and transport processes or in direct effects on water relations of colonized plants. Hardie (1985) studied transpirational flux in non-mycorrhizal and mycorrhizal Allium porrum and Trifolium pratense that were not nutrient limited. Mycorrhizal clover had slightly higher transpirational fluxes and lower stomatal resistances than the non-mycorrhizal plants. Removal of external hyphae did not affect the stomatal resistance but appeared to reduce the transpirational flux, although the differences were not significant. Hardie used the maximum reduction in transpirational flux caused by hyphal removal and transplanting (9.9 X 10~^ 1 m~^ root length s~^) to calculate that damage to 40 hyphal entry points per metre of colonized root would account for this reduction if they had the same water flux as Phycomyces blakesleeanus. This is a bold assumption, given the lack of the significance of the data. However, Faber et al, (1991) produced some evidence for hyphal depletion of soil water in a compartmented system and calculated an apparent water flux through the hyphae of 375 X 10~^ 1 h~^ per hypha crossing the mesh (or, using internal radii of hyphae of 5 |Lim, 1.32 1 m~^ s~^; see Table 5.3). Kothari et al. (1990) also examined the question using Zea mays. The difference in transpiration rates between mycorrhizal and non-mycorrhizal plants was considerable and could largely be attributed to increased leaf area. However, root lengths in mycorrhizal plants were reduced by 31% and in consequence the water uptake per unit length of root was much higher than in non-mycorrhizal plants (A 7.3 X 10~^ 1 m"^ s~^; a value similar to that found by Hardie, 1985). Assuming 60% colonization of the root system and 1000 entry points (of 10 |Lim diameter) per metre of colonized length (Kothari et al., 1990), the cross-sectional area of hyphae through which the water would enter can be estimated as 4.7 X 10~^ m^ m~^ and the flux of water as 0.15 1 m~^ s~^, which is an order of magnitude less than the estimate of Faber et al. (1991). Kothari et al. (1990) calculated that if P was delivered in the hyphae by mass flow of a solution of concentration 16 mmol 1~^ the inflow should have been 10 X 10~^° mol m"^ s~^. This is 1-2 order of magnitude higher than the actual inflow, which was in the range of 6.222.0 X 10~^ mol m~^ s~^. They concluded that the apparent bulk flow of water in hyphae did not seem to make an important contribution to P inflow (see also Sanders and Tinker, 1973; Cooper and Tinker, 1981). The conclusion that mass flow of solution in the hyphae did not occur to any great extent was supported by the fact that consumption of water from the hyphal compartments in their experiments was negligible. Furthermore, George et al. (1992) confirmed these findings and showed no difference in water depletion in a hyphal compartment, regardless of whether the plants were well watered or water stressed, whether or not the hyphae were cut.

Mineral nutrition, heavy metal accumulation and water relations

159

Mass flow has been proposed as one of the possible mechanisms of translocation in fungi (Jennings, 1987). In a multihyphal strand or in a hypha growing apically and with all resource allocation polarized towards the apex, this idea could have some credibility. However, it is well established that mycorrhizal hyphae translocate different nutrients in opposite directions. Organic C moves from the plant to the external mycelium and supports its apical growth in soil. Conversely, mineral nutrients are absorbed from the soil and translocated towards the plant. Any mechanism of translocation or of mass flow of solution must take this into account. It seems highly unlikely that mass flow of solution through VA mycorrhizal hyphae towards the plants takes place at rates able to account for the apparent extra water uptake by mycorrhizal roots, while at the same time permitting C transfer in the opposite direction. It remains to be seen whether the pleiomorphic vacuolar tubules, which do seem to pulsate in opposite directions, move water as well as the solutes within them at rates adequate to support the measured fluxes. The current consensus is that hyphae do not play an important part in reducing the hydraulic resistance of roots and many workers would feel that there was no need to invoke this in any case, given the complexities of measurement and the dependence of hydraulic conductivity on the (variable) rate of transpiration and size and branching of the root systems. However, there are still those who consider that a non-nutritional effect of mycorrhizal colonization on water relations may be important and research in this area continues (Druge and Schonbeck, 1993; Ebel et flZ., 1994). Mycorrhizal colonization may have effects on drought tolerance that are not directly related to water relations. Nutrients, as we have seen, become less and less available as soil dries because of the increasing tortuosity of the diffusion path. Under these conditions growth of non-mycorrhizal plants is likely to be increasingly limited by nutrient availability, and reduced root growth would limit the accessibility of water. Under these conditions the hyphal contribution to uptake of nutrients would become more and more important.

Conclusions There is excellent evidence to demonstrate that external hyphae of VA mycorrhizal fungi absorb non-mobile nutrients (P, Zn, Cu) from soil and translocate them rapidly to the plants, thus overcoming problems of depletion in the rhizosphere, which arise as a consequence of uptake by roots. Transfer across the symbiotic interface results in increased nutrient acquisition by the plant. These processes act in series and, with efficient fungi, lead to depletion of nutrients in the soil well beyond the rhizosphere. The sources of P (and other nutrients) available to the fungi are less clear. The soil solution, in equilibrium with the so-called labile fraction, must be the primary source. Hyphae are able to penetrate soil pores inaccessible to roots and may also be able to compete effectively with soil-inhabiting microorganisms for recently mineralized nutrients. Rapid removal from solution at sites of release will accelerate the use of that K fraction that exchanges rapidly with the solution.

160

Vesicular-arbuscular mycorrhizas

The evidence that mycorrhizal fungi lower the threshold concentration for uptake is slim. There is no good evidence that VA mycorrhizal fungi actually hydrolyse organic P or N sources. The use of non-labile inorganic sources or applied RP is still open to question. Mycorrhizal plants do seem to grow better and take up more P from these sources, but until methods are worked out which allow the P fractions in soil to be distinguished and their accessibility to plants (whether mycorrhizal or not) determined, little progress is to be expected in sorting out what mechanisms actually underlie the effects. At present it appears that localized alterations in pH might play a role in increased P mobilization in microsites. Production of chelating agents that would increase the availability of Fe or Al phosphates has not been demonstrated, despite the apparent differences in accessibility to mycorrhizal and non-mycorrhizal plants. The interactions between mycorrhizal colonization and accumulation of heavy metals and other toxic elements is an area of considerable interest. A number of different mechanisms may be involved, including tissue dilution of the toxic element due to interactions with P nutrition, sequestration of the toxic metal in the fimgus, and development of tolerance by the fungus. Water relations of plants are modified in some ways by the mycorrhizal interactions. The mechanisms are difficult to determine, but most of the effects can be related to changes in nutritional status. There is little evidence either for actual water transport via the fimgal hyphae or for alterations in root or shoot hydraulic properties or water potentials that are independent of increased P uptake or of changes in growth as a result of this.

Structure and development of ectomycorrhizal roots

Introduction The ectomycorrhizal root is characterized by the presence of three structural components: (a) a sheath or mantle of fungal tissue which encloses the root; (b) a labyrinthine inward growth of hyphae between the epidermal and cortical cells called the Hartig net; and (c) an outwardly growing system of hyphal elements which form essential connections both with the soil and with the fruit bodies of the fungi forming the ectomycorrhizas. In the absence, until recently, of fossil ectomycorrhizas, discussion of the origin of this type of symbiosis was based largely on conjecture. Fossil leaves and wood of families of plants, such as the Pinaceae, which are today almost exclusively ectomycorrhizal, appear for the first time in the Cretaceous around 130 million years BP (Axelrod, 1986) and molecular clock evidence (Berbee and Taylor, 1993) indicates an origin of the holobasidiomycete group, within which many of their current fungal associates occur, at around the same time. Trappe (1977) suggests that the disjunct distribution of many hypogeous fungi, which are dependent upon animals for dispersal, can best be explained by their presence in the Laurasian subcontinent before the land migration routes of the vectors between Europe and North America were separated by tectonic events some 50 million years BP. On the assumption, now widely accepted, that epigeous forms were ancestral to those of hypogeous habit, the mushroom-forming ectomycorrhizal fungi, and therefore the potential to form this type of the symbiosis, would have originated before that. Support for this contention, in the form of well-preserved ectomycorrhizas, has now been provided in fossils recovered from the approximately 50 million years BP Princeton cherts of British Columbia of the Eocene period (Le Page ei al., 1996). These provide the first unequivocal evidence of ectomycorrhizas in the fossil record. Small roots of a Pinus sp., each having a diameter of 3-5 mm, bear coralloid clusters of attenuated and thickened, dichotomously branched rootlets 0.1-0.5 mm in diameter (Fig. 6.1). Transverse sections reveal the presence of a Hartig net-like fungal tissue between the cells of the cortex up to the position of the endodermis (Fig. 6.1c). Only traces of mantle tissue remain and extraradical hyphae are scarce.

164

Ectomycorrhizas

'0

' ^^^

Figure 6.1 Fossil ectomycorrhizas of a Pinus sp. recovered from the Princeton cherts of Eocene age in British Columbia, (a) Coralloid clusters of ectomycorrhizal roots. Bar, I mm. (b) Individual rootlets showing the dichotomous branching characteristic of ftnus mycorrhizas. Bar, I mm. (c) Transverse section showing Hartig net-like fungal tissue (arrowed) surrounding root cells and showing evidence of labyrinthine structure. Bar, 20 ^ m . From Le Page et al. (1996), with permission. Copyright National Academy of Sciences, USA.

Those that are present lack clamp connections and have diameters of 1-2 ^lm. Among the small number of fungal associates of extant Pinus spp. that produce such coralloid clusters of rootlets and lack clamp connections are members of the hypogeous genus Rhizopogon. If, as proposed by Le Page et al. (1996), the fossil mycorrhizas of Pinus were formed by a Rhizopogon sp., these findings would confirm the view of Trappe (1987) that hypogeous basidiomycetes originated more than 50 million years BP and provide support for the molecular clock-based datmg of the origin of the ancestral epigeous forms, concurrently with that of the plant genus, around 130 million years BP.

Structure and development of ectomycorrhizal roots

165

Almost all of the plants upon which ectomycorrhizas develop are woody perennials. The anatomical structures of the sheath and the emanating mycelia are stable at least at the level of the fungal genus and are increasingly used to facilitate characterization of the ectomycorrhizas (Agerer, 1987-1993; Ingleby et al., 1990). This type of organ is also clearly distinguishable from all other types of mycorrhiza on the basis of the absence of intracellular penetration by the fungus. In the event of penetration of healthy root cells by a mycorrhiza-forming fungus, whether from the Hartig net or from the hyphae of the sheath, the structure is referred to as an 'ectendomycorrhiza' (see below and Chapter 10). The identity of the plant can influence the outcome. Some fungi, for example the ascomycete Wilcoxina mikolae, routinely produce ectendomycorrhizas on young plants of Finns and Larix in nursery soils while forming ectomycorrhizas on Ahies, Picea and Tsuga (Mikola, 1988; and see Chapter 10). However, most ectomycorrhizal fungi are capable of forming intracellular penetrations in senescent parts of the rdot axis, or when the nutrient balance of the association is disturbed. In these circumstances the fungus appears to be behaving in a weakly pathogenic manner. Associations of this kind were called 'pseudomycorrhiza' by Melin (1917) but the lack of precision of this term is strong justification for abandoning its use (Mikola, 1965; Harley, 1969). Many fungi that produce typical ectomycorrhizas on members of the Pinaceae and Fagaceae form extensive intracellular growths as well as Hartig net and sheath in certain ericaceous hosts (see Chapter 11). This type of colonization is recognized as being of the distinct arbutoid category. Intracellular penetration in the form of haustorium-like structures can sometimes be observed in ectomycorrhizal roots. These are produced by fungi of uncertain status that are associated with, but not involved in the formation of, the mycorrhizal mantle. Thus members of the Gomphidiaceae grow in mycorrhizal sheaths formed by Rhizopogon and Suillus spp., from which they penetrate the cortical cells of Pinus spp. (Agerer, 1990). Similarly, the ascomycete Leucoscypha leucotricha, a ubiquitous resident of sheaths formed by Lactarius subdulcis on Fagus sylvatica, does the same on that species producing 250-310 haustoria in every 2 mm of mycorrhizal root tip (Brand, 1991, 1992; Fig. 6.2). While the presence of the three structural elements signifies an ectomycorrhiza, there may be considerable variation in the extent to which Hartig net, sheath and extraradical mycelium are developed. Indeed, even structures that have only a patchy sheath as in some Asteraceae (Warcup, 1980), or that lack a Hartig net as in the roots of Pisonia grandis (Ashford and AUaway, 1982) and of Pinus spp. colonized by Tricholoma matsutake (Ogawa, 1985), have been referred to as 'ectomycorrhiza'. The danger in broadening the category to this extent is that relationships between structure and function established by study of 'typical' forms may break down. This is well illustrated in the case of the Tricholoma 'ectomycorrhiza' reported by Ogawa (1985) to be parasitic. It may be acceptable to include the association seen in Pisonia grandis as a true ectomycorrhiza because, despite the absence of a Hartig net, wall proliferations in the interface are apparently similar to those seen in some typical ectomycorrhizas. Cairney et al (1994) have shown that a basidiomycete forming 'ectomycorrhizas' on Pisonia formed a sheath and some intercellular penetration on Picea sitchensis, but not on Eucalyptus pilulans. In view of the diversity of niches and of plant-fungus combinations possible in

166

Ectomycorrhizas

4

•%.!•,,:,:: ::^r\;:^/

WW

Figure 6.2 Mycorrhizas of Lactarius subdulds on Fagus sylvaticOy showing a dual colonization with an ascomycete Leucoscypha. (a) Ascomycete hyphae (arrowed) penetrating the fungal mantle, (b) Ascomycete hyphae (arrowed) on the inner surface of a dissected mantle, (c) Haustorium of an ascomycete (h) and intercellular hypha (arrowed). From Brand (1992), with permission.

nature it is not surprising that a range of structural forms is found. However, it is important not to lose sight of the fact that the vast majority of associations defined as ectomycorrhizal conform to the basic pattern in which a sheath, Hartig net and some, if only seasonal, development of extraradical mycelium are all present, while intracellular penetration is lacking. These well-conserved features appear to have been favoured because they confer particular functional attributes which further distinguish them from mycorrhizal types that do not have all of these defining characteristics.

Structure and development of ectomycorrhizal roots

167

That is not to say that there is no diversity of form or function within the ectomycorrhizal category defined in this way. Indeed, schemes of classification based upon differences of mantle structure are now enabling us to characterize ectomycorrhizas, and in many cases to identify the fungal symbionts involved, in the absence of fruiting structures. These advances will greatly enhance the precision of analysis of field-collected material, enabling relationships between structural and functional aspects of biodiversity to be evaluated. Techniques of molecular fingerprinting of the fungi associated with individual mycorrhizal roots tips are also playing a greater and greater role in determining the fungal species composition in natural populations (Gardes et al, 1991a,b; Gardes and Bruns, 1993, 1996).

The Plants: Taxonomy and Geographic Occurrence While a relatively small number, probably around 3% (Meyer, 1973), of phanerogams (seed plants) are ectomycorrhizal, their global importance is greatly increased by their disproportionate occupancy of the terrestrial land surface and their economic value as the main producers of timber. Thus the Pinaceae, members of which form the major component of the vast boreal forests of the northern hemisphere, the Fagaceae, dominants of the northern temperate forests, and their counterparts in the temperate and subtropical regions of the southern hemisphere, the Myrtaceae, are all families of predominantly ectomycorrhizal species. Table 6.1 lists examples of families and genera within which ectomycorrhizal colonization has been reported. Some are exclusively ectomycorrhizal, but others may also form vesicular-arbuscular (VA) mycorrhizas and indeed this may be the typical mycorrhizal type for the taxon. In the tropics of south-east Asia the most important family of moist and monsoon forests, the Dipterocarpaceae, is almost exclusively made up of ectomycorrhizal species (Alexander and Hogberg, 1986; Smits, 1992). This habit is also found in certain leguminous plants of the tropics most notably in the subfamily Caesalpinioideae, members of which are characteristically not nodulated. Here, most of the tribe Amherstieae and some genera such as Afzelia, Intsia and Eperua in the tribe Detareae have this type of association (Alexander, 1989a,b). In drier savannah woodlands of the miombo type the prominent leguminous genera Brachystegia and Jubernaldia are also ectomycorrhizal (Hogberg, 1982; Hogberg and Pearce, 1986). In contrast the subfamilies Mimosoideae and Papilionoideae appear, with a very few exceptions that need to be confirmed, to be made up of species colonized by VA mycorrhizal fungi (Alexander, 1989a). There are occasional reports of 'ectomycorrhizal' colonization in species such as Acacia (Mimosoideae; Warcup, 1985; McGee, 1986) even though neither Hartig net nor sheath development are typical. As most reports suggest that such genera have VA mycorrhizas under natural conditions there seems little to be gained by describing them as being ectomycorrhizal. Some genera of shrubs and a very small number of herbaceous species of angiosperm are routinely found to be ectomycorrhizal. Of these, the shrubs Dryas (Rosaceae) and Helianthemum (Cistaceae) are of particular ecological importance. Amongst the herbaceous species the dicotyledonous herb Polygonum viviparum and the cyperaceous monocot Kobresia myosuroides have typical ectomycorrhizal short

Ectomycorrhizas

168

Table 6.1 Genera reported to contain at least one species on which ectomycorrhiza has been described. Family or subfamily

Genus

Family or subfamily

Genus

Aceraceae

B Acer

Epacridaceae

Astroloma

Betulaceae

B Alnus

Ericaceae

Arbutus Arctostaphylos

Betula

Caesalpinioideae

B Carpinus

ChirDQphila

B Corylus

Gaultheria

B Ostrya

Kalmia

B Ostryopsis

Ledum Leucothoe

B Afzelia

Rhododendror)

Aldina

Vacdnium

Anthonota Berlinia Brachystegia

Euphorbiaceae Fagaceae

Eperua

6 Fagus 6 Lithocarpus

B Julbernardia

6 Nothofagus

Monopetalanthus

B Pasania 6 Quercus

Paramacrolobium Swartzia

6 Trigonobalus

Tetraberlinia B Sambucus

Casuarinaceae

B Casuahna B Allocosuorina

Cistaceae

6 Helianthemum 6 Cistus

Cupressaceae

6 Castanea 6 Castar)opsis

B Gilbertiodendron B Intsia

Caprifoliaceae

Porar)thera

Gnetaceae Goodeniaceae

Gnetum Brur)onia* B Gooder)ia*

Hammamelidaceae Juglandaceae

Parrotia B Carya

B Cupressus

Engelhardtia B Juglar)s

B Juniperus

Pterocarya

Cyperaceae

6 Kobresia*

Mimosoideae

B Acacia

Dipterocarpaceae

6 Anisoptera

Myrtaceae

6 Angophora

Ralanocarpus

B CoWistemon

Cotylelobium

6 Campomones/o

Dipterocarpus

B Eucalyptus

Dryobalanops

B Leptospermum

Hopea

B Me/a/euco

Monotes

B Thstar)ia

6 Shoreo Valica Elaea^naceae

Shepherdia

Nyctaginaceae

B Neeo B Torrubia B Pisor)ia

169

Structure and development of ectomycorrhizal roots

Fannily or subfamily

Papilionoideae

Genus

Brachysema

Family or subfamily

Rhamnaceae

Daviesia

B Rhamnus

Dillwynia

Spyridium

Eutaxia

Trymalium

B Gompholobium B Hardenbergia

Rosaceae

B Crataegus

Kennedya

B Dryas

B Mirbelia

B Malus

B Oxylobium

B Prunus

Platylobium

B Pyrus

Pultenaea

B Rosa

B Viminaria Abies Cathaya

B Sorbus Salicaceae

Larix Picea B Pinus Pseudolarix B Pseudotsuga B Tsuga

Polygonaceae

B Platanus Coccoloba Polygonum'^

B Populus B Salix

B Cedrus Keteleeha

Chaembatia Cirocarpus

Jacksonia

Platanaceae

Cryptandra Pomaderris

Chohzema

Pinaceae

Genus

Sapotaceae Sterculiaceae

Glycoxylon B Lasiopetalum Thomasia

Stylidiaceae

B Stylidium

Thymeliaceae

B P/me//a

Tiliaceae

B T/7/0

Uapacaceae

B Uapaca

Ulmaceae

B U/mt/s

This list cannot pretend to be exhaustive but illustrates the wide range of families and genera of Angiospermae and Gynnnospermae in which ectomycorrhizas have been observed. A record of the presence of ectomycorrhizal individuals in a genus does not mean that all species are or may be ectomycorrhizal, nor does it mean ectomycorrhizal colonization is necessarily consistendy or even normally present in any species of that genus. Those marked * are herbaceous and those marked B may form both ecto- and VA mycorrhizas, with the latter in many cases being the most common mycorrhizal type observed. Modified from Harley and Smith (1983).

roots with sheath and Hartig net. Neither of these plants would normally be colonized by VA mycorrhizal fungi. One further category of woody plants is of interest because it shows the facultative ability to be either VA or ectomycorrhizal. Members of the Salicaceae fall into this group. Species in genera such as Salix and Populus, depending upon local circumstances, can be predominantly colonized by VA or ectomycorrhizal fungi. The nature of the mycorrhizal type first formed and the extent to which either type of colonization persists into the adult condition appears to depend upon local soil conditions: VA mycorrhizas are typical of Salix or Populus species growing on mineral- or nutrient-rich soils, while ectomycorrhizas predominate in organic soils. Several plants, including Eucalyptus (Lapeyrie and Chilvers, 1985; Chilvers

170

Ectomycorrhizas

et al., 1987) and Helianthemum (Read et al., 1977) may be VA mycorrhizal in the seedling stages, while Alnus is reported to be VA mycorrhizal under some circumstances and shows growth responses to inoculation with VA and ectomycorrhizal fungi (Fraga-Beddiar and Le Tacon, 1990; Jha ei al, 1993). The situation in the Pinaceae is less clear and the reports (Cazares and Smith, 1992, 1996) of colonization by vesicles and hyphae, with occasional arbuscules, are of as yet unknown significance.

The Fungi Forming Ectomycorrhizal Associations In contrast to the situation seen in the VA mycorrhizal symbiosis, a large number of fungal species have been recorded as forming ectomycorrhizas. The majority of these are in the basidiomycetes, but there is a significant representation from the ascomycetes including the so-called 'E-strain' fungi, which also form ectendomycorrhizas on Pinus and Larix (Laiho, 1965; Mikola, 1965). A few species of zygomycetous fungi in the genus Endogone are also known to form this type of association. Molina et al. (1992) estimate that between 5000 and 6000 species of fungi form ectomycorrhizas or ectendomycorrhizas. While most of these are of the epigeous types (around 4500) a number, perhaps up to one-quarter, are hypogeous. Members of the ascomycetes are particularly conspicuous in the latter group. Lists (e.g. Table 6.2) provide valuable pointers to the possible extent of mycorrhizal involvement in a given genus, but it must be emphasized that they are based largely upon observed associations between hosts and sporophores in the field, and that in only a small proportion of cases has the mycorrhizal status been confirmed. These observations do not, therefore, serve as substitutes for direct analysis of the nature of the relationships between the fungi and their hosts. These can only be established by a combination of experimental approaches amongst which tracing of connections from fruit body to plant root (Agerer, 1991a) and resynthesis of mycorrhizas under axenic conditions (Duddridge, 1987), together with molecular fingerprinting are recommended. Records of plant-sporocarp associations suggest that the majority of ectomycorrhizal fungi have a broad host range (Trappe, 1962; Molina et al., 1992; and see Table 6.2). It is certainly the case that some species, such as Amanita muscaria, Cenococcum geophilum, Hebeloma crustuliniforme, Laccaria laccata, Pisolithus tinctorius and Thelephora terrestris, have worldwide distribution on a very wide range of plants. This should not disguise the fact that specificity, at least at the level of the plant genus, can be recognized in a diverse range of fungi, hosts and habitats. Studies of the plant genera Eucalyptus (Malajczuk et al., 1982), Nothofagus (Garrido, 1988) and Pseudotsuga (Molina et al, 1992) show that each has an extensive assemblage of specific fungi. In the case of Pseudotsuga, Molina et al. (1992) estimate that there may be around 250 such genus-specific fungi. Once again, observations of this kind, which are almost exclusively based upon plant-sporocarp associations, need to be verified by synthesis of mycorrhizas. Such syntheses must, however, be established and interpreted in a sensitive manner because environmental and culture conditions can have important effects on the outcomes. The use of media containing large quantities of exogenous C, as originally recommended for synthesis of ectomycorrhizas (Molina and Palmer, 1982), can

Structure and development of ectomycorrhizal roots

171

lead to considerable distortion of the apparent host range of a fungus. Species such as Suillus grevillei which are confined to the plant genus Larix in nature, were shown (Duddridge, 1986a,b), in the presence of glucose, to form mycorrhizas on a number of other genera. Under similar circumstances, Molina and Trappe (1982b) observed that Rhizopogon vinicolor, the sporocarps of which, in nature, appear to be exclusively associated with Douglas fir, was induced to colonize species of Picea, Pinus and Tsuga. Clearly, syntheses should be carried out in such a way as to reflect the natural inoculum potential of the fungus, a situation that can best be achieved in the laboratory by enabling it to grow from a true host through natural substrates to the roots of the test plant. This provides a measure of 'ecological specificity' and reflects a situation which is perhaps as close as possible to that prevailing in nature. While ectomycorrhizal fungi can be categorized as having either a narrow or a broad host range, the root system of an individual tree, for example of Eucalyptus, Pinus or Pseudotsuga, will normally be colonized by several members of each category. The factors that may have favoured the selection of this pattern of cooccurrence in forest ecosystems is discussed in chapter 15. Furthermore, successions of fungi - both on individual roots and in communities - have been repeatedly described and are discussed in a later section. The taxonomic status of fungi forming ectomycorrhizas is normally described at the level of the genus or species, but there is increasing recognition of the extent of diversity, both structural and functional, seen within the fungal species. Understanding of the genetic basis of this variability is now being sought, not only because of the intrinsic interest in the factors which determine plasticity, but also because the potential exists for manipulation of the genome and selection of functional attributes which are likely to be favourable to the hosts.

Genetics of Ectomycorrhizal Fungi There has long been an awareness of the extent of interspecific variability between ectomycorrhizal fungi in the structure and function of the mycorrhizas that they form. Recent studies have demonstrated, however, that in some of the most widely occurring fungi such as Pisolithus tinctorius (Lamhamedi ei al, 1990; Lamhamedi and Fortin, 1991; Burgess et al, 1994,1995), Laccaria hicolor (Kropp et al, 1987; Wong et al, 1989, 1990; Wong and Fortin, 1990) and Hebeloma cylindrosporum (Debaud et al, 1986; Marmeisse et al, 1992a) the magnitude of intraspecific variability can be as great as that between species. Most basidiomycetes are heterothallic, with very complex genetic control of mating, there being several thousand mating types determined by 1-4 multi-allelic loci (Aa, A(3, Ba, Bp; Kiies and Casselton, 1992; Casselton and Kiies, 1994; Debaud et al, 1995). A sexually sterile monokaryotic mycelium is produced from germination of a basidiospore during the formation of which meiosis has occurred. In mycorrhizal basidiomycetes, such monokaryons are normally unable to produce fully developed ectomycorrhizas (see below) but the mating of two monokaryotic mycelia enables the resulting dikaryon both to produce fruit bodies and to form mycorrhizas. Two mating systems are recognized. In bipolar forms, which constitute approximately 25% of heterothallic species, compatibility between homokaryons is controlled by multiple alleles at a single locus, A. The remaining 75% are

172

Ectomycorrhizas

T a b l e 6.2 Examples of ectomycorrhizal fungal species w i t h little host restriction (broad host range) by fruiting habit, class, family and genus Habit, class, family

Genus

Species

Epigeous habit Basidiomycotina Amanitaceae

Amanita

Astraeaceae Boletaceae

Astraeus Boletus

aspera, fulva, gemmata, inaurata, muscaria, pantherina, phalloides, rubescens, solitaha, spissa, strobiliformis, vaginata, verna, virosa hygrometricus, pteridus appendiculatus, calopus, edulis, erythropus, luridus, minatJoolivaceus, pulverulentus, regius castaneus, cyanescens rhodoxanthus ravenelii chromapes, felleus, gracilis, porphyrosporus armeniacus, badius, chrysenteron, rubellus, spadiceus, subtomentosus, truncatus dbarius, infundibuliformis, tubiformis aurea, botrytis, flava, formosa, mairei, subbotrytis atrovirens sublutea byssinium, croceum, sulphureum acutus, anomalus, bicolor, bivelus, everneus, hemitrichus, leucophanes, mucosus, multiformis, obtusus, phrygianus, saniosus anthracina, cinr)amomea, malicoria, palustris, phoenicea crustuliniforme, cyliridrosporum, hiemale, lor)gicaudum, mesophaeum, mir)us, pumilum, siriapizans asterospora, bongardii, brur)nea, cincinr)ata, dulcamara, fastigiata, jurana, lacera, lanuginella, petJgir)osa, terrigena, umbrina caperata repar)dum velutinum rufescer)s capreolarius, comarophyllus, chrysodor), discoideus, hypothejus, karstenii, marzulus, pudorinus ir)volutus cristatus decipiens, fuligiriosus, helvus, necator, piperatus, repraesentar)eus, rufus, scrobiculatus, spinosulus, uvidus, vellereus, volemus aerugmea, albonigra, amoeria, anthracina, cyanoxantha, densifolia, emetica, foetens, heterophylla, lutea, nigricans, ochroleuca, odorata, oHvacea, paludosa, palumbina, parazurea, vesca, virescens, xerampelina

Gyroporus Phylloporus Pulveroboletus Tylopilus Xerocomus Cantharellaceae Clavariaceae Corticiaceae

Cortinariaceae

Cantharellus Ramaria Byssocorticium Byssoporia Piloderma Cortinarius

Dermocybe Hebeloma

Inocybe

Hygrophoraceae

Rozites DentJnum Hydnellum Hydnum Hygrophorus

Paxillaceae Polyporaceae Russuiaceae

Paxillus Albatrellus Lactahus

Hydnaceae

Russula

Structure and development of ectomycorrhizal roots

Habit, class, family

Genus

173

Species

Epigeous habit Basidiomycotina (contd) Sclerodermataceae Strobi lomycetaceae Thelephoraceae Tricholomataceae

Scleroderma

bovista, cepa, citrinum, laeve, polyrhizum, verrucosum

Pisolithus

tinaorius

Boletellus

betula, chrysenteroides

Strobilomyces

floccopus

Thelephora

ar)thocephala, atrocitrina, per)icillata, terrestris

Sarcodon

imbricatus, scabrosus

Laccaria

amethystina, bicolor, laccata, montana, proximo

Tricholoma

caligatum, columbetta, flavobrur)neum, flavovirer)s, myomyces, sapor)aceum, sulphureum

Hypogeous habit Ascomycotina Balsamiaceae

Balsamia

magnata, platyspora, vulgaris

Elaphomycetaceae

Cenococcum"^

geophilum

Elaphomyces

ar)thracir)us, grar)ulatus, muricatus, mutabilis,

Ger)abea

cerebriformis

reticulatus, variegatus Geneaceae

Ger)ea

gardneri, harknessii, intermedia

Helvellaceae

Hydriotrya

tulasr)ei

Pezizaceae

Pachyphloeus

citrir)us, ligericus, melanoxanthus

Terfeziaceae

Choiromyces

alveolatus, venosus

Tuberaceae

Tuber

aestivum, borchii, brumale, califorr)icum, excavatum, melanosporum, puberulum, rapaeodorum, rufum

Basidiomycotina Cortinariaceae

Hymenogaster

bulliardii, calosporus, citrinus, decorus, HIacmus, luteus,

Hysterangiaceae

Hysterangium

membrar)aceum

Leucogastraceae

Leucogaster

r)udus

Melanogastraceae

Melanogaster

ambiguus, broomeiarius, euryspermus, intermedius,

Russulaceae

Basmomyces

mattiroHar)us

Zelleromyces

stephensii

olivaceus, populetorum, tener, vulgaris

tuberiformis, variegatus

Sclerodermataceae

Scleroderma

hypogaeum

Strobi lomycetaceae

Gautieria

graveolens, mexicana, otthii

Endogorie

lactiflua

Zygomycotina Endogonaceae

Data modified from Molina et al. (1992). * Taxonomic status uncertain.

tetrapolar species in which there are two unlinked mating-type loci, A and B, again with multiple alleles. In the tetrapolar system, homokaryons are compatible with each other when they have different alleles at both mating-type loci. Mycorrhizal basidiomycetes are much harder to work with than the saprophytes that have been used to reveal these genetic systems. However, mating types of the bipolar kind have been identified in three Suillus species (Fries and Neumann, 1990; Fries and Sun, 1992), while H. cylindrosporum (Debaud et al., 1986) and Pisolithus tinctorius (Lamhamedi et

174

Ectomycorrhizas

Mycorrhiza

2

P. tinctorius Roots

(a)

^

Figure 6.3 (a) Cell wall polypeptides in ectomycorrhizas formed between Eucalyptus globulus and Pisolthus tinaorius 441 and in non-colonized Eucalyptus roots and cultured Pisolithus tinaorius 441. Densitograms of l-D SDS-PAGE from cell wall proteins (CWP). Note the increased accumulation of a fungal band (32-kDa CWP) in mycorrhizas (arrowed). Data of De Carvalho and Martin, unpublished. From Martin and Tagu (1995), with permission, (b) Changes in gene expression during the formation of ectomycorrhizas between Eucalyptus globulus and Pisolithus tinctorius 441. Free-living mycelium ( ^ ) and ectomycorrhizas ( • ) . From Tagu et al. (1993), with permission.

al, 1990) are of the tetrapolar type. Pairings between monokaryons of H. cylindrosporum that were derived from the progeny of six wild dikaryotic strains of disjunct geographical distribution, have demonstrated the occurrence of multiple alleles at the A and B mating-type loci in this species (Debaud et al, 1986). Within the genus Laccaria a complex pattern of mating systems is revealed. The most important ectomycorrhizal species L. amethystea, L. hicolor, L. laccata and L. proxima have a tetrapolar mating system (Fries and Mueller, 1984), but one in which all four mating types are rarely found in the progeny of a dikaryon. Doudrick et al (1990) showed the presence of a large number of alleles in L. laccata var. moellerl By pairing isolates obtained from different regions of North America, they estimated the outbreeding efficiency of this system to be 88%. However, there is also evidence that in L. laccata, genes other than those determining mating types can restrict pairings between homokaryons from morphologically similar strains. Thus Fries (1983) found two incompatible groups of the species in a restricted area of Sweden, and Mueller (1991) detected three such groups in North America that were also incompatible with the isolates of Fries. Laccaria bicolor also shows evidence of incompatibility groups (Kropp and Fortin, 1988; Doudrick and Anderson, 1989). Such analyses highlight the inadequacy of our understanding of the 'species' as a unit, and emphasize the need to characterize and describe the origin of isolates used in any experimental study Intraspecific genetic variation can be expressed at the physiological level in the form of differences in growth, production of enzymes or of auxins (Gay and Debaud, 1987), or at the level of mycorrhizal infectivity or aggressiveness (Wong

Structure and development of ectomycorrhizal roots

• u

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and Fortin, 1990; Burgess et al, 1994, 1995). Gay and Debaud (1987) measured production of indole acetic acid (lAA) by different species of Hebeloma and compared the observed rates with those of wild strains, of monokaryons produced by germination of spores from a single fruit body (sib-monokaryons) and of dikaryons synthesized from the monokaryons of H. cylindrosporum (Fig. 6.4). Variation of auxin production was as large in the intraspecific strains as between species. Analyses of differences in growth and of enzymes such as glutamate dehydrogenase (Wagner et a/., 1988), nitrate reductase (Wagner et al, 1989) and acid phosphatase (Meysselle et al, 1991) reveal relatively broad variability (Table 6.3). Similar differences in expression of the last enzyme have been observed in progenies of controlled dikaryons of L. laccata (Kropp, 1990).

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177

Structure and development of ectomycorrhizal roots

Table 6.4 Estimates of the components of the variance recorded in 50 synthesised dikaryons, which were progeny of the Hebeloma cylindrosporum HCI strain Item

Genetic variance

Compatibility

Phenotypic

Environmental

group

variance

variance

Growth in the presence of ammonium (mg protein per culture)

AIB2 X A2BI

100

22.6

33.4

44.0

AIBI X A2B2

100

15.5

51.5

32.9

GDH activity (nkat mg~' protein)

AIB2 X A2BI AIBI X A2B2

100 100

14.1 6.0

42.7 40.0

43.0 53.9

Growth in the presence of nitrate (mg protein per culture)

AIB2 X A2BI AIBI X A2B2

100 100

6.9 8.3

79.0 7.2

14.1 19.5

Nitrate reductase activity (nmol NO2 synthesized mg~' protein h~')

AIB2 X A2BI AIBI X A2B2

100 100

13.3

85.6

1.0

—

—

—

lAA synthesizing activity (nmol lAA mg~' protein h~')

AIB2 X A2BI AIBI X A2B2

100 100

14.3 57.8

43.3 26.7

42.4 15.3

Interactive Parental variance variance

GDH, glutamate dehydrogenase; lAA, indole-3-acetic acid. * Expressed as a percentage of the phenotypic variance. Data from Gay and Debaud, 1987; Wagner et al., 1988, 1989; Gay et 0/., 1993; Debaud et 0/., 1995.

Debaud et al. (1995) calculate that up to 36% of the progeny of a dikaryon are more efficient in terms of enzyme production than the parents, and point out the obvious potential which this presents for designing programmes to select improved mycelia. The problem faced by any such programme is that phenotypic expression is strongly influenced by environmental conditions. Thus measurements of growth and of enzyme or lAA production in monokaryons and wild or synthesized dikaryons of H. cylindrosporum (Table 6.3) reveal that the expression of genetic diversity is strongly influenced by nutrition. Such studies demonstrate that genotypes selected for performance under one set of conditions will not necessarily be useful in others. Variation in growth was generally less than in enzyme expression probably because the former process is controlled by more genes. The interactive nature of gene expression in the control of growth leads to generation of phenotypes in which performance is ultimately less distinctive than is predicted from initial screening of progeny. Biometrical approaches (Table 6.4) enable calculation of the percentage of variance in synthesized dicaryons that is attributable to genetic (parental or interactive) and environmental effects. The relative magnitude of the latter depends upon the parameter being measured but is an important influence upon growth, GDH, NR and lAA production. Some insights into the processes of genetic control of mycorrhiza development have been obtained by challenging potential host plants with fungal strains of known genotype. Lamhamedi et al. (1990) examined the ability of 28 sib-monokaryotic and

178

Ectomycorrhizas

78 reconstituted dikaryotic strains of P. tinctorius to form mycorrhizas with Pinus. While a few of the monokaryons showed limited ability to colonize roots, fully developed mycorrhizas and growth promotion of the plant were obtained only with dikaryons. The ability to produce rhizomorphs was also restricted to dikaryotic strains (Lamhamedi and Fortin, 1991). Strong influences of the environment upon the expression of genetic potential were indicated by the observation that development of rhizomorphs by a given dikaryotic strain was far more extensive over soil than over cellulose sheets. Here, it is interesting to speculate that the expression of hydrophobin genes may be involved. These have been detected in the population of symbiosis related (SR) proteins in Eucalyptus mycorrhizas (Martin and Tagu, 1995). Hydrophobins are also implicated in formation of hyphal aggregates and fruit bodies of the wood-decay fungus Schizophyllum commune and in that fungus some of the genes are only expressed by the dikaryons (Wessels, 1992). Using sib-selected homokaryotic cultures of L. hicolor, Kropp et al. (1987) demonstrated that monokaryotic strains could form mycorrhizas, at least under laboratory conditions. While some of the monokaryons were strongly mycorrhizal, others quickly lost the ability to colonize roots of Pinus banksiana and in some cases no colonization took place. Wong et al. (1989), also using strains of L. hicolor, showed that a given genotype could proceed only to a particular stage of mycorrhiza development, arrest being seen at defined points in the differentiation processes. Some strains did not respond to the presence of the root, others produced only a surface weft of hyphae, while others produced a Hartig net but no mantle. The strains could also be distinguished by the rate of development of these structures and the morphology of the fungus in the Hartig net (Wong et al., 1990). Observations of this kind suggest that each stage of the processes of mycorrhiza development is under separate genetic control, with completion of the sequence being dependent upon a hierarchy of gene expression in both organisms. The differences within fungal species in their ability to express physiological or morphogenetic attributes can be ascribed to genetic differences, most of which are features of DNA sequences of the nuclear genome. The use of variants should permit the description of colonization as a sequence of well defined steps which can be reasonably presumed to be under the influence of particular fungal genes. The studies of Wong et al. (1990) and Burgess et al. (1994, 1995) provide excellent starting points for analysis in Pinus and Eucalyptus, respectively. Further progress in understanding the control of these processes will be dependent upon isolating and cloning the genes coding for each attribute.

Studies of Somatic Incompatibility In the heterokaryotic phase, basidiomycete mycelia that have originated from the same genet are able to fuse, while those that are not genetically identical reject each other, thereby maintaining their genetic integrity. In those fungi that can be grown in culture, this rejection system, referred to as somatic or vegetative incompatibility, is readily seen as a zone of growth inhibition separating two isolates as they approach each other. By recording the presence or absence of rejection in pairings of isolates obtained from different fruit bodies of Suillus bovinus in Swedish forest of Pinus sylvestris, Dahlberg and Stenlid (1990) were able to determine the distribution of individual genets within populations. Iso-

Structure and development of ectomycorrhizal roots

179

lates were obtained from fruit bodies in stands of different ages and it was possible to determine relationships between the numbers of genets occurring, the area of land covered by each, and the age of the trees with which they were associated. The area occupied by any single genet and the numbers of genets per unit area both decreased with increasing age of the trees. In stands of 10-20 years old, there were 700-900 genets ha~^, and the distance between the outermost compatible fruit body of each genet was 4-5 m. In stands of 100 years old, there were only 30-100 genets ha~^, the extent of each genet being of the order of 14-20 m. Genets of such a size encompass a number of trees, all of which, therefore, are likely to be interlinked by the same genetically distinct mycelium. The significance of interlinking is further discussed in Chapter 15. The study by Dahlberg and Stenlid (1990) indicates that over time, selection operates in nature to favour some genets at the expense of others. It will be important to understand the mechanistic basis of this selection. It might operate at the level of compatibility with the plant, with the soil or in terms of the ability of one genet to compete with others of the same or different fungal species. None of these possibilities is mutually exclusive. Using the same tests Sen (1990) confirmed the occurrence of distinct genotypes within Suillus spp, but analysis of their isozymes showed considerable intraspecific similarities, suggesting their functional differences between clones may be small. Fries (1987) has shown variation between ectomycorrhizal fungi in the extent to which they demonstrate somatic incompatibility. Paxillus involutus, Pisolithus tinctorius and Thelephora terrestris do not show the phenomenon, Suillus luteus and S. variegatus show it to varying extents, whereas in Amanita muscaria, Hebeloma mesophaeum and Laccaria proxima heterokaryons from different genets are routinely incompatible.

The Formation of Ectomycorrhizas The early stages of development of the ectomycorrhiza will obviously be influenced by the source of the inoculum. Two distinct patterns can be envisaged. In nature on established trees, first, second and further orders of lateral roots, all having restricted potential for extensive growth, will be produced, usually seasonally, from the axis of the long roots of unlimited growth. These laterals will become colonized either from where the Hartig net is developed on the long root, as in the case of Pinus spp. (Robertson, 1954; Wilcox, 1968a,b), or from the inner mantle of the parent root, as in the case of Eucalyptus (Massicotte et al., 1987b). In both of these circumstances the colonizing fungus will be the same as that forming mycorrhizas on the parent root. In contrast, where the lateral root emerges through a portion of the uncolonized long root, or where a seed germinates to produce a completely new root system, the potential arises for colonization by different fungi from propagules in the soil. In this case the processes of mycorrhiza formation are determined by phenomena such as recognition, compatibility and inoculum potential. The diversity of mycorrliizal types and fungal species observed in nature on a single tree reflects the complexity of temporospatial events determining the colonization process. In order to elucidate such complex events most research to date has employed

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simplified systems in which plants, usually seedlings, are challenged by a single fungal species in monoxenic culture. By sequential analysis in such systems some of the major events in development of the symbiosis from approach to contact and differentiation have been evaluated. Precolonization Events It is well known that plant roots release compounds into their immediate environment and the importance of such exudates, mainly as nutrients for the general microbial population, is evidenced by enhancement of this community in the rhizosphere. The challenge remains to determine which, if any, of the array of compounds so far identified is sufficiently specific in its effects to exert selective impacts and so enable approach of potentially intimate partners. That specific root exudates may be involved in the ectomycorrhizal situation is suggested by experiments using compatible and incompatible isolates of Pisolithus tinctorius and Paxillus involutus (Horan and Chilvers, 1990). By interposing a permeable membrane between plant and putative symbiont they showed that hyphae of compatible symbionts were attracted towards the membrane while those of the incompatible species were not. The likelihood is that specific signal molecules are in very much lower concentration than those which exert nutritional effects. There are parallels in the flavonol compounds involved in signalling in Agrobacterium and Rhizobium interactions with plants (Peters and Verma, 1990). Debate continues on the chemical nature of such attractants. It is known that hormones including cytokinins (Gogala, 1991) and lAA (Gay and Debaud, 1987) can influence hyphal branching and growth, but again there is little evidence for specificity. However, exudates of plant roots can stimulate germination of spores of ectomycorrhizal species (Fries, 1987; Ali and Jackson, 1988) but often such effects can be obtained using roots of non-hosts (Wong and Fortin, 1990). The possible importance of volatile compounds as triggers has been stressed (Koske and Gemma, 1992). Albrecht et al. (1994) have gone so far as to suggest that peroxidases induced in the root on contact with ectomycorrhizal fungi could be involved in triggering developmental processes in mycorrhiza formation. The demonstration (Garbaye, 1994) that the rate and extent of mycorrhiza formation can be enhanced under some circumstances by the presence of fluorescent pseudomonads, so-called 'mycorrhiza helper bacteria' (see Chapter 15), indicates the possibility that trigger compounds are released by organisms other than the mycorrhizal fungus itself. Contact between Fungus and Root Molecular Events New techniques in which seedlings are placed in contact with a developed mycelial inoculum and then harvested sequentially in the hours or days following contact and formation of ectomycorrhizas, have enabled elucidation of the responses of the partners at the molecular level. The symbiosis between Eucalyptus (E. globulus and £. grandis) and Pisolithus tinctorius has proved to be particu-

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F i g u r e 6.5 Synthesis of mycorrhizas on Eucalyptus by Pisolithus tinctohus, using the cellophane-over-agar technique. Bar, 2 cm. See Table 6.5. Photograph courtesy of B. Dell.

T a b l e 6.5 Sequence of the formation of Eucalyptus ectomycorrhiza obtained using the cellophane-over-agar technique (see Fig. 6.5) Time

Developmental stage

Anatomical features

0-12 h l2-24h 24-48 h

Preinfection Symbiotic initiation Fungal colonization

48-96 h

Symbiotic differentiation

96 h-7 days

Symbiotic function

Hyphal contacts with the root Fungal attachment to the epidermis Initial layers of mantle Hyphal penetration between epidermal cells Rapid build-up of mantle hyphae Hartig net proliferation Mantle well developed and tightly appressed to epidermal cells End of Hartig net growth

Data from Malajczuk et al. (1990).

larly amenable to experimental manipulation. The paper-sandwich method of Horan et al (1988) enables synchronous colonization of lateral roots over a period of days and the cellophane-over-agar method (Fig. 6.5) of Malajczuk et al (1990) enables the same sequence of events to be completed on the primary root within hours (Table 6.5). Establishment of the symbiosis must be under the control of genes of both partners and changes of gene expression, revealed in the form of changes of protein biosynthesis and mRNA populations, have provided valuabje insights into the events occurring at the subcellular level. Hilbert and Martin (1988) established the E. globulus-P. tinctorius association in paper sandwiches and used twodimensional polyacrylamide gel electrophoresis (2-D PAGE) to compare the protein profiles of mycorrhizal roots harvested over several weeks of development, with

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those of uncolonized roots and of free-living mycelium. In the established mycorrhizas they observed a large decrease (down-regulation) of those polypeptides present in the free-living partners and of a total of 520 polypeptides found, only 10 were unique to the symbiotic condition. These symbiosis related (SR) proteins were termed 'ectomycorrhizins'. By harvesting the primary roots of seedlings grown in the colonized and uncolonized condition, Hilbert et al. (1991) subsequently showed that major changes of polypeptide synthesis occurred within hours of colonization by the fungus. They detected the accumulation of seven ectomycorrhizins during the early stages of synthesis. This synthesis was accompanied by a marked decrease of several plant and fungal polypeptides, the loss being referred to as 'polypeptide cleansing'. Studies of the pattern of incorporation of labelled ^^S-methionine into protein (Hilbert et al. 1991) showed that whereas there was extensive down-regulation of polypeptides resulting from inhibition of synthesis, intense labelling of ectomycorrhizins occurred. This is indicative of acceleration of current biosynthesis of SR proteins during symbiotic development. Such changes were observed only when roots were challenged by compatible races of P. tinctorius. Subsequently, Burgess et al (1993, 1994, 1995) investigated the extent of specificity and aspects of the possible function of ectomycorrhizin production by screening the impacts of different isolates of P. tinctorius upon protein synthesis in another eucalypt mycorrhiza, that of E. grandis. Three isolates were chosen: one very aggressive, one moderately so, and another incapable of forming mycorrhizas. Only during colonization by the most aggressive isolate, which was also shown to enhance seedling growth, was there a dramatic up-regulation of biosynthesis of fungal proteins and this occurred within four days of contact. During mycorrhizal development there was a marked inhibition of polypeptide synthesis by the plant, accompanied by enhanced accumulation of some fungal polypeptides as well as appearance of SR proteins. The most important changes were in a group of fungal, acidic polypeptides which increased as the symbiosis became established up to four days after contact. The effects were most marked in the presence of the highly aggressive isolate, H2144, and were also observed with the moderately aggressive one, H441. A strongly up-regulated fungal polypeptide with an apparent molecular mass of 32 kDa accumulated in the fungal walls (32 kDa CWP; see Fig. 6.3a) and was apparently composed of isoforms of an acidic polypeptide (AP 32), accumulating during the early stages of colonization (De Carvalho and Martin, in Martin and Tagu, 1995). Changes in polypeptide profiles have also been observed in mycorrhizas formed between Betula pendula and Paxillus involutus (Simoneau et ah, 1993) and between Finns resinosa and Paxillus involutus (Duchesne et al., 1989), but in Picea abies-Amanita muscaria mycorrhizas no ectomycorrhizins were detectable, although there were changes in protein concentration (Guttenberger and Hampp, 1992; Guttenberger, 1995). At present, the relative importance of different plant-fungus combinations or of methodological differences in explaining the discrepancies is a matter for debate. The enhanced production and localization of the specific SR protein as an insoluble component of the cell wall indicates the possibility that it is involved in recognition or adhesion and its change from the soluble state in submerged conditions to insoluble in the mycorrhizal wall is suggestive of a hydrophobin-like molecule. It is known that the extracellular matrix in which the hyphae of the

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183

mantle are embedded has hydrophobic qualities and the recent cloning of a cDNA that has sequence homology to hydrophobin genes from Eucalyptus mycorrhizas (Martin and Tagu, 1995) lends support to the hypothesis that some SR proteins may act as hydrophobins. Hydrophobins are cysteine-rich polypeptides which, in Schizophyllum commune, are secreted to the culture medium but later become polymerized into the walls of hyphae emerging into the air, covering them with a waterproof coat (Wessels, 1992, 1994a,b; Wosten et al, 1993, 1994). It has been suggested that the deposition of water-repellant proteins in the walls of hyphae behind the growing apices will restrict nutrient absorption or loss to the apical region (Rayner et al, 1994, 1995). Hydrophobins have also been strongly implicated in hyphal recognition, attachment to insect cuticle and in the walls of dikaryotic mycelium forming fruit bodies (St Leger et al, 1992; Talbot et al, 1993). Some of the Sc genes coding for hydrophobin-like proteins are, in S. commune, fruit body specific, appearing only in dikaryotic mycelium. The properties and expression of hydrophobins provide important pointers to future work on their roles in mycorrhizas. While studies of polypeptide profiles indicate that a major reprogramming of protein synthesis by both symbionts takes place during early stages of mycorrhiza synthesis, there has as yet been very little progress towards understanding the function of ectomycorrhizins. A complementary molecular approach is to identify fungal genes that are changed in expression in a more direct way. This involves sorting out which mRNAs are differentially accumulated at different stages of the colonization process. As discussed in Chapter 3, the methods have considerable potential for investigation of genes involved in development. The targeted approach requires some knowledge, or at least educated guesses, of the expected changes in function. So far, probing ectomycorrhizal material with specific ribosomal sequences from Pisolithus tinctorius (ITS spacers of the ribosomal genes) has indicated very high ribosomal activity in the hyphae of the fungal sheath (Martin and Tagu, 1995). Probing with hydrophobin genes, or genes for specific membrane transport proteins or host defence responses would be likely to prove valuable, especially using plant-fungus associations with different levels of compatibility. Where there is no information on the function of the genes involved, a nontargeted approach to identify changes in mRNA abundance followed by sequencing of the clones of interest should reveal novel genes involved in the symbiosis. Considerable change in the relative accumulation of fungal mRNAs has been revealed (see Fig. 6.3b). As the cDNAs are sequenced and also used for in situ hybridization, much more functional and temporospatial information on the gene expression can be expected (Tagu et al, 1993; Martin and Tagu, 1995). At present, the challenge is to establish functional links between gene expression and the key events of recognition and mycorrhiza synthesis. Events at the Whole-Root Level

Root hairs proliferate behind the apices of growing uncolonized roots and provide a large surface area for potential contact with any ectomycorrhizal mycelium in surrounding soil. It appears that on making contact with root hairs, ectomycorrhizal hyphae can alter their orientation of growth to the surface of the root and partially envelope the hairs (Massicotte et al, 1989a; Thomson et al, 1989). When

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hyphae first make contact with the root surface they may show morphological changes prior to the production of mantle or Hartig net; these include increased branching and fusion of hyphal tips (Jacobs et al, 1989). The mechanisms which enable attachment of compatible hyphae to the roots and provide discrimination against the incompatible, remain unclear. That they are likely to be subtle is indicated by the marked differences in compatibility revealed when hosts are challenged with different isolates of ectomycorrhizal fungus (Malajczuk et ah, 1990; Lei et al., 1991; Burgess et al, 1994). There is evidence that among the plant defence processes increased deposition of phenolic materials at the point of contact with incompatible species or strains may be involved (Ling-Lee et al., 1975; Malajczuk et al, 1984; Horan et al, 1988). Campbell and Ellis (1992a,b) observed an increase in lignification of the walls of cultured cells of Pinus when they were exposed to extracts of Thelephora terrestris, a feature which was associated with enhanced polypropanoid metabolism. However, Miinzenberger et al (1990) showed that soluble phenolics decreased in roots of Picea abies colonized by Amanita muscaria. Clearly, change of phenolic metabolism are likely to be only one of many interacting effects, the most critical of which will be occurring at the functional level. Albrecht et al (1994) found that the induction of chitinases and peroxidases in Eucalyptus was related to the aggressiveness of the fungal strain, with only good colonizers inducing a strong response. This calls into question the widely accepted roles of these enzymes in plant defence. The involvement of a lectin-carbohydrate recognition system has been demonstrated in the formation of a number of biotrophic plant-fungus associations (Mengden et al, 1988), and plant pathogens have been shown to secrete glycoproteins to facilitate adhesion to lectins on plant cell wall surfaces (Anderson, 1992; Xiao et al, 1994). Ultrastructural studies of the plant-fungus interface of ectomycorrhizas have now demonstrated the presence of fibrillar material, probably made up of glycoproteins (Lei et al, 1990a,b), extending from the fungal wall towards that of the plant (Piche et al, 1983; Lei et al, 1990a,b,c). A layer of extracellular fibrillar polymers is present on the surfaces of free-living mycelium of Laccaria bicolor and Pisolithus tinctorius (Lei et al, 1990a,b,c) before contact with a root and it seems likely that reorientation of these towards the surface of the plant cell is one of the important initial steps in mycorrhiza formation. Giollant et al (1993) detected binding sites on the root surface of spruce (Picea) for lectins isolated from the hyphal walls of Lactarius deterrimus. These were different from those of L. deliciosus in symbiosis with Pinus. Recently the possibility has emerged that receptor sites are present on both partners in the symbiosis, but that they are masked by unreactive materials. Lapeyrie and Mengden (1993) found that in the case of the fungal wall these compounds could be removed by the enzymes laminarase or protease, raising the possibility that recognition was through a programmed process of enzyme release, leading to unmasking of the critical receptor sites on the contiguous surfaces of both partners and establishment of the symbiosis. A possible role for unmasking processes was suggested in the RhizobiumAegume symbiosis by Solheim and Fjellheim (1984). Now it seems that proteins released by Rhizobium, following activation of nodulation genes by exposure to flavonols, may be involved in the unmasking of specific root hair receptors in legumes (Krishnan and Pueppke, 1993). In view of such observations it might be rewarding to carry

Structure and development of ectomycorrhizal roots

185

out integrated studies of the interaction between flavonol-type compounds as possible triggers and hydrolytic enzymes as unmasking agents, in the course of induction of the ecton\ycorrhizal symbiosis.

Development of Ectomycorrhizas Use of the pouch system of Fortin et ah (1980) has enabled detailed studies of the development of mycorrhizas formed by symbionts with roots of known age. Amongst the plant-fungus combinations studied are those between Alnus crista and Alpova diplophoeus (Massicotte et al, 1986, 1989a,b), Eucalyptus pilularis and Pisolithus tinctorius (Massicotte et al, 1987b,c) and Betula alleghaniensis and Pisolithus tinctorius (Massicotte et al, 1990). In the case of the A. crispa-A. diplophoeus association, colonization of first-order laterals by rhizomorphs leads to mycorrhiza formation within 2-4 days of contact. Fungal hyphae first contact the growing root at a position immediately proximal to the root cap, from which point they grow both basipetally and acropetally to produce a covering of hyphae that keeps up with root elongation. This stage is reached within 24-48 hours of initial colonization and concomitantly a swelling of the root appears proximal to the root cap. The final morphology of each mycorrhiza is dependent upon the stage of lateral root outgrowth at which colonization is initiated. The swollen tip is a feature of a mycorrhiza formed after a lateral has elongated, whereas colonization at an early stage of outgrowth results in a mycorrhiza of uniform thickness. Longitudinal sections reveal that hyphae of the inner mantle start to penetrate between cells of the root cap immediately behind the apex, and within a very short distance proximal to this, penetrate between epidermal cells to form the Hartig net. The Hartig net develops in an acropetal direction but never penetrates beyond the outer tangential wall of the first layer of cortical cells. The epidermal cells enveloped by hyphae show only slight radial elongation. A combination of light and electron microscopy has elicited a schematic composite drawing of the AlnusAlpova mycorrhiza (Fig. 6.6) which reveals the progressive development as the mycorrhiza matures behind the apex. The diagram indicates the proliferation of the hyphal walls of the Hartig net produced by repeated branching in the intercellular position (Fig. 6.6c,d), the production, typical of this particular association, of ingrowths on the epidermal cell walls (Fig. 6.6d), and the concentration of rough endoplasmic reticulum and mitochondria in fungal and epidermal cells (Fig. 6.6e), the latter becoming progressively more vacuolate as they mature (Fig 6.6e). The development of the Eucalyptus-Pisolithus mycorrhizas (Massicotte et al, 1987b,c) follows a broadly similar pattern to that described in the case of Alnus. As in that plant, the final morphology of the individual ectomycorrhiza depends on the stage of lateral root elongation at the time of colonization. Some differences are, however, observed. In Eucalyptus there is a well defined zone behind the apex in which Hartig net formation is lacking - the so-called apposition or pre-Hartig net zone (Fig. 6.7). The epidermal cells of Eucalyptus show a rapid response to the presence of ectomycorrhizal fungi in the form of considerable radial enlargement which is not accompanied by normal apical elongation. Such changes of epidermal cell development are thought to be specifically induced by fungal colonization

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Ectomycorrhizas

100/xm Figure 6.6 Schematic drawings of an ectomycorrhiza formed between AInus crispa and Alpova diplophloeus. (a) Three-dimensional drawing of a mycorrhizal root, showing the mantle (Ma), paraepidemal Hartig net (HN) and epidermal cells (E), which become progressively more radially enlarged from the apex back, (b) Root cap region. Hyphae (Hy) have begun to penetrate between the cells of the root cap (RC). The densely cytoplasmic epidermal cell (E) is also shown, (c) Hartig net region. Fungal hyphae (Hy) with rough endoplasmic reticulum and labyrinthine wall branching are penetrating between the epidermal cells (E). (d) Mature Hartig net region. Fungal hyphae (Hy) have reached as far as the modified wall of the hypodermis (*). Epidermal cells (E) show wall modifications, including ingrowths, (e) Older Hartig net region. Fungal hyphae (Hy) show reduced numbers of cysternae of endoplasmic reticulum and mitochondria. Epidermal cells (E) show modifications including deposition of wall material, vacuolation and a decrease in number of mitochondria. The hypodermal cell (*) is also more highly vacuolated. From Massicotte et al. (1986), with permission.

Structure and development of ectomycorrhizal roots

^

187

.c.

Figure 6.7 Light microscopy of mycorrhiza formed between Eucalyptus pilularis and Pisolithus tinctorius. Longitudinal section showing zonation. Zone A, root cap-meristem; zone B, apposition (pre-Hartig net); zone C, young Hartig net; zone D, older Hartig net. Bar, 100 |Lim. From Massicotte et al. (1987c), with permission.

since, as shown previously by Chilvers and Pryor (1965), they are not seen in nonmycorrhizal roots. A further feature which appears to distinguish the £. pilularis-P. tinctorius association is that the plant-fungus interface in the Hartig net is relatively simple. There are no fungus-induced wall ingrowths in the root cells involved in forming the interface and only a small number of labyrinthine branches in the fungal tissue. Compensation for simplicity in the interface at the cellular level may be obtained by the precocious development of lateral roots to form extensive clusters and tubercles (see below) which would have the effect of increasing the total surface area of interface per root system (Chilvers and Gust 1982; Dell et al, 1990). Longitudinal sections of Eucalyptus roots viewed under the light microscope (Massicotte et al. 1987b,c) reveal that there are inherent anatomical differences between primary roots and first-order laterals even in the absence of mycorrhizal colonization. The primary roots are pointed (Fig. 6.8a) and have a complex organization with a root cap consisting of a distinct columella, an extensive meristem, a cortex of three or four layers, a differentiated stele and a single-layered epidermis. In contrast, first-order laterals (Fig. 6.8b) have a rounder, blunt apex, a reduced apical meristem, a subapical construction, three or four layers of cortical cells and a poorly developed stele. Cortical cells and tracheary elements are seen to mature closer to the apex in these laterals than in the primary root (Fig. 6.8a). The distinctive structure of the two types of root is a basic feature of heterorhizic systems and has been described also in Fagus (Clowes, 1951; Warren et al., 1983) and in Pinus (Hatch and Doak, 1933; Wilcox, 1964,1968a). The important functional difference between the two types of structure is that while the primary root has the capacity for continuous growth, the laterals have little such ability and may abort if not colonized by a mycorrhizal fungus. In the case of Eucalyptus seedlings growing in pouches with inoculum of P.

Ectomycorrhizas

188

50 /im

Figure 6.8 Longitudinal sections of non-mycorrhizal roots of Eucalyptus pilularis. (a) Primary root, showing well-developed root cap and apical meristem (AM), giving rise to well defined tissue layers, (b) A young lateral root, showing limited root cap, reduced apical meristem and subapical constriction (arrowed). C, cortex; E, epidermis; VC, vascular cylinder. Bars, 50 |Lim. From Massicotte et al. (1987b), with permission.

Structure and development of ectomycorrhizal roots

189

Figure 6.9 Sections of mycorrhizal first-order lateral roots of Eucalyptus pilularis (a) Longitudinal section showing lateral root primordia (arrowheads). Surface hyphae are present, but hyphal penetration between the cortical cells has only just begun (arrow) (b) Transverse section, showing a young lateral root primordium (arrowheads). Hartig net (arrowed) is completely formed and the hypodermis (H) is collapsing, (c) Transverse section showing growth of the lateral primordium (arrowheads) up to the hypodermis. (d) Lateral prinrjordium haS completely penetrated the cortex and pushed out the epidermis Wall thickenings m the cortex are becoming evident (arrowheads), (e) Higher magnification of the lateral root primordium shown in (d) surrounded by collapsed cells (arrowheads) and showing initiation of root cap (arrows). From Massicotte et al. (1987b). with permission

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Ectomycorrhizas

tinctorius, first-order laterals become colonized by hyphae or rhizomorphs extending from the plug of inoculum. A thin mantle is formed over the lateral, sometimes within 12-24 hours of contact. In longitudinal sections of such a root, four zones each representing a distinct type of colonization, can be recognized (zones A, B, C, D, Fig. 6.7). In zone A, a thin but continuous sheath is seen to form over the blunt meristematic area. In zone B the hyphae do not penetrate between the cortical cells while in contact with the surface; this is referred to as the apposition or pre-Hartig net zone. In zone C the Hartig net begins to form and the cells of the epidermis begin to show radial extension. In the mature Hartig net zone, D, the epidermal cells are fully extended in a radial direction and all are surrounded by the net. Second-order root primordia appear in the pericycle of first-order roots very close to the meristem (Fig. 6.9a). Here the endodermis may already have differentiated, and de-differentiation occurs at the sites of lateral root initiation (Fig. 6.9a). At this level, hyphal penetration between cells of the epidermis of the parent long root is commencing (Fig. 6.9a,b). Transverse sections taken proximally to this (Fig. 6.9b,c,d,e) show stages of emergence of the second-order laterals into the cortex of the parent root and eventually into the epidermis which is pushed out (Fig. 6.9d). At higher magnification (Fig. 6.9e), early stages of differentiation of a root cap are seen around the meristem of the primordium but there is still no sign of fungal penetration of its tissues. Soon after this an apical meristem and vascular cylinder are seen (Fig. 6.10a) and longitudinal sections (Fig. 6.10b,c,d) show proliferation of hyphae over the apex of the emerging root, radial extension of epidermal cells and stages in the formation of the Hartig net. Transmission electron microscopy (TEM) enables the interaction between fungus and plant to be visualized in each of the four zones shown in Fig. 6.7 (Massicotte et al, 1987c). In zone A (Fig. 6.11a) a thick mantle made up of densely cytoplasmic hyphae is seen and some of the hyphae have penetrated between the root cap cells despite the presence, between the cap and the mantle, of an elaborate electrondense layer. In the young Hartig net zone (C) densely cytoplasmic hyphae penetrate between vacuolate epidermal cells up to the hypodermis (Fig. 6.11b). At higher magnification the epidermal cells can be seen to contain a diffuse electron-dense matrix or peripheral deposits of electron-dense materials (Fig. 6.10c). Hyphae of the Hartig net are densely cytoplasmic (Fig. 6.lid) and contain numerous lipid droplets as well as mitochondria. The walls of the epidermal cells do not produce ingrowths (Fig. 6.11d) in this association, which thus appears to be of a simpler type than that seen in Alnus, Pisonia (AUaway et al, 1985) or Pinus (Duddridge and Read, 1984a,b). In zone C contiguous walls between epidermal and hypodermal cells show suberin lamellae in the hypodermal cell walls (Fig. 6.11e) and plasmodesmatal connections between the cells. TEM micrographs of the mature Hartig net zone (Fig. 6.12a,b,c) show that penetration by now vacuolate Pisolithus hyphae between the epidermal cells is far more extensive. They still, however, do not pass into the hypodermis. There are extensive formations of suberin lamellae in the hypodermal cell wall (Fig. 6.12b). In some cases hyphae that have reached the hypodermis appear to disrupt the middle lamellae (Fig 6.12c). When Betula alleghaniensis was challenged by the same strain of P. tinctorius in pouches (Massicotte et al., 1990), mycorrhizas were formed on first-order laterals within 4-10 days. Prior to sheath formation preferential growth of hyphae is

Structure and development of ectomycorrhizal roots

Figure 6.10 Development of lateral root primordia in Eucalyptus pilularis. (a) Transverse section of a primordium (*) protruding into the mantle (M). There is no hyphal penetration of tissues. Vascular cylinder (VC). (b) Longitudinal section of primary root (PR) and primordium (*). Aggregations of hyphae at either side of the primordium (arrowheads) and radial enlargement of epidermal cells (E) are evident, (c) Uter stage In v/hich hyphal penetration between epidermal cells of the young lateral is evident (arrowheads) (d) Longitudinal section of a fully emerged lateral showing paraepidermal Hartig net (double arrowheads) and mantle (M). From Massicotte et al. (1987b), with permission

observed between root hair papillae in the subapical region of the root (Fig. 6.13a) Ihe mantle becomes progressively thicker (Fig. 6.13b) until eventually root hairs are no longer visible (Fig. 6.13c). A pre-Hartig net zone is again detectable and where this Hartig net is formed, it is, like that of Alnus, of the para-epidermal kind A feature of this mycorrhizal association is that while there is no ingrowth of the epidermal ceU walls adjacent to the Hartig net, the fungus branches prolificaUy to torm a very complex system of labyrinthine growths in which the spaces between the hingal branches are barely wide enough to accommodate the elongated mitochondria. °

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The Structure of the Mature Hartig Net Relationships between Plant and Fungus at the Tissue Level The zone of contact between the symbionts is of key importance in mycorrhizal root function. In this zone the Hartig net is produced by hyphae that penetrate between the outer cells of the root axis. This penetration is normally from the inner mantle (see Figs 6.6, 6.7, 6.9) but can occasionally, for example in the case of Picea abies (Nylund and Unestam, 1982), occur as soon as hyphae reach the root surface and hence before the mantle is formed. The depth of this penetration differs in angiosperms and gymnosperms. In the majority of angiosperms penetration is confined to the epidermal layer, so forming what is referred to as an 'epidermal' Hartig net (Godbout and Fortin, 1983). Examples of this type are seen in Eucalyptus (Figs 6.7, 6.12), Alnus (Fig. 6.6) and Betula (Fig. 6.14). Within the epidermal type two variants are recognized: the so-called 'para-epidermal' type, in which there is a partial encircling of the epidermal cell as described earlier in Alnus; and the 'periepidermal' structure, in which hyphae encircle the whole cell (Godbout and Fortin, 1983). Of the two types, the former appears to be most common, although Godbout and Fortin (1985) report that in Populus tremuloides the peri-epidermal state develops from the para-epidermal under certain culture conditions, indicating that there is possibly a continuum in which the final state is determined by age or environmental conditions. In ectomycorrhizal gymnosperms the Hartig net typically penetrates beyond the epidermis to enclose several layers of cortical cells (Fig. 6.15), sometimes extending even to the endodermis. This type of structure, best described as a 'cortical Hartig net', is also seen in a few genera of angiosperms such as Cistus (Giovannetti and Fontana, 1982) and Dryas (Alexander and Bigg, 1981; Debaud et al, 1981) and is occasionally reported in genera, for example Populus, which normally have a net of the more superficial kind. It,is of interest that the radial elongation of epidermal cells appears to be restricted to those mycorrhizas which have an epidermal Hartig net. When hyphae penetrate the cortex, whether in the gymnospermous hosts or in angiosperm genera such as Dryas, Cistus and Populus, no such radial elongation is observed. This suggests that increased surface contact between the symbionts is achieved by penetration of the fungus in the cortical type and by extension of the plant cell wall in the epidermal type. Figure 6.1 I Transmission electron microscopy of zones of a mycorrhizal root of Eucalyptus pilularis (refer to Fig. 6.7). (a) Root cap (zone A), showing thick mantle (M) and penetration of hyphae (H) between the cells of the root cap (RC). (b) Young Hartig net (zone B). Cytoplasmic hyphae (arrowed) are present between the vacuolated epidermal cells (E), up to the hypodermis (*). (c) Higher magnification of region similar to (b). (d) Hyphae between the epidermal cells (E), the walls of which (arrows) do not show structural modifications, (e) Contiguous walls between an epidermal (E) and hypodermal (*) cell, showing suberin lamellae (arrowheads) in the hypodermal wall and plasmodesmata (arrows) between the cells. From Massicotte et al. (1987c), with permission.

Structure and development of ectomycorrhizal roots

F i g u r e 6.11 (Caption opposite)

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Ectomycorrhizas

Figure 6.12 Electron microscopy of mycorrhizas formed between Eucalyptus pilulahs and Pisolithus tinctorius in the older Hartig net zone D. (a) A well developed Hartig net between epidermal cells (E), showing vacuolation of the hyphae in the the inner mantle and Hartig net (arrowheads). The Hartig net does not pass the hypodermis (*). (b) Contiguous walls between epidermal (E) and hypodermal (*) cells showing suberin lamellae (arrowheads) but no plasmodesmata. (c) Hartig net adjacent to the hypodermis (*), where the hyphae appear to disrupt the middle lamella between the epidermal and hypodermal cells. From Massicotte et al. (1987c), with permission.

Figure 6.13 Scanning electron microscopy of mycorrhizas formed between 8etu/o alleghaniensis and Pisolithus tinctorius. (a) Early stages of mycorrhiza formation, in which a few hyphae are present on the root surface and numerous root hairs (arrowed) are evident, (b) A thin mantle (arrowed) has been formed on the root surface, (c) A compact mantle covers the root and root hairs are no longer evident. Bars, 100 jim. From Massicotte et al. (1990), with permission.

Structure and development of ectomycorrhizal roots

F i g u r e 6.13 (Caption opposite)

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Ectomycorrhizas

Microscopic Structure Intercellular penetration induces profound morphogenetic change in the mycorrhizal hyphae. A repeatedly lobed, fan-like hyphal front advances across the radial surfaces of the plant cell. A structure of this kind was observed under the light microscope by Mangin (1910) who referred to the fungal lobes as 'palmetti'. It is now generally accepted that the lobes are a product of repeated and prolific hyphal branching producing a labyrinthine structure (Figs 6.14, 6.15). In addition to proliferation of the hyphal structures, in some mycorrhizas the presence of the fungus can induce the plant to form wall ingrowths into the adjacent epidermal cells. These have been observed (Fig. 6.6) in Alnus-Alpova mycorrhizas (Massicotte et al, 1986) and were apparently induced in Finns sylvestris-Suillus bovinus mycorrhizas by the presence of exogenous C sources (Duddridge and Read, 1984c). In the case of mycorrhizas formed on roots of Pisonia grandis, wall ingrowths are produced in the absence of a Hartig net (Ashford and Alia way, 1982; Ashford et al, 1988; Caimey et al, 1994). Whether or not wall ingrowths are produced by the cells of the plant, the prolific branching of the fungus as it encircles the epidermal or cortical cells yields a structure of immensely enlarged surface area. The hyphal walls are so closely associated with those of the plant that the two appear to be

Figure 6.14 Transmission electron microscopy of the mature Hartig net of a mycorrhiza formed between B>etu\Q allegt)aniensis and Pisolithus tinctorius. Multibranched hyphae, showing mitochondria (M), endoplasmic reticulum (arrowed, ER) and vacuoles (V). From Massicotte et al. (1989b), with permission.

Structure and development of ectomycorrhizal roots

197

fused into a joint structure which has been termed the 'contact zone' (Strullu and Gerault, 1977) or 'involving layer' (Scannerini, 1968; Duddridge and Read, 1984c). The architecture of a typical mature Hartig net is revealed diagrammatically (Fig. 6.15a) and in an ultrathin tangential section (Fig. 6.15b) of a mycorrhiza of Picea abies formed by Amanita muscaria and viewed under TEM (Kottke and Oberwinkler, 1987). The main growth direction of the hyphae, indicated in the inset (Fig. 6.15c) by an arrow, is transverse to the axis of the root, the ultimate finger-like branches being extremely fine and packed together so that there is little or no space between them. These parts are densely cytoplasmic, non-vacuolate, and contain large numbers of mitochondria which, together with the extensive endoplasmic reticulum, appear to be stretched in the direction of hyphal growth. In more proximal parts, the Hartig net hyphae are seen to be of larger diameter and some are vacuolate. Since more than two nuclei can sometimes be observed (Fig. 6.15b) to occur in a non-septate part of the mycelium in this type of mycorrhiza, Kottke and Oberwinkler (1987) regard the structure as having a coenocytic construction. Flowever, Massicotte et al. (1989b) found only one or two nuclei in the Hartig net compartments of B. alleghaniensis mycorrhizas and a similar situation was reported in association of A. rubra with A. diplophloeus (Massicotte et ah, 1989b). More investigations of nuclear migration patterns and mitotic events are required to enable the nuclear organization of the Hartig net to be evaluated. The similarities between the elaborate structures produced by the proliferation of hyphae in the Hartig net, and those seen in transfer cells which increase the surface area for exchange of solutes in many physiologically active plant tissues, has been recognized (Duddridge and Read, 1984a,b; Kottke and Oberwinkler, 1987). The extent to which these two types of structure are functionally comparable is discussed in Chapter 14.

Cellular Interactions in Hartig Net Formation Restriction of Hartig net formation to a specific zone closely proximal to the root cap (zones B and C in Fig. 6.7) is indicative of the fact that intercellular penetration can occur only at a specific stage of differentiation of the epidermal cell. The conventional view is that this penetration is achieved by mechanical means (Foster and Marks, 1966; Nylund and Unestam, 1982; Piche et al, 1983; Duddridge and Read, 1984a,b) although it has been suggested (Duddridge and Read, 1984a,b; Nylund, 1987) that in conifers the walls of cortical cells in the zone susceptible to penetration have a higher pectin:cellulose ratio than fully mature walls which, at this early stage in their development, might make them more susceptible to penetration by the fungus. The possibility of involvement of fungal enzymes in the penetration process is suggested by observation of lysis of the m i d d k lamella of epidermal cells in advance of the hyphal tips in the A. crispa-A. diplophloeus mycorrhiza (Massicotte et al, 1986). Likewise, disruption of the middle lamella was seen in the epidermal cells of E. pilularis being colonized by P. tinctorius (Massicotte et al, 1987c). Further support for enzymic involvement in the process of Hartig net formation comes from analysis of the mature interface between the partners, in which adjacent plant and fungal walls become indistinguishably fused to form the interfacial matrix or

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'involving layer'. Duddridge and Read (1984b) interpreted this fusion as a loss of integrity of the plant wall, a view supported by Nylund (1987) who showed by histochemical means that pectic polysaccharide components of the plant wall and of the interfacial matrix were similar. Harley (1985) interpreted these temporospatially related events in terms of interference by the fungus with the processes of deposition of the plant primary wall and middle lamella of the plant cell. He proposed that there may be impairment of the supply of precursors for wall assembly, possibly as a result of copolymerization of fungal proteins with those plant enzymes responsible for assembly of the plant wall. The possibility of direct attack by fungal enzymes upon the plant cell wall cannot be discounted. The potential of ectomycorrhizal fungi to express enzymes necessary to degrade non-lignified walls under some circumstances is demonstrated by their ability to penetrate epidermal cells in the production of arbutoid and ectendomycorrhizas (see Chapters 10 and 11). Indeed, in ectomycorrhizal roots themselves, the fungus is widely observed to penetrate the epidermal or cortical cells of the plant in older parts of the root (Nylund et ah, 1982a; Downes et ah, 1992) and this is very likely to require the expression of wall-degrading enzymes. A continuum may thus be envisaged in the zones shown in Figure 6.7 from a situation in zones A and B where epidermal cells are resistant to penetration, to a balanced interaction in zones C and D which facilitates the production of a joint structural entity, probably involved in nutrient exchange, to the interfacial matrix, and leading finally to a situation where the fungus breaks through the wall into the now moribund cell. According to Downes et al. (1992) this sequence of events may occur over a period of about 80 days. By this stage the sheath may still be present as a moribund structure or it may be progressively lost (Abras et al, 1988). While evidence for the involvement of cell wall degrading enzymes in the formation of the Hartig net itself is largely circumstantial, there are increasing indications from pure culture studies using model compounds that ectomycorrhizal fungi have the ability to produce the necessary suites of enzymes. The primary cell wall of the plant is made up mainly of cellulose, pectin and P(l,3) to P(l,4) glucans. Of particular interest in this connection is the demonstration by Cao and Crawford (1993a,b) of the production of cellulolytic enzymes by P. tinctorius, including three distinct isoforms of P-glucosidase in each of four different isolates Figure 6.15 Development of the Hartig net. (a) Block diagram showing typical structure of the Hartig net in different sectional aspects and of a pseudoparenchymatous mantle. The main growth direction of the hyphae in the Hartig net is transverse to the root axis, (b) Transmission electron microscopy of a mycorrhiza formed between Picea abies and Amanita muscaha. Ultrathin section through the intercellular space and several cortical cells showing fully developed, mature Hartig net Extensive branching leads to the formation of narrower and narrower hyphae (fh). Numerous mitochondria (m) and nuclei (n) can be seen. The presence of two dikaryons (arrowed) indicates the coenocytic nature of the tissue. (FV) fungal vacuole; (n) nucleus; (cv) epidermal cell vacuole, (hw) host wall. Bar, 2 |Lim. (c) Outline of the fungal hyphae in (b). Main growth of the hyphae is in the direction of the full arrow. Dolipore septum and dikaryons are marked. From Kottke and Oberwinkler (1987), with permission.

Structure and development of ectomycorrhizal roots

Figure 6.15 (Caption opposite)

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of the fungus. These isozymes were present in static as well as actively growing cultures and their presence indicates a tightly regulated cellulolytic capability in this fungus. Endoglucanase activities have been reported in isolates of Suillus hovinus, Paxillus involutus, Amanita muscaria (Maijala et ah, 1991) as well as in Cenococcum geophilum and Hebeloma pusilum (Linkins and Antibus, 1981). It is known that some ectomycorrhizal fungi will grow upon pectin as a sole source of C (Giltrap and Lewis, 1982) and pectinase (polygalacturonase) activity has been observed in culture filtrates of Piloderma croceum and Suillus variegatus (Nylund and Unestam, 1982). A wall-bound endo-polygalacturonase (endo-PG) activity was demonstrated in three out of six ectomycorrhizal fungi examined by Keon et al. (1987), who point out that endo-PG activity alone might facilitate localized, non-destructive cell wall breakdown by solubilizing pectins during the morphogenesis of the Hartig net. These recent studies have transformed our appreciation of the potential of ectomycorrhizal fungi to influence the receptivity of the plant to penetration. We should abandon the view based largely upon earlier comparisons of mycorrhizal and non-mycorrhizal strains of a given fungus (e.g. Lindeberg, 1948; Norkrans, 1950; Lindeberg and Lindeberg, 1977) that ectomycorrhizal fungi, as a group, lack the ability to degrade complex C-containing polymers. The challenge now is to determine the extent of expression of newly revealed enzymic potentials in the ectomycorrhizal root. Recent advances in affinity labelling techniques have produced specific molecular probes for individual components of the plant cell wall. These include monoclonal antibodies against pectic polysaccharides. The use of these in conjunction with more conventional histochemical techniques should enable clarification of the processes whereby compatible associations between plant and fungus lead to the production of a stable interface for nutrient exchange.

The Mycorrhizal Mantle Whereas the Hartig net forms the most extensive interface between fungus and plant, its biomass in most ectomycorrhizas is small relative to that of the overlying mantle. By separating the mantle from the core of selected mycorrhizas of Fagus, Harley and McCready (1952; and see Fig. 7.2c) were able to calculate that 40% of the weight of the colonized root was due to the fungus. This value does not, of course, include the weight of the Hartig net and has been widely, and often loosely, used by later workers even though they were studying other plant-fungus associations growing under different conditions. Vogt et al (1982, 1991) have reported values similar to those obtained for Fagus from Ahies amabilis growing in subalpine forest. However, they observed that in low altitude forest of Pseudotsuga menziesii the sheath constituted only 20% of the root weight. Bearing in mind that Harley and McCready were specifically collecting relatively large ectomycorrhizas, probably those formed by Lactarius suhdulcis, with fleshy sheaths, it is reasonable to assume that 40% may be a high value for fungal weight and that values between 20% and 40% would be more commonplace.

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Use of Mantle Structure for Classification of Ectomycorrhizal Types The need for a system of classification of mycorrhizal types which enables identification of the fungal associates involved has long been recognized. Apart from the intrinsic interest in the question of diversity of species present on a given plant, a rational system for identification and selection of defined types of the symbiosis is an essential prerequisite for rigorous classification of the functional differences between them. Early attempts to define mycorrhizal types (Melin, 1927; Dominik, 1969), which were based upon differences of macroscopic and microscopic characteristics, did little to facilitate identification of the fungi involved. Trappe (1967) emphasized the need to identify fungal partners and suggested various hyphal and other microscopic characteristics that could be used. In an analysis of Pseudotsuga mycorrhizas, Zak (1971a, 1973) used gross morphological characters and emphasized colour as an important distinguishing feature. Chilvers (1968a) added descriptions of mantle structure, based upon surface characteristics. All of these systems offered only limited scope for the determination of the fungal partners involved in the association. There are circumstances in which it is possible to identify the causal organism by careful exposure of hyphal connections between sporophore and mycorrhizal mantle. However, in many genera such as Lactarius, Russula and Inocybe tracing of this kind is very difficult. The ephemeral nature of fruit bodies and the fact that many important mycorrhizal types are not, as yet, attributable to a given species of fungus, are features which have further advanced the need for a method of classification based upon stable, easily recognizable characteristics of the fungal mantle and its associated mycelial structures. One such system has been developed by Agerer and the results are assembled in an 'Atlas' of mycorrhizal types (Agerer, 1987-1993). Classification requires preliminary recording of colour, using daylight-quality film, and of morphology of the colonized root and its emanating hyphae. This level of analysis, or 'morphotyping', widely used in the past as the sole method of description, is supplemented by anatomical characterization of the mantle, using Nomarski interference contrast microscopy. While the morphology of the mycorrhizal root is largely under the control of the plant, the construction of the mantle is a well-conserved feature apparently largely determined by the fungus. The known types of ectomycorrhizal mantle, as revealed by simple scrapings of the surface, can be divided into two main groups. In the first the hyphae can be discerned as individual structures which form a loose plectenchymatous or prosenchymatous (Chilvers, 1968a) construction, while in the second they lose their identity as individual structures being packed, normally as irregularly shaped cells, in diagnostic patterns that produce a pseudoparenchymatous structure. Agerer (1991a, 1995) recognized nine types of plectenchymatous (A-I) and seven (K-Q) of pseudoparenchymatous construction (Table 6.6; Fig. 6.16). The assembled morphological and anatomical details of a given type are presented by Agerer (1987-1993) in the form of colour plates showing the characteristic appearance of the whole mycorrhizal root, and of halftone plates which selectively demonstrate features of the mantle, revealed by scraping and sectioning, and of emanating hyphae. Following the investigations of Brand and Agerer (1986) and Brand (1991), some

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of the most important ectomycorrhizal types of Fagus are now described in the Atlas, and can be distinguished to the level of the fungal species. Thus, for example, the robust and apparently smooth mycorrhizal type which was selectively collected by Harley and co-workers for use in studies of nutrient absorption is described (Plate 3; Figs 6.17, 6.18) and the fungus responsible for its formation, Lactarius suhdulcis, has been identified. Colour plates (Plate 3) accurately reflect the overall appearance of the roots and demonstrate the presence of rhizomorphs, difficult to observe without the most delicate dissection, which provide connections to the soil and to fruit bodies of the fungus. Views of the surface and of varying depths in the mantle (Fig. 6.17), the latter as scrapings, obtained using Nomarski optics, reveal its pseudoparenchymatous nature, the characteristic angularity of its individual cells and the presence of laticiferous hyphae which are a diagnostic feature of the genus Lactarius. While the specific identity of this type of mycorrhiza was originally obtained by tracing connections between fruit bodies of L. subdulcis and the root, there are many types in which, to date, no such connections have been established. Gronbach and Agerer (1986) propose that in those cases where the identity of the fungus forming a widely distributed and well-characterized mycorrhizal type is not known, a binomial system of nomenclature can usefully be retained. In such cases the name is made up of the genus of the host plant and a characterizing epithet, e.g. Fagirhiza setifera. The major features of the gross morphology (Plate 4) and of the anatomy of the mantle and emanating hyphae, are again described. When the identity of the fungal partner is discovered the artificial name of the ectomycorrhiza can be replaced by that of the fungus. Comparable progress has been made with characterization and identification of the mycorrhizas of Picea (Agerer, 1986, 1987-1993). One of the most widely occurring mycorrhizal types on Picea abies in Europe is that produced by Russula ochroleuca (Plate 5). A combination of features including the presence of bright yellow-green patches revealed by colour microscopy and of angular cells in the mantle (Fig. 6.160) packed with yellow granules enables identification. Structural

Table 6.6 Examples of ectomycorrhizal fungal species which show the types of mantle construction depicted in Figure 6.16 Pseudoparenchymatous

Plectenchymatous

C

Lactarius glyciosmus, L pubescens

D

Russula aeruginea, Thelephora terrestris

E

Rhizopogon luteolus

K L M N O P

F

Boletinus cavipes, Leccinum scabrum

Q

G

Ceriococcum geophilum

H

Russula xerampeliria

1

Lactarius picinus

A

Amanita muscaha, Boletus edulis. Scleroderma citrinum

B

Cortiriarius armillatus, Hebeloma crustuliniforme, Laccaria laccata, Paxillus involutus, Pisolithus tir)ctorius

The letters A - O refer to Figures 6.16. Data from Agerer (1991 a).

Russula fellea

Lactarius pallidus

Tuber aestivum T. melanosporum, Lactarius rufus Russula emetica Russula ochroleuca Lactarius subdulcis

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features of the mantle and Hartig net provide further information to assist in identification.

Tuberculate Mycorrhizas Many of the plant species which produce individual ectomycorrhizal roots exposed to surrounding soil also support compound structures in which clusters of such roots, each with its own mantle and Hartig net, are enclosed in a globular rind of fungal tissues. The often densely packed complex of roots can have a diameter of up to 20 mm, and is termed a tuberculate mycorrhiza. Such aggregates, observed by Frank (1988), MoUer (1903), Miiller (1903), Tubeuf (1903), McDougall (1922) and Melin (1923) in Pinacease, but first described in detail by Masui (1926) when occuring on Quercus, appear to be of quite uniform anatomical structure across a wide range of plant families and genera. These include Pseudotsuga (Pinaceae; Zak, 1971b, Photina (Rosaceae; Grand, 1971), Eucalyptus (Myrtaceae; Dell et al, 1990), Castanopsis (Fagaceae) and Engelhardtia (Juglandaceae; Haug et ah, 1991). They are normally formed by basidiomycetous fungi. Analysis of the fungal rind in a Eucalyptus tubercle (Dell et al., 1990) reveals a two-layered structure, the outermost being 150-200 |im thick and composed of densely packed hyphae cemented together by a matrix of carbohydrate and lipid-rich material to yield a tissue likely to be highly impermeable. The inner layer is relatively thin and loosely packed. Masui (1926) and Haug et al. (1991) observed that within this rind were two types of ectomycorrhizal root. While one was of the normal structure, the other had a particularly heavy colonization characterized by a very thick hyphal mantle around root tips that were invariably dead. Just below the tip of such roots, a constricting ingrowth of fungal hyphae penetrated to the vascular cylinder, forming what has been called the strangulation zone. All cells basipetal to this zone were living and had a nominal epidermis with Hartig net. The inner tangential walls of the outermost cortical cells in this region were shown by Haug et al. (1991) to form a prolific wall ingrowths, each with extensive endoplasmic reliculum and numerous mitochondria. These workers emphasized that such structures have transfer-cell like properties suggesting that as in normal individual mycorrhizal roots they are involved in nutrient exchange. However, the isolation of the tuberculate mycorrhizal roots from soil means that they are unlikely to have an absorptive function. Both Zak (1971) and Haug et al. (1991) observed that an extensive system of fungal rhizomorphs radiates outwards from the rind of the tubercle into the surrounding soil. Indeed, Zak describes rhizomorphs, believed to be produced by Rhizopogon vinicolor, up to 1000 |Lim in diameter, with their outer tissues continuous with those of the rind but in which the central core of hyphae enters the tubercle to branch within it. Dell et al. (1990) observed branching rhizomorphs within tubercles of Eucalyptus. These observations suggest that the roots contained within the tubercle are primarily involved with storage of nutrients and their exchange between plant and fungus. Some evidence for a storage function was provided by Gobi (1967) who showed that concentrations of P and K in the tubercles of Pinus cemhra were greater than those in single ectomycorrhizal roots. Tuberculate mycorrhizas thus exemplify a situation, which may also be a feature of the individual ectomycorrhizas

204

5-n'n.

Figure 6.16 Schematic drawings of surface plan views of plectenchymatous (A-l) and pseudoparenchymatous (K-Q) mantles, based on surface scrapings. From Agerer (1991), with permission.

Structure and development of ectomycorrhizal roots

F i g u r e 6.16

Continued.

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Figure 6.17 Different layers of the mantle fornned in a Lactarius subdulcis mycorrhiza. (a) Hyphal reticulum forming mantle surface, (b) Surface view of the very tip of the mycorrhiza. (c) Plan view of outer pseudoparenchymatous layer of the mantle, (d) As (c), but in an older region of the mantle, (e) Plan view of inner, plectenchymatousi mantle, showing lactiferous hyphae. (f) Inner surface of the mantle, (g) Tangential section through the outermost cortical cells and Hartig net. Bars, 10 |im. From Agerer (1987-1993), with permission.

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Figure 6.18 Characteristics of a Lactarius subdulds mycorrhiza. (a) Cross-section of the Hartig net in plan view, (b) Longitudinal section, showing young emanating hyphae. (c) Cross-section of the Hartig net in plan view, (d) Rhizomorph, close to mantle, (e) Crosssection of emanating hyphae. (f) Young rhizomorphs, showing anastomoses, (g) Branching rhizomorph. Bars, 10 |im. From Agerer (1987-1993), with permission.

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formed by some fungi, in which there is a distinct compartmention of function: those of acquisition and transport of resources being fulfilled exclusively by the fungal mycelium in soil, while the mantle, into the which nutrients are delivered by this mycelium, is effectively an outwardly sealed compartment solely involved with storage and exchange between the symbionts (see also Chapters 14 and 15).

The Structure of Emanating Hyphae and Rhizomorphs While the bulky tissue of the mycorrhizal mantle may provide a structure suitable for nutrient storage and, through its intimate contact with the root surface, play a key role in control of nutrient transfer between fungus and plant, it does little to increase the surface area of the colonized root in contact with the soil (see below). This function is served by the extraradical mycelium which, as single hyphae or linear aggregates of such hyphae, extend from the mantle. Structural attributes of these mycelial systems are of additional importance, because singly and collectively their constituent hyphae form the connection between mantle and soil, and so provide the pathways for nutrient exchange. Recognition of the critical role played by this component of the mycorrhizal system constitutes an important change of emphasis in research on the symbiosis in recent years (Read, 1984, 1992). The simplest level of organization seen in ascomycete associates such as Cenococcum geophilum and Tuber spp., and in some of the more widespread basidiomycetes, is one in which hyphae emanating from the sheath retain their individuality, growing as single elements into the soil. The majority of basidiomycetous associates of ectomycorrhizal roots do, however, produce structures in which hyphae aggregate and grow in parallel to some extent, as they leave the mantle, so forming a linear organ. Some confusion has arisen over nomenclature applied to these multihyphal linear aggregates and a number of terms have been used to describe them, including bundle, cord, strand, rope and rhizomorph. The term 'rhizomorph' has the combined advantage that it appears to be the first used to describe this type of structure and that it highlights their root-like morphology. Earlier reluctance to employ this term for mycorrhizal organs was based upon the view that rhizomorphs were structures which extend, by the action of initials, in a well defined apical meristem (Garrett, 1963; Motta, 1969), in contrast to the hyphal aggregates of mycorrhizal fungi which have a loose, apically spreading mode of growth. It is now known (Rayner et al, 1985) that rhizomorphs of Armillaria spread, not as a result of meristematic activity, but as a looser front of apically extending hyphae which interdigitate to produce a structure which superficially resembles a meristem. There is thus a continuum of structural differentiation (Fig. 6.19) from apically dominant organs (Fig. 6.19a) such as those of Armillaria at one extreme, to the apically diffuse and spreading systems typical of many ectomycorrhizal fungi at the other (Fig. 6.19e). In view of this, Caimey et al. (1991) made the sensible recommendation that the term rhizomorph be used to describe all linear aggregates of hyphae. Agerer (1991a, 1995) recognized six categories of rhizomorph (A-F, Fig. 6.20). The simplest structures are undifferentiated aggregations of loosely woven hyphae of equal diameter, the whole having an ill-defined margin (type A, Fig. 6.20). A still undifferentiated but more compactly arranged structure (type B, Fig. 6.20) with a

Structure and development of ectomycorrhizal roots

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(c)

Figure 6.19 Stages in the development of types of rhizomorph from systems of the Armillaria kind with organized apices (a), to those typical of most ectomycorrhizal fungi (d and e), in which a loose and usually fan-shaped hyphal front explores the medium. From Rayner et al. (1985), with permission.

smooth margin is produced by Laccaria and Lactarius spp. (Table 6.7). Some enlargement of central hyphae within the aggregate yields a somewhat more complex structure (type C, Fig. 6.20). Considerable increases in complexity are seen in types D, E and F (Fig. 6.20) where internal hyphae have much enlarged diameters. In type D the thicker hyphae are randomly distributed while in the most highly organized types (E and F) there is a central core of thick hyphae, those in type F showing dissolution of transverse septa. The well defined structural differences between rhizomorph types provide supplementary features which could assist in classification of mycorrhizas and also be associated with functional differences. The basic processes of growth and differentiation of the extraradical mycelia of rhizomorph-forming ectomycorrhizal fungi have been described and quantified by observing their development in transparent observation chambers containing nonsterile natural substrates (Brownlee et al, 1983; Read et ah, 1985; Finlay and Read, 1986a,b,c; Fig. 6.21). Under controlled conditions of temperature (15°C day, 10°C night) and day length (18 h), fungi such as Suillus bovinus extend from colonized roots of Pinus spp. as an undifferentiated fan-like front of hyphae at a constant rate of 2-4 mm d~^. Coutts and NichoU (1990a) measured the extension rates of Thelephora terrestris and Laccaria laccata growing from roots of Picea sitchensis through a

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growing season, under natural conditions of temperature and day-length. A mean extension rate of 3 mm d~^ was recorded for T. terrestris mycelium in July while L. laccata achieved 2 mm d~^ (Fig. 6.22). Between June and November the fans had extended from colonized roots to reach a distance of 0.24 m. The mycelia of T. terrestris continued to grow, albeit at a much slower rate of 0.44 mm d~^, through the winter. During the growing season the primary roots of spruce grew at a slightly faster rate than the advancing hyphal front (Fig. 6.22c,d) but because the mean distance from the tip of such a root to the youngest short root to emerge from its axis is normally greater than that to the hyphal front, such laterals are rapidly colonized as they break through the cortex into the domain occupied by the fungus. Whereas extension growth of roots ceases during the winter months, the mycelium of T. terrestris continued to extend from December to March. In contrast, the mycelium of L. laccata was not evident after October (Coutts and NichoU, 1990a). When the mycelial network of T. terrestris was subjected to a waterlogging treatment in winter, the undifferentiated parts of the network senesced but the rhizomorphs retained their structure and thus provided a skeletal framework from which new hyphae could be regenerated when soil conditions ameliorated (Coutts and NichoU, 1990b). The extent of development of extraradical mycelia has been quantified in several studies (Read and Boyd, 1986; Jones et al, 1990; Francis and Read, 1994; Rousseau et al., 1994) enabling their contribution to the potential absorbing surface of the plant to be estimated. Read and Boyd (1986) determined hyphal lengths of a number of ectomycorrhizal fungi after they had grown for three months from seedlings of Pinus sylvestris across non-sterile unfertilized peat. Values ranging from 2000 to 8000 m m~^ of colonized root were obtained. Pisolithus tinctorius growing for eight weeks into sterilized and fertilized quartz sand from seedlings of Pinus taeda, had hyphal lengths of 504 m m~^ root (Rousseau et al, 1994). This value is similar to the 289 m m"^ and 308 m m~^ reported by Jones et al (1991) for Laccaria proxima and Thelephora terrestris, respectively, after growth for 12 weeks, again in a sterilized and fertilized sandy matrix, but from Salix viminalis. Differences between experiments are to be expected when measuring hyphal lengths, especially where substrate conditions range over such extremes. It is arguable that the high values obtained in organic matter of low fertility are likely to be more representative of those seen in nature. Whatever their cause, however, the differences in hyphal length recorded between experiments cannot disguise the main feature of these results which is the enormous increase in potential absorption area which the root system gains from the presence of the extraradical mycelium. It is difficult to emphasize the importance of this component sufficiently. In his early experiments on ectomycorrhiza. Hatch (1937) had observed increases in nutrient absorption efficiency of colonized roots, the reasons for which lay, according to him, in changes in the geometry of the absorbing root surfaces which became increasingly branched as mycorrhizas developed (see Chapter 9). Rousseau et al (1994) compared the increases of absorptive area gained by branching of this kind with that provided by the mycelium of Pisolithus and Cenococcum colonizing Pinus taeda (see Table 9.4). They found a fourfold increase of root area in the case of P. tinctorius, but no increase in seedlings colonized by C. geophilum. In contrast, extraradical mycelium of the former fungus provided a 47-fold increase of surface

Structure and development of ectomycorrhizal roots

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;

F i g u r e 6.20 Schematic drawings of different types of rhizomorph f o r m e d by ectomycorrhizal fungi. See t e x t f o r descriptions. From Agerer (1991), w i t h permission. T a b l e 6.7 Examples of ectomycorrhizal species having different types of r h i z o m o r p h construction. A B C D E F

Cortinarius obtusus, Dermocybe cinnamomea, Tricholoma sulfureum Loccaria amethystina, Lactarius deterrimus, Lactahus vellereus Gomphidius glutinosus, Thelephora terrestris Cortinarius hercynicus, Cortir)arius vahecolor Tricholoma saponaceum Leccinum scabrum, Paxillus ir)volutus, Scleroderma citrinium, Suillus bovmus, Xerocomus chryser^teror)

The letters A-F refer to Figure 6.20. Data from Agerer (1991 a).

area and that of the latter an approximately 28-fold increase. The extent to which such relationships are translated into increased efficiency of nutrient use is discussed in Chapter 9.

The Processes of Rhizomorph Construction On artificial media, such as agar gels or liquids, the mycelia of ectomycorrhizal fungi show a dense 'fluffy' but largely undifferentiated growth form, which is unrepresentative of that seen in nature. In heterogeneous natural substrates, a considerable morphogenetic plasticity is revealed both at the leading edge of the hyphal front and in the maturing regions behind the front. When hyphae of the advancing front make contact with particles or aggregates of a particular quality they can be induced to branch repeatedly, to increase in diameter by up to fourfold

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Figure 6.21 Development of extensive mycelium and rhizomorphs by Suillus bovinus in symbiosis with a seedling of Pinus sylvesths (A). Note the fan-like spread of the extraradical fungal system, with which the germinating seedlings (B) have already formed mycorrhizas (arrowed). From Read (1991c), with permission.

and to develop thick gelatinous walls. Such morphogenetic changes have been described in association with mineral and organic materials (Read et al, 1985; Ponge, 1990; Agerer, 1992). Intensive hyphal proliferation can be induced by introducing organic substrates of a particular quality to sparsely growing mycelial systems (Read, 1991a; Unestam, 1991; Bending and Read, 1995a; Fig. 6.23). There is evidence (see Chapters 8 and 15) that these proliferations effect nutrient mobilization from the added substrate. Behind the leading edge of the mycelial fan, hyphae growing in parallel approach each other more and more closely, to form the linear aggregates which eventually achieve dimensions of sufficient magnitude to be visible as rhizomorphs. Construction of, and cohesion within, the rhizomorph is achieved by a number of mechan-

213

Structure and development of ectomycorrhizal roots

A

M

J

J

A

S

O

N

Figure 6.22 Cumulative growth of three clones ( • , • , • ) of Picea sitchensis and their mycorrhizal fungi during 1987. In (c) the open symbols are Laccaria proximo and the closed symbols are Thelephoro terresths. (a) Seasonal variations in temperature, (b) Shoot extension, (c) Root extension, (d) Mycelial extension. From Coutts and Nicholl (1990a), with permission.

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Figure 6.23 Development of mycelium of Suillus bovinus growing from a mycorrhizal plant of Pinus sylvestris in an observation chamber, (a) Uniform colonization of the peat substrate and the proliferation of mycelium in one of the trays (arrowed), (b) Autroradiograph of the same chamber after feeding CO2 to the shoot. Note the intense labelling in the highly colonized tray (arrowed). From Bending and Read (1995a), with permission.

isms which can be seen individually or in combination to produce structures of the kinds shown in Fig. 6.20. Amongst these, interhyphal bridges formed by anastomoses, adhesion between hyphae facilitated by gelatinous wall materials and the production of backward and forward growing ramifications have been identified by Agerer (1992) as being the most important. Bridges may provide open connections between the attached hyphae or may become secondarily 'closed' by the formation of clamps or simple septa. Since the septa of higher fungi have a central pore, such 'closure' does not necessarily constrain either cytoplasmic continuity or transfer of materials (see Chapter 14). However, complete closure of the pore may be advantageous under some circumstances in that it would facilitate isolation of any segments of the rhizomorph, in which, for whatever reasons, cytoplasmic continuity has been disrupted. The production of backward or forward growing elements, often from clamp connections on the main hyphae, has been known to be a prominent feature of construction of these organs since the studies of Butler (1958) of the ontogeny of rhizomorphs of the wood-decay fungus Serpula lacrymans. These secondary elements, referred to as 'tendril hyphae', ensheath tKe larger central structures. A similar type' of construction is seen also in the rhizoidal rhizomorphs of the moss Polytrichum juniperinum (Agerer, 1991c). Its independent selection in the plant and fungal kingdoms suggests favourable attributes that probably include increased mechanical strength.

Structure and development of ectomycorrhizal roots

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The tendrils ensheath the main hyphae from which they have developed, become densely cytoplasmic and produce an extrahyphal matrix that helps to cement the structure together. In the most complex mycorrhizal rhizomorphs, such as those produced by Suillus bovinus (Duddridge et al, 1980; Read, 1984), Leccinum scabrum (Fox, 1987), Xencomus chryseuteron (Brand, 1989), these thinner structures encase a group of central hyphae of much larger diameter. The central elements lose their cytoplasm and show progressive breakdown of transverse septa, to form pipe-like structures bearing some superficial resemblance both in their axial location and tubular conformation to the xylem vessels of plants. For this reason they have been referred to as 'vessel hyphae'. Microscopy reveals that fully differentiated vessel hyphae first appear in the rhizomorph well behind the advancing front. They occupy the full length of thfe rhizomorph but do not enter the mantle of ectomycorrhizal root. The junction between rhizomorph and mantle surface is normally formed by loose aggregates of undifferentiated hyphae, which may fan out to produce a delta-like confluence with the ensheathing system. Analysis of the zone of conjunction by light microscopy indicates that direct continuity between any differentiated components of the rhizomorph and the mantle is lacking. Agerer (1990) described ramification of the vessel-like hyphae of Suillus bovinus as the rhizomorph approaches the mantle surface, the narrower elements arising from it crossing the transition zone as a delta- or fan-like structure to intertwine with the mantle hyphae. In the less well differentiated rhizomorphs of Sarcodon imbricatus, the slightly thicker central hyphae again ramify proximally, and their ultimate branches grow into the inner mantle where they lie in close contact with the root surface (Agerer, 1991b). There is evidence, in this area, of elaborate anastomosis between the mantle hyphae and those entering from the rhizomorph. The basic structure thus seems to be one which can give polarized transport to and from the mantle and dispersed distribution within it. At the distal end of the rhizomorph in soil, a fan-like arrangement of fine hyphae is again seen, the pattern of distribution of the individual elements apparently being ultimately determined by the nature of the resources they encounter (Fig. 6.23). These features are of key importance both in scavenging resources from the soil and distributing these and organic C derived from the root throughout the fungal biomass. Longevity of Ectomycorrhizal Roots The initiation, maturation and senescence of individual mycorrhizas and of the whole mycorrhizal root system are clearly dynamic processes, and the developmental changes influence productivity of the root systems, turnover of the interfaces involved in nutrient transfer, as well as the succession of different fungi associated with the roots. Studies of annual productivity of forest ecosystems have necessitated measurements of rates of fine root turnover and, in so doing, provided estimates of the longevity biomass and of individual short roots. Living and dead roots have been distinguished largely by morphological criteria, a transition being recognized from the light coloured turgid juvenile structures to those that are dark and wrinkled and

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which retain pale coloration only at the apex. Darkening of this apical region has been taken as an indication of death (Vogt et al., 1981). Using these criteria, the life span of individual fine roots (<1 mm diameter) of Picea sitchensis has been estimated to be 3.8 months (Alexander and Fairley, 1983), and of Pinus sylvestris 2.5 months (Persson, 1979). Santantonio and Santantonio (1987) examined fine roots of <2 mm diameter in stands of P. radiata and estimated a life span range of 2-6 months. The values derived from biomass estimates of living and dead roots contrast with those obtained in earlier anatomical studies, in which Orlov (1957, 1960) investigated fine roots of 100-year-old trees of Picea abies. On the basis largely of the appearance of tissues of the stele, he calculated that over 40% of these roots retained vitality for more than three years. It is clear that from a functional standpoint the conditions of the hyphal mantle and of the plant-fungus interface are likely to be of greater significance than features of either gross morphology of the root or of the structure of the stele. When the interface has been examined and its condition related to observations of changes in root morphology, a life span closer to that suggested by biomass studies has been indicated. Downes et al (1992) grew P. sitchensis in observation chambers, with Tylospora fibrillosa or Paxillus involutus as mycorrhizal associates, and sequentially harvested short roots over a period 0-140 days. They found that most of the mycorrhizas formed by both fungi had darkened and become wrinkled by 50 days, although many retained a light and turgid apex even to the final sampling occasion. It was apparent that a morphological appearance of the mycorrhiza was not a good indication of its chronological age, or functional lifespan, the latter being probably not more than 35 days. Ultrastructural analyses revealed changes at the plant-fungus interface in mycorrhizas produced by both fungi which are indicative of a shorter life span than that suggested by morphological observations. The process of senescence of cortical cells invested by the Hartig net is initiated early and some such cells are already dead within 24 days of their formation. The senescence of the plant cell is closely followed by that of the Hartig net itself. Death of outer cortical cells was observed by Downes et al (1992) to proceed from proximal to distal regions of the root so that the longevity of the whole short root is ultimately determined by the length of time over which the meristem continues to add new cortical cells at the distal position. Senescent meristematic cells were seen in some 60-85-day-old mycorrhizas of Tylospora and in some of those of P. involutus at 25-50 days. It was confirmed by the use of fluorescein diacetate as a vital stain, that physiological activity in the region of the plant-fungus interface declined markedly in mycorrhizas over 85 days old. The situation is complicated by the fact that some apparently senescent meristems can periodically produce new bursts of growth, each of which yields a young turgid cortex. This activity gives rise to a 'beaded' mycorrhiza (Thomson et al, 1990c). According to the observations of Downes et al (1992), degeneration of the stele occurs proxiinally from the meristem and after that of the cortex and meristem. Since lateral roots are initiated in the pericycle, this enables the parent to retain the potential to produce new roots. The observed sequence of senescence is thus in the order cortex, meristem, stele. This involves loss first of the capacity to'extend the

Structure and development of ectomycorrhizal roots

217

life of the existing organ and only later of the ability to produce a new mycorrhiza either apically or as laterals. Abras et al. (1988) related morphological changes in Picea mycorrhizas, which had probably been formed by Hebeloma spp., to carbohydrate respiration as the roots aged up to three years. The ageing process appeared to involve the disappearance of the fungal sheath, but the sheathless mycorrhizas were metabolically active although less so than young complete mycorrhizas. Both sheathless and complete mycorrhizas had higher respiration rates than non-mycorrhizal roots. Absence of a sheath, and any mycelial connections with the soil, would presumably eliminate any functional significance in terms of nutrient acquisition by that part of the root system. Since tissue degeneration takes place sequentially in an individual root it is difficult to provide a finite point of death. In functional terms, death can best be considered to have occurred when no active plant-fungus interface remains. In the study of Downes et al. (1992), which was carried out using roots grown in transparent observation chambers, this stage was reached in some mycorrhizas by 30 days after their first appearance but, in the majority, by 85 days. The maximum longevity was predicted to be 170-240 days. Clearly, these values are closer to those obtained in biomass studies than those suggested by Orlov (1957,1960). They were, however, obtained under controlled conditions and there is a need to extend such studies to mature trees growing in the field. It is necessary to bear in mind, also, that while the life of the individual short root may be restricted to periods of the order of 2-3 months, the long root continues to develop through the growing season and from its axis new short roots can be continuously initiated. The length of time the cells at the plant-fungus interface remain alive is most important with respect to nutrient transfer between the symbionts. Dead cortical cells cannot, except for a very brief period of senescence, be expected to provide organic C to the fungus. Nor can they, conversely, absorb nutrients from the intercellular apoplast. Lei and Dexheimer (1988) have demonstrated the distribution of membrane-bound ATPases in the interface of 32-day-old Pinus sylvestrisLaccaria laccata mycorrhizas. The fungal membrane showed high activity in the Hartig net region where hyphae were adjacent to living root cells, but not where the root cell cytoplasm had become disorganized during senescence. This point will be discussed further in Chapter 14.

Succession of Ectomycorrhizas and Mycorrhizal Fungi Community Level Studies F.T. Last's group analysed the temporal and spatial patterns of sporocarp production around individual trees of birch (Betula) in Scotland (Mason et al, 1982, 1983; Deacon et al, 1983; Last et al, 1983). They observed that the fruit bodies of some fungi (e.g. species of Hebeloma, Inocybe and Laccaria) appeared within the first four years of planting and were produced progressively further from the stem with time (Fig. 6.24). They were succeeded and eventually replaced by species of Lactarius,

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year 6

year 5

H B Hebeloma SDD

^

Laccariasp

Figure 6.24 Sucession of fruit bodies of ectomycorrhizal fungi around trees of BetulOy showing the mean distance of production from the trunk during the six years after planting. From Last et al. (1983), with permission.

Leccinum and Russula. Comparable sequences have been described on Pinus radiata in New Zealand (Chu-Chou, 1979) and on P. hanksiana in Canada (Danielson, 1984). From observations of this kind, the concept of successions of ectomycorrhizal fungi arose, those fungi appearing first being referred to the category 'early-stage', those that replace them to 'late-stage' (Mason et al, 1982,1983; Deacon et al, 1983). The terms 'early' and 'late' stage may have been originally intended to denote the times at which fruit bodies first appeared in age-related sequences, rather than suggest the fungi were exclusively restricted to that stage, but the terms have led to some misconceptions. Arnolds (1991) argued that the terminology was misleading because so many so-called late-stage species could be abundant in young forest stands, while early-stage fungi are by no means restricted to seedlings or even young trees. The situation was to some extent clarified by Danielson (1984), who described as 'multi-stage' those fungi that were present throughout the life of the stand. A further difficulty with the sequence of fruit body production described by Mason et al (1982,1983) is that it took place on a brown-earth soil previously under agriculture and so reflected an ecologically unrealistic circumstance (Read, 1991a; Molina et al, 1992; Newton, 1992). This weakness was highlighted by Fleming (1983, 1984), who showed that when birch seedlings were planted into forests supporting mature trees, they were colonized largely by the late-stage fungi associated with the adults. This observation is of particular importance because in a forest, or even around free-standing individual trees, the majority of seeds of a given species normally fall within a small distance of an adult. It appears that in nature, seedlings are colonized by multi-stage or late-stage fungi growing from

Structure and development of ectomycorrhizal roots

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established trees and that the fungi of the early-stage type will have a role primarily where ectomycorrhizal trees are pioneer colonists of natural sites, old agricultural land or in nurseries. Problems over the use of terms should not disguise the fact that real biological differences can be found between fungi that occur as pioneers and those of established forests. Recognition of these differences may permit a more broadly based classification. The differences between the two groups of fungi recognized by Mason et al. (1983) appear to be largely derived from their relative abilities to disseminate as spores or small fragments of inoculum and to use these to colonize new food bases in the form of roots. Competitive replacement of one fungus by another is recorded infrequently (see below). Fox (1986) observed that only earlystage fungi were able to colonize seedlings from spore inoculum when plants were growing on mineral soil. Similarly, Deacon et al. (1983) found that when seedlings were sown into soil cores collected from beneath fruit bodies of fungi representative of the different stages, early-stage fungi alone were able to colonize roots. In broad terms, the attributes of early-stage fungi are those of ruderal or 'r' selected organisms, and those of late-stage are of 'k' selected forms (Gadgil and Solbrig, 1972). The distinction is sufficiently realistic to be used to discriminate between early-stage fungi which are amenable for use in commercial inoculation programmes, from late-stage which are not (Marx and Cordell, 1989). Whether further discrimination between groupings will be profitable is doubtful. So little is known about interactions between species of mycorrhizal fungi growing from their hosts in natural substrates that attempts to classify them on the basis of strategy theory (Grime, 1979) into ruderal, competitive or stress-tolerant forms is premature. The presence of large intraspecific variability in terms, for example, of resistance to metals and hence stress tolerance (Colpaert and Van Assche, 1987a) further suggests the need for caution. Under these circumstances the major requirement is for experiments to determine the nature and extent of the successions and their underlying causes. Even elaborations beyond simple discrimination between 'r' and 'k' strategists can be fraught with difficulty. Last et al (1987) elaborated the model of Dighton and Mason (1985) of succession in even-aged plantations of trees in which a population of ruderal fungi lacking host specificity increased in diversity up to the stage of canopy closure and thereafter declined as these organisms were replaced by 'k' or 's' (stress tolerant) strategists showing greater host-specificity (Fig. 6.25). This process was seen to be driven by decreases in quality and increases in quantity of litter. According to Molina et al. (1992), experience in the Pacific north-west contradicts this model. Outplanting trials (Castellano and Molina, 1989) and soil bioassays (Amaranthus and Perry, 1989; Borchers and Perry, 1990) indicate that Pseudotsuga menziesii seedlings are colonized by genus-specific Rhizopogon spp., in particular R. vinicolor, at the outset and that these fungi persist in mature and oldgrowth stands up to 1000 years of age irrespective of changes in amount or form of organic matter on the forest floor. They are clearly multi-stage fungi. Whatever the merits of any classification based upon production of fruit bodies, it is evident that it can provide only an indirect indication of the population structure of fungi forming ectomycorrhiza on the roots themselves. This point was made in a study of mycorrhizal populations of Picea sitchensis plantations (Taylor and Alexander, 1989) which showed that despite the presence of a distinctive flora of mycorrhizal hymenomycetes as epigeous fruit bodies, over 70% of

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Figure 6.25 Diagrammatic illustration of a proposed successional sequence of fungal species. With decreasing litter quality, selection is seen as progressively favouring stresstolerant (s) species at the expense of those showing ruderal (r) strategies. From Dighton and Mason (1985), with permission.

mycorrhizal roots were colonized by the fungus Tylospora fibrilosa which was not represented in the sporophore population. A recent study by Visser (1995) is enlightening in this respect. She investigated changes of ectomycorrhizal morphotypes on roots, as well as fruit body production in P. banksiana stands of a wide range of age cohorts (6, 41, 65 and 121 years) which had established by natural regeneration on pine sites after fire. Assessment based upon analysis of both fruit bodies and mycorrhizal morphotypes suggested that there was a sequence of mycorrhizal fungi with stand age and that the succession was broadly similar, in terms of the identity of the fungi, regardless of the methods used. Early-stage fungi such as Thelephora terrestris and those of the E-strain type were followed, but not completely replaced, by species of the late-stage category among which Cortinarius spp., Lactarius spp., Russula spp. and Tricholoma spp. were strongly represented. In addition there was a population of fungi which did not dominate at any particular stage, amongst which Inocybe spp., Suillus brevipes and Cenococcum geophilum were recognized. In so far as evidence for complete extinction of early colonizing species was weak, it is arguable that succession, as a strict species replacement process, was not observed. Rather, an increase in complexity of species composition of the community arises. The species abundance distribution in the six-year-old stand resembled a geometric series, typical of that seen in plant and animal communities of low species richness, dominated by 'r' selected species (May, 1981). The community increased in complexity within 6-41 years as more species were added, shifting the species abundance from geometric to lognormal. The lognormal abun-

Structure and development of ectomycorrhizal roots

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dance is representative of a more stable community, where a relatively large number of species is more equally distributed in complex interactive situations. Whereas results of Dighton et al (1986) and Last ei al (1987) indicated a decline in species diversity after canopy closure in forest stands at about 17 years, the study of Visser (1995) over the much greater age range showed no evidence of such decline. A marked increase of species richness was observed as stands aged between 6 and 41 years, but thereafter both the structure and the composition of the ectomycorrhizal community had largely stabilized (Fig. 6.26). All of the abundant root morphotypes present at 41 years also occurred at 65 and 112 years. It is important to consider the mechanisms driving the sequential development of the mycorrhizal flora up to the time stability is achieved. Some (e.g. Dighton and Mason, 1985; Last et al 1987) have suggested relationships between accumulation of organic matter on the forest floor and increasing diversity (Fig. 6.25). It is logical to postulate that changes in the quality and quantity of substrates in which the symbiosis develops will lead to selection favouring fungi with the functional attributes that enable them to obtain essential nutrients under the changed circumstance. In the case of the P. banksiana stands studied by Visser (1995), however, the possibility of such a relationship was largely discounted because mycorrhizal roots

25

50 75 Stand age (yr)

100

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10 15 20 Species rank

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F i g u r e 6.26 (a) Species richness; and (b) species relative abundance for ectomycorrhizal fungi colonizing root tips of Pinus banksiana in an age sequence of P. banksiana stands. O , 6 years; • , 41 years; V , 65 years; T , 122 years. From Visser (1995), with permission.

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of this plant, in common with those of some other Pinus spp., proliferate almost exclusively in the mineral soil just below the organic horizon. Changing patterns of C allocation associated with tree age were considered by Visser to provide the most likely driving force for shifts in the fungal population, although the additional possibility that the nature of leachates from thickening organic horizons might change sufficiently to contribute to such shifts was acknowledged. There is a need here to explore the pattern of foraging of the extraradical mycelium as well as distribution of roots (see Fig. 6.23). Even where, as in the case of P. banksiana, mycorrhizal roots themselves appear to be localized in the mineral soil, the fact that the majority of their fungal colonists form rhizomorphs indicates that they have the potential to explore the overlying litter resource for nutrients. If, as laboratory studies suggest, preferential foraging in organic substrates does occur, changes in the physicochemical nature of the organic horizon could still be influencing the vigour, hence inoculum potential, of the colonizing fungi even though the colonized roots were in a different substrate.

Molecular Approaches to the Study of Ectomycorrhizal Communities As we have already mentioned, molecular tools have introduced new possibilities for identification of fungi involved in mycorrhiza formation from small amounts of vegetative material. The data in a recent application of these techniques at the community level (Gardes and Bruns, 1996) have provided new insights into the occurrence and succession of fungi in their vegetative stages, which can be usefully compared with information derived from studies based upon 'morphotypes' or fruit bodies. Over a four-year period. Gardes and Bnms (1996) collected and identified all fruit bodies of ectomycorrhizal basidiomycetes produced above ground in plots established in a stand of Pinus tnuricata. They simultaneously sampled mycorrhizas occurring in soil cores under some of the most frequently occurring fruit bodies, such as those of Amanita franchetii and Suillus pungens and, after initially separating them on the basis of morphotype, attempted identification using polymerase chain reaction (PCR)-based molecular tools. Nearly all of the morphotypes could be identified using these approaches, at least to generic level. In general, despite the fruit body-biased sampling, correspondence between identity of fungi producing the fruit body and the predominant mycorrhizal type was not good. In cores taken both under Amanita franchetii and Suillus pungens (Fig. 6.27a) the dominant mycorrhizal types were produced by the fungi Russula amoenolens, a Boletus type and Tomentella sublilacina and R. xerampalina, whether the results were expressed as frequency of occurrence (Fig. 6.27b) or as a percentage of total mycorrhizal tips produced (Fig. 6.27c). While the small, resupinate fruit bodies of the thelephoroid fungus T. sublilacina could have been overlooked, those of the Russula and Boletus types are conspicuous, and the absence of a relationship between their occurrence as tips and appearance on fruit bodies is striking. Likewise, the failure of S. pungens to appear as anything but a rare component, both in terms of frequency and occurrence, below ground despite its ability to fruit prolifically is of considerable interest. The reasons for the discrepancy between frequency and abundance of fruit bodies and mycorrhizas are not clear. Gardes and Bruns (1996) speculate that in

223

Structure and development of ectomycorrhizal roots 50 r 1/3

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Ectomycorrhizas

the case of S. pungens, which has a narrow host range extending only to P. muricata and P. radiaia, there may be a particular efficiency in C transfer between roots and fruit bodies enabling production of the latter, despite the small resource base. There is a possibility, conversely, that the process of C transfer between mycorrhizal tips and mycelium of R. amoenolens is inefficient. An alternative explanation at the population level is that those species such as R. amoenolens which fruit so rarely are made up of a large number of genotypes, the clonal sizes of which are too small to obtain sufficient C to support fruit body production. Whatever the genetic and physiological bases for the lack of correspondence between fruit body and mycorrhiza formation, the study of Gardes and Bruns (1996) is enlightening in a number of ways. It draws attention, as did that of Taylor and Alexander (1990), using conventional methodologies to the danger inherent in attempts to determine below-ground population structures from patterns of fruit body production. Further, and perhaps of greater long-term importance for the understanding of processes at the population level, it indicates that molecular tools have the power to provide the rapid and unequivocal identification of mycorrhizal types, upon which accurate determination of population structure depends.

Succession and Replacement of Fungi on Roots and Root Systems Replacement on Individual Roots: Interactive Replacement The mechanisms by which species of fungi replace each other on a root system will play important roles in any community interactions and successions in the field. Once established on a short root the mycorrhiza formed by a given fungus is resistant to replacement. Coexistence of two or more associations of ectomycorrhizal fungi can occasionally be observed on individual roots, but this is the exception rather than the rule, although dual associations have been reported to occur on as many as 1-4% of roots of Pinus radiaia (Marks and Foster, 1967). In Pinus hanksiana, Danielson and Visser (1989) observed the overgrowth of the mantle formed by early-stage fungi, particularly in the distal part of the short root, by fungi of the Suillus type. They refer to this process as one of interactive replacement and contrast it with the far more commonly seen phenomenon, so-called 'non-interactive' replacement, in which certain species die, apparently spontaneously, after a period of occupancy of the short roots. There is evidence that interactive replacement is more likely to occur as roots resume growth after dormancy. Wilcox (1968a,b) observed that the root axis of Pinus sometimes extended prior to regrowth of the fungal mantle, so that its uncolonized apex was exposed in soil to other fungal symbionts. Similarly, Betula roots initially forming mycorrhizas with Hebeloma spp., became colonized by Lactarius pubescens after dormancy (Fleming, 1985). Clearly, in view of the restricted longevity of a mycorrhizal association on a single root (see above), there is a possibility that interactive replacement is facilitated by senescence of the original colonist. There appears, as yet, to be no experimental evidence for replacement on this basis.

Structure and development of ectomycorrhizal roots

225

Replacement on Whole Root Systems In the majority of cases fungal species on root systems appear to change in composition by a process of non-interactive replacement in which one population dies, the short roots either dying with the fungi or persisting in the non-mycorrhizal condition (Danielson and Visser, 1989), while a new suite of species colonizes the next generation of short roots. A relationship between age of the plant and its root system and the susceptibility to colonization by different fungi is suggested by observations such as those of Fleming et al. (1984). They showed that whereas some fungi, such as Hebeloma spp., colonize container-grown plants of Betula in the first year, others, for example Lactarius pubescens, do so only in the second year despite the persistent presence of inoculum of both fungi. This situation mirrors that seen in the field in newly afforested sites where Hebeloma spp. are recorded as early-stage and L. pubescens as being among the first of the late-stage species. By growing 2-year-old saplings of birch in transparent troughs containing soil pre-inoculated with H. crustuliniforme and L. pubescens, Gibson and Deacon (1988) were able to follow the sequence of development of mycorrhizas formed by the two fungi as the plants aged. They found that whereas H. crustuliniforme could form mycorrhizas in any region of the root system, L. pubescens did so only on short roots emerging in the oldest parts of the system. Since the soil was of uniform quality throughout the length of the trough, it is logical to postulate that some age-related feature of the plant is the primary factor determining susceptibility to colonization. If, as is almost invariably the case, the short roots are produced in the current year, the observation of Gibson and Deacon (1988) carries the implication that their ability to accept and sustain colonization by L. pubescens is determined by an age-related feature of that part of the root system which supports them. Among the various features of the root system that have been considered as possibly contributing to age-related differences in susceptibility to colonization, the supply of C in sufficient quantities to sustain the relatively robust and highly differentiated mycelial systems of late-stage fungi emerges as being of probable importance. Gibson and Deacon (1990) compared the ability of a number of earlyand late-stage fungi to grow in pure culture and to produce mycorrhizas on Betula, using agar media containing different concentrations of glucose. Whereas representatives of the early-stage category grew continuously across agar in the absence of glucose, the late-stage fungi, including L. pubescens, required glucose at a concentration of at least 0.1% to sustain growth. Similarly, while early-stage fungi colonized Betula roots in the absence of exogenous glucose, most of the late-stage fungi failed to do so. Clearly, these experimental conditions are unrealistic and may affect the outcome of plant-fungal interactions as noted above. They nonetheless demonstrate that mycorrhizal fungi depend to different extents upon their environment for C, and raise the possibility that those in the late-stage category colonize laterals in older parts of the root system because these provide superior access to endogenous sources of organic C. The possibility of different patterns of C allocation or of C turnover in different regions of sapling root systems is amenable to investigation using ^^C02, but the necessary experimental analyses do not appear to have been

226

Ectomycorrhizas

carried out. Circumstantial evidence in favour of a carbon-related explanation for the observed colonization sequences comes from studies using transparent observation chambers (Finlay and Read, 1986c). In these, late-stage fungi are seen to have the ability to colonize the short roots in any part of a seedling root system, provided that they are growing from an established plant. The pattern is repeated when seedlings are sown in the field under established trees already colonized by late-stage fungi (Fleming, 1985). To the extent that their inoculum potential is closely related to the size of the food base from which they are growing, fungi of the late-stage types respond to C availability in the same way as their rhizomorph-producing saprotrophic counterparts such as Serpula lacrymans, Phanerochaete laevis and Armillaria mellea. The larger the base, the more aggressively can they forage for new resource units.

Physiological Processes Involved in the Regulation of Ectomycorrhiza Formation This chapter on ectomycorrhiza formation cannot be considered complete without a discussion of two theories occupying centre stage in the ongoing debate over the nature of physiological processes involved in the initiation and regulation of the ectomycorrhizal symbiosis. These, the so-called 'carbohydrate theory' of Bjorkman (1949) and the 'auxin theory' of Slankis (1973), have been critically re-examined by Nylund (1988). Both suffer from similar methodological and conceptual limitations. The methods available to Bjorkman for the analysis of tissue carbohydrate status and to Slankis for evaluation of the effects of auxins, were not sufficiently sensitive to enable reliable conclusions to be drawn from their experiments. Furthermore, in conceptual terms the view that the complex and, by definition, interactive processes involved in the formation of mycorrhizas could be controlled by single factors, is inherently unsatisfactory. The Bjorkman carbohydrate theory, reduced to its bare essentials, was that mycorrhizas develop only under circumstances where the roots of the plant contain a 'surplus' of soluble carbohydrates. He acknowledged the earlier observation of Hatch (1937), using Pinus sirobus, that mineral nutrient deficiency, especially of N, was a prerequisite for mycorrhiza formation, but believed that the effects were mediated through the higher carbohydrate concentrations in nutrient deficient plants. Some support for the view of Bjorkman was later provided by Marx et al. (1977), who grew P. taeda inoculated with P. tinctorius with 10 different combinations of fertilizer treatment and determined the interactive effects of nutrients and sugar concentration using more reliable analytical techniques. Differences in sucrose concentration accounted for 85% of the variation in mycorrhiza development and there was a strong negative correlation between high concentrations of N and sucrose concentration. However, when Rudawski (1986) carried out similar experiments she found only weak correlations between sugar concentrations and mycorrhiza formation. In an attempt to re-evaluate the interactive effects of N and sugar concentrations in the process of mycorrhiza formation, Wallander and Nylund (1991) grew Pinus sylvestris seedlings with Laccaria laccata in a balanced, circulating nutrient medium.

227

Structure and development of ectomycorrhizal roots 500 400 300

200

lOOH

16

1-8

20

2-4

2-2

N concentration in shoots {%)

1

400-

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300-1

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O

100-

O

o

•

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LU

04

- t

I

5

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7

6

j

8

Carbohydrate concentration in roots {%)

Figure 6.28 (a) Relationship between ergosterol concentration in roots of Pinus sylvestris colonized by Laccaha bicolor and the internal N concentration In the shoots. Y = 1889 X""^' (r^ = 0.73). (b) Relationship between ergosterol and sugar concentrations in the roots of Pinus sylvestris colonized by Laccaha bicolor. Pooled samples from five seedlings. Concentrations of N in the medium in mg N I " ' . A , 10 mg N; O, 100 mg NH4; • , 100 mg NO3; Q . 300 mg NH4; • , 300 mg NO3. • , non-mycorrhizal control. From Wallender and Nylund (1991) with permission. Table 6.8 Soluble root sugar and needle nitrogen concentration in Scots pine seedlings before inoculation with Laccaha bicolor Nitrogen treatment (mgNJ-')

0-10 N-b 200 NH4

Ergosterol (juig per grann of root) (after Inoculation)

Nitrogen (%) needles (before inoculation)

Root sugar (%) (before Inoculation) Whole roots

Lateral roots

1.5 (0.2) a 2.7(0.1) b

4.6 (0.1) a 4.9 (0.3) a

3.6 3.2

214 (38) a 112(16) b

Standard errors in parentheses. Different letters indicate statistically different values (P < 0.05) using Student's t-test (three replicates). Values without letters (lateral roots) represent pooled samples of three seedlings. N-b indicates N balanced with other ions. Data from Wallender and Nylund (1991).

228

Ectomycorrhizas

containing high or low levels of N. They assessed mycorrhiza formation quantitatively, in fungal biomass on the roots, using the ergosterol method of Salmonowicz and Nylund (1988). Regression analyses applied to their data (Fig. 6.28a) showed that variations in N concentration in the shoots could alone explain differences in fungal colonization and they concluded (Table 6.8) that N had an effect on mycorrhiza formation that was not mediated through carbohydrate concentration (Fig. 6.28b). This does not mean that C availability can be ignored as a factor in determining colonization. It is abundantly clear from experiments carried out on the impact of light deprivation (Bjorkman, 1970) and from many others examining C movement from plant to fungus (see Chapter 7) that the heterotroph has an absolute requirement for C derived from the plant. It is the notion of 'surplus' C that should be abandoned, especially as it is increasingly evident that the fungus has considerable independent power to influence the extent and polarity of C allocation within the plant. The hypothesis, proposed by Slankis (1973), that mycorrhiza formation was regulated through the controlled production of auxins by the fungus appears to be somewhat better supported by evidence now, than it was when Harley and Smith subjected it to critical examination in 1983. The development of superior analytical methods has enabled the ability of several ectomycorrhizal fungi to produce lAA in monoxenic culture, to be convincingly demonstrated (Ek ei al., 1983; Frankenberger and Poth, 1987). However, analyses of the lAA content of mycorrhizal roots have produced conflicting results. A study by Mitchell et al. (1986) showed significantly more lAA in roots of Pinus echinata colonized by Pisolithus tinctorius, while another (Wallander et ah, 1994), using Pinus sylvestris associated with Laccaria hicolor, reported a 40% reduction of lAA in colonized, relative to non-colonized, roots. The prospect of some progress towards resolving the question of the role, if any, of auxins in regulation of the symbiosis, is provided by genetic manipulation that enables the production of fungal mutants expressing an ability to metabolize endogenous tryptophane and so release lAA in greater quantities than the wild type, independently of tryptophane supply from the plant. These mutants have been produced from wild-type monokaryons of Hebeloma cylindrosporum by a twostep procedure using fluoroindole resistance (FIR) as the selection factor (Durand et al, 1992). The mutants were stabilized by re-selection after passing through a meiosis, at which time their segregation ratio indicated that lAA overproduction resulted from a single gene mutation. Gay et al. (1994) have subsequently shown that the high overproduction of lAA of these mutants gives them an advantage in the formation of mycorrhizas: 3-6 times more colonized roots are produced on Pinus pinaster seedlings by FIR mutants than by the wild type. At first sight this might be interpreted as evidence in favour of the Slankis hypothesis, but in the_ course of this work Gay et al. (1994) observed that there was a lack of correlation between the lAA synthesizing ability of the mutants measured in pure culture (Durand et ah, 1992) and their mycorrhizal activity. Clearly, a number of plant, fungal and environmental factors interact to determine the outcome of the association between fungi and plant. Despite these complications, it was widely observed that mycorrhizas formed by overproducer mutants could be characterized by the presence of a thicker Hartig net made up of multiseriate files of fungal cells (Fig. 6.29) in contrast to the thinner uniseriate

Structure and development of ectomycorrhizal roots

229

Figure 6.29 Extensive development of a multiseriate Hartig net in a mycorrhiza formed between Pinus pinaster and a mutant of Hebeloma cylindrosporum overproducing lAA (see text). CC, Cortical cells; N, nucleus; M, mitochondria, L, lipid droplets; P, globules of continuous phenolic deposits; cy, cytoplasmic layer. Arrows point to polyphosphate granules. Bar, 2 jim. From Gea et al. (1994), with permission.

system formed by the wild type (Fig. 6.30a). Detailed structural comparisons of roots of P. pinaster colonized by overproducer and wild-type strains of H. cylindrosporum (Gay et al, 1994) have confirmed these differences. The mutant type (Fig. 6.30b) produced a more highly developed Hartig net, up to seven layers of hyphae in width, which reached the endodermis. One further feature of the mycorrhizas produced by mutant fungi, not seen in wild-type associations, was the occurrence of intracellular hyphae surrounded by plant plasma membrane and tonoplast. Despite these extreme manifestations of invasiveness there was no indication of pathogenesis. While thus appearing to produce a balanced association with the

230

Figure 6.30 Light microscopy of transverse sections of mycorrhizas formed between Pinus pinaster and Hebeloma cylindrosporum. (a) Wild-type H. cylindrosporum monokaryon hi. Note the uniseriate Hartig net (HN) and mantle (M), surrounding cortical cells (CC), endodermis (e), vascular cylinder (vc). (b) lAA over-producing mutant monokayron hi FIR4 FI 331. Note the extensive development of the Hartig net (HN). Letters defined as In (a). Bars, 13 jim. From Gea et al. (1994), with permission.

host, the plants colonized by the mutants did not show advantages in the form of growth increases relative to those associating with the wild type. Perhaps this is what would be expected in view of the greater fungal biomass to be sustained by the autotroph. Such mutant fungi clearly hold out the prospect for considerable advances in our understanding of the role of auxins in the symbiosis. Studies of seedling hypocotyls (Rayle and Cleland, 1992) suggest that auxins, by inducing localized acidification, facilitate loosening of wall components to permit cell elongation. They may also cause polymerization of cellulose components of the cell wall (Fry, 1985). As both processes appear to be involved in Hartig net formation (see above), there is the possibility of using over-producer mutants to elucidate mechanisms of auxin action as the symbiosis develops.

Structure and development of ectomycorrhizal roots

231

While it is sensible to consider the role of auxins as one of many agents contributing to the regulation of ectomycorrhiza formation, the notion of an 'auxin theory' of regulation clearly represents an oversimplification. Specific biochemical roles for auxins in the regulation of cell metabolism have been identified in higher plant tissues; notable examples amongst these are the switching on of 'G' proteins by lAA, the triggering of transmembrane ion fluxes in guard cells by abscisic acid and the aforementioned wall loosening in cell growth. The search for defined biochemical mechanisms, involving auxins, in the mycorrhizal symbiosis is to be encouraged, and the development and use of lAA overproducer mutants is a step in the right direction. However, until such time as a specific role for auxins is identified there would seem to be no cause to change the view, expressed by Harley and Smith (1983), that the plexus of hypotheses elaborated by Slankis lack full credibility.

Conclusions A combination of rigorous descriptive and experimental approaches is providing new insights into the development, mature structure and dynamics of ectomycorrhizas both at the levels of the individual root and the comanunity. These insights are essential prerequisites for further understanding the functional attributes of the association. The use of light and electron microscopy in combination has enabled the development of the symbiosis to be studied in detail in a number of plant-fungus partnerships from precolonization to maturity. The main structural features of the three diagnostic components of ectomycorrhizas - the Hartig net, mantle and extraradical mycelium - have been revealed. The Hartig net is seen to be a structure with the properties of transfer cells, in which effective exchange of solutes between partners would be facilitated, albeit over a relatively short life span. Its conformation is similar in all plant-fungus combinations examined, however, the extent of penetration of the root is more superficial in most angiosperms than in gymnosperms. The hyphal mantle, in contrast, has a structure diagnostic of the fungus species forming it. Recognition of its distinctive anatomical features is enabling a rigorous classification of types and, increasingly, the ability to identify them to the level of fungal genus or even species in the absence of fruit bodies. Molecular tools are beginning to be applied to questions of identity of mycorrhizal types and have progressed to allow consideration of population changes in the roots themselves. Structural and molecular methods provide complementary, rather than competing, approaches to analysis of the structure of ectomycorrhizal communities. Ideally, the two methods might be applied simultaneously to a given population to provide the potential for cross-checking of results. Undoubtedly, however, there will often be circumstances where logistics, in particular the availability of the necessary skills or equipment, will dictate the approaches to be taken in a particular study. Studies at the community level have revealed successions of fungal species on roots as trees age. These were first recognized in terms of appearance of fruit bodies, but have progressed to consideration of population dynamics of the mycorrhizal roots in the field. Analysis of root populations, whether using morphotyping.

232

Ectomycorrhizas

anatomy or molecular methods, reveal that discrepancies can occur between aboveground and below-ground assessments of fungi symbiotic with plants, indicating the need for thorough analysis of roots themselves in studies at the community level. Some of the mechanisms causing successional developments on roots have been elucidated. They appear to involve ageing of individual roots and of root systems, and are complicated by seasonal effects such as dormancy. Much more information is required on plant-fungus interactions and their changes in time before a full mechanistic understanding will be achieved. In relation to the capture of resources, there has been a major shift of emphasis away from the ectomycorrhizal short root itself, to consideration of the extraradical mycelium as the structure priniarily involved in exploration and exploitation of soil. There is recognition of the need to examine intact systems, and to evaluate spatial and temporal aspects of mycelial development with soils as sources of nutrients and plants as sinks. These studies are in their infancy and are still to some extent constrained by methodological difficulties, but progress is being made. We are still, unfortunately, a long way from being able to investigate the vegetative mycelia of ectomycorrhizal roots in a non-destructive manner in the field. While past emphasis in ectomycorrhizal research has been placed on attributes of fungal species, there is increasing realization that structural and functional diversity within species can be as large as that at the interspecific level. Using a small number of ectomycorrhizal species that can be induced to fruit in culture, and thus produce offspring of known genotype, the genetic basis of intraspecific variability is being examined, in particular as it relates to compatibility phenomena, population structure and function. The ability to produce biochemical mutants also promises to increase our understanding of the processes involved in formation of the symbiosis and its function under particular nutritional conditions.

Plate 2. Effects of mycorrhizal inoculation of a range of crop plants in fumigate soil. Right-hand block, inoculated with VA mycorrhizal fungi. Left-hand block, not inoculated. Crops (front to back): Allium, Catalpa, Pisum, Vicia, Zea. Non-host plants trimmed. Photograph courtesy of V. Gianinazzi-Pearson.

Plate 3. Lactahus subdulcis mycorrhiza, formed between L subdulcis and Fagus sylvotica. (a) Irregular pyramidally branched mycorrhizal system (X5.5). (b) Irregular pyramidally branchged mycorrhizal system ( X I 0.6). (c) Tip of mycorrhizal axis (X44). (d) Rhizomorph ( X 4 4 ) . From Brand (1987), with permission.

Plate 4. FagirhizQ setiferOy formed on Fagus sylvatica by an unknown fungus, (a) Ectomycorrhizal lateral roots with different levels of branching (X5.4). (b) Several nnonopodial ectomycorrhizal systems interconnected by hyphae ( X I 0.6). (c) Two monopodial ectomycorrhizas with rough mantle surface and hairy emanating hyphae (X22). (d) Ectomycorrhiza densely covered by long spines. From Brand (1991), with permission.

Plate 5. Russula ochroleuca mycorrhiza, formed between R. ochroleuca and Picea abies. (a) Different mycorrhizal systems (X5.5). (b) Pinnate mycorrhizal system with conspicuous rhizomorph ( X I 0.6). (c) Mycorrhizas of different ages (X22). (d) Mycorrhiza showing typical greenish-yellow patches (X44). From Agerer (1987, 1993), with permission.

Growth and carbon economy in ectomycorrhizal plants

Introduction Frank (1894), conducted experiments on the effects of ectomycorrhizal colonization on the growth of seedlings of Pinus. Those which were grown in unsterilized soil developed mycorrhizas and grew faster than those in sterilized soil. Although this experimental design is faulty, because heat sterilization may release both nutrients and toxic substances, the results have been, in principle, confirmed repeatedly. Refinements aimed at obviating the effects of heat sterilization have been used and the results can be accepted with confidence. In addition, observations on the establishment of exotic species of ectomycorrhizal trees in many parts of the world have shown that artificial inoculation is usually essential to success, although nonmycorrhizal seedlings may be grown if provided with adequate fertilizers. Generally, emphasis has been placed upon growth of the plant but, increasingly, as the extent and importance of the external mycelium is becoming recognized, attention has been given to the C demands of the fungus in intact systems. These, often seen as a 'cost' of the symbiosis, are being quantified in intact systems and assessed in relation to the 'benefits' seen to accrue to the plant from improved access to the nutrient resources of the soil. When considering the effects of mycorrhizal colonization in terms of growth and yield, it is necessary to bear in mind that although these analyses are much easier to carry out than are many other determinations of response, they do not provide a direct measure of 'fitness'. Measurements of survival in the regeneration phase, and of fecundity in the adult plant, would provide direct indications of impacts of the symbiosis upon fitness but these, especially the latter, are difficult to obtain in long lived ectomycorrhizal plants. It is therefore largely for reasons of experimental convenience that responses to colonization have normally been assessed in terms of vegetative growth.

Plant Growth Surprisingly, while there have been innumerable reports of growth enhancement as a result of ectomycorrhizal colonization, relatively few reports have provided

234

Ectomycorrhizas

unequivocal evidence, in the form of nutrient response curves, of the benefits accruing to a plant from colonization when its growth is limited by a single nutrient. Some such studies are now available (e.g. Heinrich and Patrick, 1986; Bougher et al, 1990; Jones et al, 1990). The study of Bougher et aL, (1990) is particularly instructive in the overall context of growth response. Eucalyptus diversicolor plants, whose small seeds have little iiutial nutrient reserve, were grown in a sandy soil of low organic content which was given mild sterilization unlikely to produce unnatural side-effects. Furthermore, the experiments examined responses to inoculation with four fimgi (two isolates, A and B, of Descolea maculata, and one each of Laccaria laccata and Pisolithus tinctorius) and to one element, P, the deficiency of which was known to be a major factor limiting growth in soils supporting the plant in nature. Ecological relevance was thus added to good experimental design, and the results provide a useful example of the type of growth response that might be expected in the field. The growth response curves of non-mycorrhizal plants in relation to soil P are of the sigmoid type, as seen typically in other coarse-rooted plants (Bolan et ah, 1983; Fig. 7.1a). In these there is a threshold P concentration in soil below which growth will not occur. Colonization by D. maculata A and B and L. laccata modified the sigmoidal growth curve by removing the threshold effect and allowing plants to grow in extremely nutrient-poor soil (Fig. 7.1a). This effect is believed to result from the ability of extraradical mycelium to exploit nutrients, in this case P, beyond depletion zones surrounding the root (see Chapter 5). The fungi may also have a lower threshold concentration for absorption of the element, or may have the ability to release P associated with complex substrates which would add to their ability to increase nutrient uptake (see Chapter 9). In the range of P supply in which a growth response to mycorrhizal colonization occurred (2-12 mg P kg~^ soil) fungal isolates differed in their ability to promote seedling growth (Fig. 7.1a). Dry weight of seedlings inoculated with L. laccata was significantly greater than that of plants colonized by D. maculata at 2 and 4 mg P kg"^ soil. At the latter concentration, biomass of L. laccata seedlings was nine times that of seedlings mycorrhizal with D. maculata and 21 times that of nonmycorrhizal plants. The pattern of P accumulation by plants in relation to soil P supply was similar to that of dry weight (Fig. 7.1b) and again mycorrhizal colonization removed the threshold seen in uncolonized plants. However, seedling dry weight reached a maximum at around 28 mg P kg~^ soil, whereas P content increased linearly above this level of P application (Fig. 7.1a). Mycorrhizal plants, in addition to showing no threshold, had higher tissue P concentrations than non-mycorrhizal plants even with no added P. This example, in addition to demonstrating that mycorrhizal colonization has the potential to enhance growth, shows that it can fundamentally change the nature of growth response curves of plants at low nutrient concentrations. It further emphasizes that there may be considerable differences both between and within fungal species in their ability to acquire nutrients and promote growth. This biologically important observation also indicates that response curves comparing the performance of different mycorrhizal fungi can be used to predict more accurately their potential for application and effectiveness for nursery and afforestation

235

Growth and carbon economy in ectomycorrhizal plants

1 (a) / •^ /•

Vu 7 1 // -

/

3

0

10

20

30

40

50

Soil phosphorus applied (mg P kg"^ soil)

0

10

20

30

40

50

Soil phosphorus applied (mg P kg~^ soil)

Figure 7.1 Growth and P uptake of Eucalyptus diversicolor in response to P application and mycorrhizal colonization by two strains of Descolea maculatOy (D) A and (O) B or by ( A ) Laccaria laccatOy compared with ( • ) uninoculated controls, (a) Dry weight, (b) P content. From Bougher et al. (1990), with permission.

practices (see Chapter 17). However, it must be borne in mind that this selected example describes responses to P when other nutrients were supplied in adequate amounts. Depending upon the soil or ecosystem in which a plant occurs, other elements, in particular N (see Chapter 8), may be more important as growthlimiting factors, and often P and N may be co-limiting. Experiments investigating dose response curves in these different situations would be extremely instructive. The difficulties of carrying out experiments with trees under controlled conditions have precluded extensive research except during a small part of their early life. In consequence, comparisons between mycorrhizal plants and controls have been made over one or a few seasons of seedling life at most. Much work has also been done in open beds of soil, sometimes inoculated with soil, humus or chopped mycorrhizas, with controls treated with sterilized inoculum (examples are given by

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Ectomycorrhizas

Harley, 1969). In some cases the soil has been sterilized by drenching or fumigation before planting. The published conclusions are simple: mycorrhizal seedlings are usually taller and have larger root systems, both shoots and roots are of greater dry weight but the ratios of root weight to shoot weight are frequently smaller. The change in the root .shoot ratio needs further investigation because, irrespective of mycorrhiza formation, the ratio diminishes during the early growth of the young seedling and is lower in soils of high nutrient availability, especially of N, as well as being sometimes reduced in conditions where photosynthesis is reduced. Although a lower ratio might be expected in mycorrhizal plants, both because of a greater ability to absorb nutrients and an increased concentration of nutrients in their tissues, it would be of great value to know more about the extent to which greater size and age themselves contribute to C allocation in the seedlings. Alexander (1981) grew Picea sitchensis seedlings in monoxenic culture with Lactarius rufus for 14 weeks. The results are shown in Table 7.1. The usual increase of height and dry weight of both shoots and roots was observed but the root:shoot ratio was greater (although below P=0.05 significance) in mycorrhizal plants. In discussing this, Alexander observed that if the fungal sheath were to comprise 20% of the weight of the roots, which is not impossible, the slight rise in root:shoot ratio might be explained by the fungal colonization directly. Bougher et al., (1990) also provided data relating root:shoot ratio of £. diversicolor to total seedling dry weight, which showed no differences in response to the fungi they employed and, further, no differences between mycorrhizal and non-mycorrhizal plants. This again emphasizes that effects on root:shoot ratio may be related less to colonization than to plant size. Other observations of Alexander (1981) seem to confirm the much older results (e.g. McComb, 1938) that colonization increases the total number of short roots per seedling. In this case the form of the root system changes, as there are more short Table 7.1 Growth of Picea sitchensis in axenic culture with Laaarius rufus during 14 weeks Control

Mycorrhizal colonization (%)

0

Inoculated

Significance of difference P <

58.2 ± 9.9

Shoot height (cm) Shoot dry weight (mg) Root dry weight (mg) Total dry weight (mg) Root:shoot ratio

5.8 47.1 25.5 72.6 0.59

9.2 100.2 74.0 174.2 0.76

0.05 0.01 0.001 0.001 NS

Number of lateral buds Length of lateral roots (cm) Total number of root tips Short roots per cm of lateral roots Short roots per mg of root weight

4.4 98.2 60.8 0.64 2.43

5.9 219.0 236.7 1.17 3.31

NS 0.01 0.05 NS NS

Substrate: vermiculite-peat with nutrient medium. The roots were kept below 25°C in a greenhouse. Midday irradiance was approximately 25 W m~^. NS, not significant. Data from Alexander (1981).

Growth and carbon economy in ectomycorrhizal plants

237

roots both per cm of root length and per mg of root weight on the mycorrhizal seedlings. There is a great need to disentangle the normal changes in form due to increase in size, those dependent on changes in nutrient supply and absorption, and those arising from mycorrhizal colonization and its physiological consequences. Moreover, although mycorrhizal colonization is known to increase growth rates of plants supplied with sub-optimal levels of a major nutrient such as P (Bougher et ah, 1990), relatively few studies have examined the consequences of ectomycorrhizal development in plants receiving optimal supplies of nutrients. On theoretical grounds it is evident that under these circumstances the benefits of colonization in the form of increased nutrient capture would be eliminated, yet the costs to the plant in terms of C supplied to the fungus for growth and respiration (quantified below) would remain. In fact several studies (Molina and Chamard, 1983; Ingestad et ah, 1986; Rousseau and Reid, 1991) indicate that while mycorrhizal plants suffer little or no growth reduction at adequate or supra-optimal nutrient supply, such reductions are experienced under lower nutrient regimen. A possible explanation for this apparent anomaly has been sought by Rousseau and Reid (1991). They point out that plants in the higher nutrient regime generally have lower root:shoot ratios relative to those under low regime. By definition plants with lower root:shoot ratio have more tissues that are able to gain rather than consume C. Therefore, even if equal quantities of root tissue were colonized in seedlings with different root:shoot ratios, those with the higher ratio would have proportionately more mycorrhizal development per plant than their counterparts with a low ratio of root to shoot. Thus, if a mycorrhizal fungus constituted 20% of the root system of a seedling with a 1:1 ratio of shoot to root, it would make up 10% of the dry weight, in cases where that ratio was 3:1 the weight of fungus would contribute only 5%. The relative C costs of supporting the fungus in a seedling with a 1:1 shoot:root ratio could thus be double that of a seedling with a 3:1 ratio, even though the fungal biomass was the same in both cases. There are data to show that in reality the extent of C costs imposed by the fungus under high nutrient regime may be further reduced by inhibition of development of the extraradical mycelium (Jones et ah, 1990; Wallander and Nylund, 1992; and see Chapters 4 and 5). Results which show no effect of mycorrhizal colonization or even a decrease of growth rate may be found scattered through the literature, but are not often stressed. They are important for two reasons. First, they show that ectomycorrhizal colonization does not invariably increase growth of the plant in size and weight, and that there is frequently an initial phase, sometimes inexplicably prolonged, when the growth of mycorrhizal plants is similar or slower in rate than that of nonmycorrhizal ones. Second, they often show that different combinations of plant and fungus are differently effective in growth, as already discussed for Eucalyptus diversicolor. Although different species of fungus were compared on a single species of plant, there is good evidence that different strains of a single fungal species also differ in their effects on a plant. Table 7.2 gives one example from the work of Marx (1979a) of the effect of different strains of Pisolithus tinctorius on Quercus rubra seedlings. Variability of this kind is exactly what would be expected from the genetic, physiological and biochemical variability of ectomycorrhizal fungi. In a similar way, different biotypes of a plant may react differently with a single strain of a fungal species (Marx and Bryan, 1971; Marx, 1979b), although research on this aspect has not yet gone far. Clearly, further information on both these topics is

238

Ectomycorrhizas

Table 7.2 Grovs^h of Quercus rubra seedlings with different strains of Pisolithus tmctorius Fungal strain

Height (cm)

Fresh weight (g)

Ectomycorrhizal percentage of short roots

138 136 145 Control

13.3 13.5 11.5 12.5

8.5a

72a

6.5c

15b

7.0b

Ic

6.7c

0

Day temperature 28°C, night 22°C. Day length 14.5 h. Growth period 4 months. Height differences insignificant at P = 0.05. Within columns, common letters denote insignificance. Percentage of ectomycorrhizas estimated visually. Data from Marx (1979a).

essential to the full elucidation of the physiological relationships between the symbionts and to the determination of the most efficient combinations of fimgus and plant for the production of forest crops. Experimentation should aim at describing in detail the structure of mycorrhizas which appear to be associated with greater or lesser growth rate. Information concerning Hartig net development, sheath thickness and particularly the quantity and extent of extraradical mycelium needs to be collected from plants grown in near-natural conditions, in order to calculate the C demand of the fungus (see below). Some advances in this area have been discussed in Chapter 6. Equally important are physiological properties of the fungal strains, in particular economic coefficients such as mass of C used per mass of mycelium formed. By such analyses the recognition of effective symbionts could be put on a firm basis, as has been done for single plant-fimgus combinations by Jones et al., (1991) and Rygiewicz and Anderson (1994; and see below). In all this work the growth of the plant has been the main preoccupation of the experimenters because they have been concerned, however distantly, with its ecology or its use for human benefit. From the point of view of understanding the physiology of the symbiosis, much more knowledge is needed concerning the physiological activities of the fungus, in symbiosis and in culture, and the factors that affect them. The decreases in dry weight of the plant which sometimes follow colonization appear to be exactly similar to those observed with vesicular-arbuscular (VA) mycorrhizal plants (see Chapter 4). A decrease in growth might be expected in a system where one symbiont depends on the other for C compounds, and the other symbiont depends on the first for nutrients essential for growth and photosynthesis. Decreases in growth rate would be expected in conditions of irradiance that limit the rate of photosynthesis but not the intensity of mycorrhizal colonization, or where the supply of soil-derived nutrients is adequate for growth but is not high enough to decrease the intensity of colonization. Similar examples where mycorrhizal colonization results in no increase or sometimes a decrease in growth rate of the plant are also found in ericoid mycorrhizas. Having discussed in general terms the growth of ectomycorrhizal plants, normally regarded as being a process of C accumulation, it is now appropriate to

Growth and carbon economy in ectomycorrhizal plants

239

consider details of C requirements of the fungi before returning later to examine C distribution in intact mycorrhizal systems. Carbon Supplies for Ectomycorrhizal Fungi Unlike the glomalean VA mycorrhizal fungi, many of the fungi of ectomycorrhizas have been isolated into culture and the physiology of their growth has been studied. However, the results have been disappointing. Much has been learned about factors that affect mycelial growth but little about intermediary metabolism or biochemistry. No positive characteristic that might explain the mycorrhizal habit has yet been discovered. Few of the early experiments even considered intermediary metabolism, respiration, secretion of metabolites and similar activities except in the sphere of the production of auxin-like substances. Frank (1894) assumed from the first that the source of organic nutrients for mycorrhizal fungi was the photosynthetic plant, although he recognized that these might be supplemented by supply from soil. Much later, Melin (1925) began to examine experimentally the requirements of these fungi for organic C. He found that most strains had little ability to grow on complex polymers, such as might be found in litter and humus, and that they could not use lignin or cellulose. They therefore seemed to be dependent on simple sugars such as might be produced by, or released from, the roots with which they were associated. Rommell (1938,1939a) showed that a number of ectomycorrhizal fungal species depended upon being associated with living roots to produce fruit bodies, a reasonable expectation if they depended on the roots for C supplies. The positive relationship between irradiance and carbohydrate concentration in the root system, and the intensity of mycorrhiza development in Picea and Pinus shown by Bjorkman's experiments (1942-1956) further appear to be consistent with this view. From a survey of much work on growth of ectomycorrhizal fungi in culture, Harley and Smith (1983) concluded that most of the ectomycorrhizal fungi have, at most, a limited ability to use lignin and cellulose as substrates for growth. Despite reports of slight lignase and cellulase activity in some such fungi, their ability to degrade polymers of this kind is much less than that of wood decomposers or even of some ericoid mycorrhizal fungi (Trojanowski et ah, 1984; Haselwandter et ah, 1990; Caimey and Burke, 1994). They vary within and between species in the ease with which starch, glycogen and inulin, the simpler oligosaccharides and the disaccharides sucrose and trehalose are used. The monosaccharides glucose, mannose and fructose are usually good sources of C for growth, whereas pectic substances can be used for growth by some but not by others. This has proved important in studies of the mechanisms of transfer of C from plant to fungus (see below and Chapter 14). The potential to use different sources of C clearly varies between fungal species and strains, and could possibly be related to fungal survival in soil and to colonization strategies. However, cellulose- and pectin-degrading abilities might be related not to use of litter as a source of organic C, but rather to penetration of root tissues, where enzyme production need only be localized and the degradation linked to softening the cell walls during Hartig net development. Much lower activity would be required for penetration than for releasing monosaccharides from complex

240

Ectomycorrhizas

polymers in amounts likely to affect the growth of the fungi. This point was clearly appreciated by Lindeberg and Lindeberg (1977), and research directed towards clarifying the extent of production of enzymes within plant roots and their roles in colonization processes is discussed in Chapter 6. The abilities of the free-living fungi to use organic C sources in soil has only limited relevance to the nutritional interactions of the associated symbionts. Bjorkman's conclusions (1970) that high internal carbohydrate supply in the roots favoured fungal colonization, although subject to recent criticism (Nylund, 1988), seem to have been upheld by Marx et al., (1977). However, the fact that the root system is more susceptible to colonization when it contains high concentrations of soluble carbohydrates such as sucrose is not in itself proof that the fungal symbiont derives its supply of C in whole or in part from the plant. Proof of this was given by Melin and Nilsson (1957) who fed ^ CO2 to the leaves of axenically grown Pinus sylvestris seedlings in combination with either Suillus variegatus or Rhizopogon roseolus. Photosynthetic products were translocated through the seedlings and were found in the roots and in their sheaths. Although this experiment demonstrated very clearly that the products of photosynthesis were translocated directly and rapidly to the fungal symbiont in ectomycorrhizas, it did not show anything about the quantities involved. Nor did it prove that all C compounds in the fungus were derived from the plant. Moreover, the controls in these experiments were decapitated mycorrhizal plants which also accumulated ^^C in their tissues. The quantities at the end of the experiment amounted to 6, 11 and 15% of that in the photosynthesizing plants in stem, non-colonized roots and mycorrhizas, respectively. Lewis (1963) pointed out that this was explicable in terms of dark fixation of CO2, which commonly occurs in plant material. Much was learned about the C metabolism of ectomycorrhizas of Fagus using excised roots and some of this work is certainly relevant to a discussion of the supply of organic C to the fungal sheath by the plant (Harley and Jennings, 1958; Lewis, 1963; Lewis and Harley, 1965a,b,c). The monosaccharides glucose and fructose are readily absorbed by excised mycorrhizas from aerated solutions, but glucose is selected preferentially from mixtures. In mycorrhizas sucrose is hydrolysed by an invertase attached to the plant wall, and glucose preferentially absorbed from the products. The rate of absorption of hexoses is temperature and oxygen dependent, and inhibited by metabolic inhibitors of the cytochrome oxidase pathway and of oxidative phosphorylation. The analysis of the tissues after uptake shows that the change in concentration of glucose and other monosaccharides is small and absorbed hexoses are rapidly converted to other compounds. Amongst the storage carbohydrates the mycorrhizas contain those typical of both the plant and the fungus (Table 7.3). Glucose and fructose are common to both. Trehalose, mannitol and glycogen are fungal, whereas sucrose and starch are from the plant. It may be assumed that sucrose is totally or dominantly a plant sugar. The typically fungal carbohydrates, trehalose and mannitol, are absorbed by nonmycorrhizal roots at rates only one-tenth and one-twentieth of that of mycorrhizas (Table 7.4). The disaccharides, sucrose and probably trehalose, are hydrolysed before absorption, and glucose and fructose have different destinations in the tissues. Since they compete in uptake they probably have a common transporter on the plasma membrane. However, glucose is mainly converted to trehalose and fructose to mannitol amongst the soluble carbohydrates of the mycorrhizal sheath.

241

G r o w t h and carbon economy in ectomycorrhizal plants

Table 7.3 Carbohydrate content of excised mycorrhizas and changes after storage for 20 h at 20°C in water or in 0.5% (w/v) solutions of glucose, trehalose, sucrose, fructose or mannitol Initial

Changes after storage in:

sample Total soluble Total reducing sugars Sucrose Trehalose Mannitol Insoluble (glucose units) Total carbohydrate present

Water

Mannitol

Fructose

Sucrose

Trehalose Glucose

14.49

-5.36

-5.66

-1.53

-0.10

+2.02

+2.91

4.86 5.07 4.74 Nil

-1.02 -2.34 -2.00 Nil

-0.22 -3.20 -2.24 +7.69

+0.34 -0.99 -0.88 +6.36

-0.43 -0.80 + 1.13 +3.86

-0.69 -0.06 +2.77 +2.27

+0.06 +0.28 +2.57 +3.86

25.94

-2.84

+3.87

+6.83

+5.20

+8.10

+ 11.14

40.63

32.43

38.84

45.93

45.73

50.75

54.68

Values as mg per gram fresh weight (= 150 mg dry weight approximately). Data from Lewis and Harley (1965a).

Table 7.4 Relative rates of uptake of carbohydrate from solution by mycorrhizal and non-mycorrhizal roots of Fagus Sugar supplied

Ratio of uptake rate mycorrhizal: non-mycorrhizal

Glucose Fructose Sucrose Trehalose Mannitol

3.1 2.7 2.3 11.8, 10.8 19.6,23.6

Data from Lewis and Harley (I965b,c).

Lewis and Harley (1965c) conducted experiments in which they fed the cut stumps of Fagus mycorrhizas with ^^C-sucrose by placing agar blocks on them (Fig. 7.2) in order to stimulate C supply in intact roots. The analysis of the apical region of the mycorrhizas after separation of the sheath and core tissue by dissection (Fig. 7.2c) showed movement of C into the sheath. Between 55 and 76% of the ^^C that had been translocated through the plant tissues to the tip region and not released as CO2 was found in the fungal sheath where it was present mainly as trehalose, mannitol and glycogen. The uptake and destination of monosaccharides has been confirmed in intact systems (Soderstrom et ah, 1988). The inhibitory effect of fructose on glucose absorption has also been confirmed and has been suggested, together with regulation of apoplastic invertase activity, to be a factor important in the control of transfer of C from plant to fungus (Salzer and Hager, 1993; and see Chapter 14). Amanita muscaria, Hebeloma crustuliniforme and Pisolithus tinctorius are unable to

242

Ectomycorrhizas

(a)

3mm

1.5% agar + CC) sucrose

1^— 3mm- -H<-

10mm

(b)

I

3mm

2mm

Sheath

1.5% agar + [^*C) sucrose

^

Core Sheath Core

i:^^

} '''!^!"^ i '^V'J'::!l^V^'^^?'l'M'!J/^-v'

ssssssss

Figure 7.2 Schematic drawings to show the method of feeding mycorrhizal roots with '"^C-labelled sugars in agar blocks in studies of '^C transfer from plant to fungus (see text), (a) The agar block abuts on both sheath and core, (b) A collar of sheath tissue has been removed to prevent contact between the fungus and the agar block, so that direct transfer of ['"^Clsucrose to the fungus is prevented, (c) Dissection of the fungal sheath from the root core at the end of the feeding period. Illustrations modified from Lewis and Harley (1965c).

use sucrose in culture because they lack a wall-bound invertase that would enable them to hydrolyse the disaccharide to glucose and fructose (Taber and Taber, 1987; Salzer and Hager, 1991; Schaeffer et al, 1995). Protoplasts of A. muscaria can absorb both these monosaccharides but, whereas the uptake of fructose is strongly inhibited by glucose, the converse was not found and nor did sucrose inhibit uptake of either monosaccharide. The uptake system had a much higher affinity for glucose than for fructose (X^ 1.25 and 11.3 mM, and y^ax 18 and 30 pmol per 10^ protoplasts per min, for glucose and fructose, respectively; Chen and Hampp, 1993). It appears that the only way that A. muscaria can use sucrose is in symbiosis, when the

Growth and carbon economy in ectomycorrhizal plants

243

disaccharide is hydrolysed by an apoplastic or wall-bound invertase derived from the plant (Salzer and Hager, 1991). The characteristics of acid invertases have been investigated in Picea abies in relation to their possible role in nutrient supply to the mycorrhizal fungus. Using cell suspension cultures of this species, Salzer and Hager (1993) showed that there were two important acid invertases associated with the apoplast. Both had relatively low pH optima, with the ionically bound form showing high activity between pH 3.5 and 4.5, and the tightly bound form having a sharp optimum at pH 4.5. Above pH 6 neither form showed significant activity. Both invertases had relatively high Km values with respect to sucrose (16 and 8.6 mM for the ionically and tightly bound forms, respectively) and were competitively inhibited by fructose but not by glucose. These authors suggested that control of carbohydrate supply might be regulated by the fungus, via changes in apoplastic pH and uptake of fructose. Using whole Picea abies roots, Schaeffer et al. (1995) showed an acid invertase with a pH optimum of around 4.0 and a Km (sucrose) of 5.7 mM, which is rather less than for the suspension cultures. By comparing different segments of mycorrhizal and non-mycorrhizal long roots it was shown that although overall the activity of invertase was lower in mycorrhizal roots (in agreement with findings of Lewis and Harley, 1965b), when the weight of fungal tissue was taken into account, invertase production by the plant was unaffected by fungal colonization. Around 75% of the invertase activity was associated with cells of the root cortex, rather than the stele, and was thus in the tissue accessible to the mycorrhizal fungus. Interestingly, Schaeffer et ah, (1995) could find no evidence for increase in plant invertase activity associated with fungal colonization and they highlighted the difference between this situation and that in some parasitic symbioses. Figure 7.3 shows a hypothetical scheme for sugar transfer from plant to fungus, based on the ideas presented by Salzer and Hager (1993), Schaeffer et al., (1995) and Hampp and Schaeffer (1995). The overall picture is one in which, following sucrose efflux to the apoplast and hydrolysis by plant invertase, glucose would be preferentially absorbed by the fungus, llie mechanisms by which net transfer of sugars to the fungus is maintained seem cumbersome according to present information. Fructose released by the invertase would exert feedback inhibition on invertase activity, whereas glucose would inhibit fructose uptake by the fungus. As the concentration of glucose fell, the fungus would absorb fructose, releasing invertase inhibition and permitting further sucrose hydrolysis. One important gap appears in this reasoning and that is the possible need to reduce or eliminate uptake by the plant of both sucrose and the hexoses from the interfacial apoplast. Here again proton-symports are likely to operate and net flux in the direction of the fungus could only be maintained by a mechanism favouring net efflux (as sucrose) from the plant and net uptake (of monosaccharides) by the fungus. Perhaps fructose also competes with reabsorption of glucose by the plant or perhaps plant hexose transporters are down-regulated. This area clearly requires continued investigation. Monosaccharide uptake by the fungus might be passive, with a concentration gradient maintained by metabolic conversion within the fungus. However, it seems just as likely that active proton co-transport would drive the inward flux, especially as low apoplastic pH controlled by the membrane-bound H'*^-ATPase (of either organism) is necessary to maintain invertase activity. ATPase activity has been demonstrated cytochemically on both fungal and plant plasma membranes in the

244

Ectomycorrhizas

INTERFACIAL APOPLAST

FUNGUS

TREHALOSE GLYCOGEN

A

PLANT

• H^ ' GLUCOSE

-SUCROSE ^ T^ I pH4.5 % [MANNITOL]

\

O

FRUCTpSE H*

^^,

ATP - ^

^\

ADP ^ ^ ^

y^

^ - ^

ATP

ADP

GLUCOSE H* H* FRUCTOSE

FPM

¥ PPM

Figure 7.3 Diagrammatic representation of the plant-fungus interface in an ectomycorrhiza, showing processes that may be important in sugar transfer from plant to fungus. Hydrolysis of sucrose effluxing from the plant occurs via the activity of a plant invertase, when the pH of the apoplast is at or below 4.5. Glucose is preferentially absorbed by the fungus and also inhibits (X) uptake of fructose by the fungus. Fructose may inhibit invertase activity (X). Mechanisms reducing or preventing reabsorption of sugars by the plant are not understood (?). D, H'*^-ATPases; O, sugar transporter. See text and Chapter 14.

Hartig net zone of Pinus sylvestris-Laccaria laccata mycorrhizas and shown, by its DES sensitivity, to be, in all probability, an H'^-ATPase (Lei and Dexheimer, 1988; and see Chapter 14). The importance of maintaining a net flux of organic C in favour of the fungus has been the subject of considerable discussion. Since trehalose and mannitol were only very slowly absorbed by non-colonized roots it was suggested that they, with glycogen, constituted a sink in the mycorrhizal sheath into which carbohydrates were accumulated in a form not readily available to the adjacent plant tissues even if they were present in the apoplast. This idea was elaborated in a review by Smith

Growth and carbon economy in ectomycorrhizal plants

245

et al. (1969) in which they consider allied features of carbohydrate movement between the partners in lichens, pathogenic associations and other symbioses. The hypothesis of a 'biochemical valve', ensuring movement in the direction of the fungus has proved to be very attractive and is discussed further in Chapter 14. There are several additional mechanisms by which organic C might pass from the plant to the fungus at different stages of colonization. First, there is the possibility that the cells of the root cap and epidermis, together with the mucigel, might provide the colonizing fungus with organic C in the same way as general rhizosphere microorganisms are supplied from non-mycorrhizal roots. The fungus may also, as it penetrates to form the Hartig net, alter the behaviour of the plant cells with respect to the production of cell wall polymers, and use their precursors as a source of C. The fungus may also alter the condition of the cells surrounded by the Hartig net, such that they release increased amounts of sucrose or other C compounds. Lastly, when the fungal hyphae penetrate senescent cortical cells, especially in the late Hartig net region, the soluble contents of the cells are presumably absorbed. The suggestion that the fungus might produce a compound that makes the plant cells more 'leaky' requires critical evaluation. Whatever occurs must be specific to the transfer of organic C from plant to fungus. It must not be so general an increase in permeability or loss of membrane integrity that it precludes transfer of solutes (N, P and so on) derived from the fungus to the plant. As we discuss in Chapter 14, bidirectional transfer between the fungal and plant symbionts requires the maintenance of specific uptake processes and energized membranes of both symbionts. Whatever the mechanisms involved, it is clear that the establishment of mycorrhizas leads to significant enhancement of C flow to colonized roots. Lewis and Harley (1965b) observed that when a low concentration of ^^C-glucose was fed to excised Fagus mycorrhizas, a very large part was incorporated into ionized compounds rather than neutral carbohydrates (Table 7.5). The labelled carbohydrates had been metabolized in the C turnover of the tissue and much smaller amounts were incorporated into storage carbohydrates. It therefore follows that if the quantities of carbohydrate synthesized by the leaves in a ^^C02 labelling experiment are low, there is much less chance of being able to identify labelled fungal sugars in the root system, even if the mycorrhizal and non-colonized root tips themselves are compared separately. A similar conclusion was reached when low concentrations of sucrose were fed through agar blocks to excised mycorrhizas of Fagus (see above). These experiments may help to explain the lack of success some observers have had in identifying the fungal carbohydrates when analysing whole root systems rather than mycorrhizal rootlets. Although sugars from the plant certainly move to the fungus, movement of organic C is not exclusively in that direction. Harley (1964) showed, again using excised roots, that the rate of dark fixation of CO2 by the mycorrhizas of Fagus was greatly increased if NH4 was being absorbed. Using [^^C]-bicarbonate he was able to show, and Carrodus (1967) later confirmed, that the main destination of the increased fixation was into glutamine. Since this method provided a simple way of labelling glutamine, Reid and Lewis (see Lewis, 1976) used it to observe the movement of glutamine from the fungal sheath to the plant, demonstrating how C compounds may return to the plant as the C-skeletons of amino compounds. Subsequently, the pathways for assimilation of inorganic N and the mobilization of

246

Ectomycorrhizas Table 7.5 Distribution of radioactivity in mycorrhizal roots of Fagus when ['"^Clgiucose is absorbed from solutions of high (0.5%) and low (0.5 X 10"'*%) concentrations, in 18 h at 20°C Sugar concentration

High Low

Soluble fraction (%)

Insoluble fraction (%)

Neutral

Ionized

Neutral

Ionized

83.1 26.6

16.9 83.4

96.5 82.1

3.5 17.9

Data from Lewis and Harley (1965b).

organic N sources in soil have recently received considerable attention (see Chapter 8). There is as yet little evidence on the extent to which C-skeletons of the organic N sources utilized by the fungi contribute to the C budgets of the plants. Abuzinadah and Read (1989) calculated that in mycorrhizal seedlings of Betula pendula supplied with N exclusively in the organic form as much as 8% of total plant C could be derived from the fungus. The movement of C and N compoimds synthesized in the fungus certaiiJy provides a route for 'cycling' of organic C between the symbionts. The relative fluxes in the two directions have not been measured and this will certainly be an important challenge in assessing the potential of the 'cycling' of organic N through the interface to support net interplant transfer of C (see Chapters 8 and 15). The fixation of CO2 in the dark is a process which maintains the pool of tricarboxylic acids as they are used in synthesis of amino acids and the synthesis of glutamine places a drain on a-ketoglutarate (see Fig. 8.1a,b). The detailed pathway of fixation has not been worked out in mycorrhizas, but it commonly involves phosphoenol pyruvate (PEP) which, with CO2/ forms oxaloacetate by a reaction mediated by PEP carboxylase. This is powered by the high-energy phosphate bond of PEP. Hence one-quarter of the C of the oxaloacetate is derived from CO2 and the rest from carbohydrate via glycolysis. This reaction does not, therefore, produce organic C compoimds which can be an additional source of energy, but is an energy-requiring process which synthesizes essential compounds using energy and C compounds produced in glycolysis. In the fungal sheath this would constitute a drain on the sugars obtained from the plant.

C Distribution in Intact Plant-Fungus Systems In their study of distribution of ^^C-labelled photosynthetic products between mycorrhizal and non-mycorrhizal roots on the same plants of Pinus radiata, Bevege et al. (1975) found that 15 times more C was allocated to the colonized roots, 45-50% of the label being in trehalose and 1-22% in mannitol. A similar study using individual plants of Eucalyptus pilularis, some roots of which were colonized by Pisolithus tinctorius, showed that the mean ratio of ^^C-accumulation in mycorrhizal versus non-mycorrhizal roots was 18:1 (Cairney et ah, 1989). The ability of these ectomycorrhizas to attract photosynthate was greatest soon after their formation and there was a progressive reduction in the amount translocated to them with age.

Growth and carbon economy in ectomycorrhizal plants

247

SO that 90 days after inoculation all translocation had ceased. Caimey and Alexander (1992) compared allocation of ^^C to younger and older mycorrhizas of Picea sitchensis formed by Tylosporafibrillosaand growing on peat. Although some ^Relabelled compounds were translocated to older mycorrhizas in all plants, the ratio of activity in yoimg to older mycorrhizas, which was initially around 2:1, became progressively greater as the whole root system aged, and reached 54:1 38 weeks after transfer of the newly colonized seedlings to the peat substrate. In the studies quoted above, the extent of onward transport of fixed C from root to extraradical mycelium was not determined. The importance of this mycelium as a potential sink for fixed C is evident from studies in which intact systems develop over semi-natural substrates (see Chapters 6 and 15). Autoradiographic techniques have shown clearly that ^^C fed to assimilating shoots of Pinus is rapidly transported to the extraradical network (see Figs 6.21 and 6.23) which, in addition to exploring the soil, provides interconnection between individual mycorrhizal short roots in the same and on adjacent compatible plants (Finlay and Read, 1986a). Thus the fungus supplies a series of potential pathways for the flow of C from structures of small or decreasing sink strength, such as old roots, to younger tissues. A question of fundamental importance concerns the amount of C allocated by the plant to support the growth and maintenance of its ectomycorrhizal fungus. The first studies of respiratory activity specifically of the mycelial phase of the ectomycorrhizal association (Soderstrom and Read, 1987) demonstrated that approximately 30% of total respiration was attributable to the mycorrhizal mycelium. This respiration was shown to be highly dependent upon the supply of current assimilate and, if mycelial connections to the roots were severed, there was a reduction in respiration rate of at least 50% within 24 hours (Fig. 7.4). A similar dependence upon current assimilate for production of sporophores has been demonstrated in an association of Pinus strobus with Laccaria bicolor (Lamhamedi et al, 1994; Fig. 7,5), Rygiewicz and Anderson (1994) fed P. ponderosa seedlings, either colonized by Hebeloma crustuliniforme or non-mycorrhizal, with ^'*C02 and examined the distribution of label in different fractions after incubating the plants in microcosms for 72 hours. Both sets of seedlings retained around 40% of labelled C in their shoots, but the presence of the fimgus (representing only 5% of total seedling weight) increased the below-ground allocation of ^^C by 23% relative to that in non-mycorrhizal plants. Dry matter allocation to roots and mycorrhizas was similar, the difference being due to increased respiration by mycorrhizal roots and mycelium. The greater allocation below groimd led to a small reduction of seedling biomass in the mycorrhizal plants. Jones et al, (1991) considered C allocation to mycorrhizas as a cost to the plant. They examined efficiency in terms of amoxmt of P taken up per imit of C expended below ground, using cuttings of Salix viminalis, either colonized by Thelephora terrestris (M) or grown in the non-mycorrhizal (NM) condition. Efficiency was calculated according to the formula AP/ACb of Koide and Elliott (1989; see Chapter 4), where AP is the amount of P taken up by the plants over a defined interval, and ACb is the total amount of C allocated to the below-groimd system during the same interval. Cb includes the C incorporated into root or fungal tissue, lost in respiration and deposited in the soil. Cb was calculated for both M and NM plants, which had

Ectomycorrhizas

248

-2

24 48 96 124

0

Hours Figure 7.4 Effect of cutting the mycelium (and hence detaching It from a source of carbohydrate from the plant) on respiration of ectomycorrhlzal mycelium. The curves represent the relative respiration rate (percentage of value before cutting) for nine separate combinations of plant and fungal species. From Soderstrom and Read (1987), with permission.

2

3

Net photosynthesis (|xmol i

Figure 7.5 The influence of rate of net photosynthesis of Pinus strobus seedlings on the biomass of fruit bodies produced by associated Laccaria bicolor after 20 days. Bars are standard errors of means. From Lamhamedi et al. (1994), v/ith permission.

Growth and carbon economy in ectomycorrhizal plants

249

been pulse-labelled prior to each harvest, for the intervals up to the first harvest (0-50 days) and from the first harvest to the final harvest (50-98 days). Two methods were used to obtain C^. In the first method, it was calculated as: Cb(Pn) = Pn ^^^5^ 57600 ^(i'-) ^ 100 - % CsR where Cb(Pn) is the amount of C allocated below groimd, in mmol of C in a 24-hour period; Pn is the net rate of photosynthesis, as mmol C s~^ for a whole shoot system; % CBG is the percentage of the total ^^C02 absorbed allocated below ground over a 9-day period; % CSR is the percentage of the absorbed ^^C02 released as shoot respiration; and 57600 is the length of the daily light period, in seconds. The term 100 - % CSR is a correction for the fact that Pn is exclusive of shoot respiration. A curve was then constructed of Cb(pn) against time and the resulting equation integrated over the interval between each harvest to give the total amount of C allocated below ground over these intervals (ACb(i>n))In the second method, shoot weight was used as an integrated measure of the amount of C deposited in tissue over a harvest interval. The relationship between C deposited in shoot tissue and C allocated below ground as determined by the ^Relabelling was then used to calculate ACb directly: ACb(wt) = AW,

^ /o CsT

where AWs is the mean increase in shoot weight over the interval; and % CST and % CBG are the mean percentages of the ^^C fixed, allocated to shoot tissue and to the below-ground compartments, respectively. For the first interval, % CST and % CBG from the first harvest were used. For the second interval, a weighted (i.e. based on the length of time between harvests) average of the % CST and % CBG value for harvests two, three and four were used. Dry weight was converted to g C using a correction factor of 0.5 g C g~^ dry weight. The amount of C allocated below ground was consistently greater in mycorrhizal than in non-mycorrhizal plants throughout the experimental period (Fig. 7.6). When the data shown in Figure 7.6 were integrated it was foimd (Table 7.6) that over the interval up to the first harvest mycorrhizal plants allocated 2.5 times more C (ACb(Pn)) below ground than did those not colonized by T. terrestris. Expressed on a weight basis, C allocation (ACb(wt)) was 1.75 times greater in mycorrhizal plants. Since colonized plants absorbed three times as much P from soil over the same period they had a higher P use efficiency according to both methods of calculation. Because, over the second half of the experiment, the difference in P uptake between mycorrhizal and non-mycorrhizal plants was less, the efficiency of P acquisition was higher in the non-mycorrhizal than in the mycorrhizal plants. Cost-benefit analysis shows the costs, expressed as C required to produce and maintain the nutrient-absorbing structures, are lowest at the earliest stage of growth while the benefits, in the form of P acquisition, are highest. This is a pattern which, Jones et al., (1991) suggest, will match the requirements of field-grown perenrual plants where, in temperate and boreal climates, maximum demands for nutrients may be experienced in spring. The seasonality of C allocation is generally overlooked. The literature (e.g.

Ectomycorrhizas

250

100

Figure 7.6 Below-ground carbon allocation (Cb(Pn)) (m mol C d~') of mycorrhizal ( • ) and non-mycorrhizal (D) Salix viminalis during a 98-day growth period. Values are means of three replicates at 50, 60 and 85 days, and 4 (M) or 5 (NM) at 98 days ± standard error of means. From Jones et al. (1991), with permission. Table 7.6 P uptake, C translocated below ground and P acquisition efficiency of mycorrhizal Salix viminalis over a 98-day growth period Mycorrhizal

Growth

AP

treatment

period

(^mol)

ACb(Pn)* (mmol)

^Cb(Wt)t (mmol)

(days)

AP/ACb(Pn)* m m o l ~ * C)

AP/ACb(wt)t (|imol P m m o l " ' C)

(^mol P

Mycorrhizal

0-50

50.1

14.2

17.0

3.52

2.94

Non-mycorrhizal

0-50

17.1

5.7

9.7

2.97

1.75

Mycorrhizal

50-98

25.6

0.55

0.37

50-98

17.9

46.6 22.0

69.8

Non-mycorrhizal

21.2

0.82

0.84

ACb is the amount of C translocated below ground during a given Interval, including that deposited in root and fungal tissues, that respired by roots and soil, and that deposited in the soil. AP is the amount of P taken up during a given interval. * Values were calculated using photosynthetic measurements. t Values were calculated using weight measurements. Data based on the 3-5 plants labelled with ''^C prior to each harvest From Jones et o/., (1991).

Shiroya et al, 1966; Gordon and Larson, 1968; Glerum and Balatinecz, 1980) suggests that the main surge of C allocation to below-ground systems occurs not in spring but towards the end of the growing season, after stem elongation and bud set are complete. This is also the time at which the main flush of root growth occurs in many of the tree species which are characteristically ectomycorrhizal (Lyr and Hoffman, 1967), and it coincides with the late summer flush of fruit body production by epigeous mycorrhiza-forming mushrooms. Since these are, as shown above, dependent upon current assimilate for their development, it is implicit in such observations that this is also a time during which the extraradical mycelium must be particularly active. There is evidence (Langlois and Fortin, 1984) for a distinct seasonality in the pattern of absorption of P (see Chapter 9) by ectomycorrhizal

Growth and carbon economy in ectomycorrhizal plants

251

roots of Abies balsamea, with maximum rates agairi observed in August, after bud set. This feature may also be a reflection of greater below-ground C allocation. A requirement for enhanced nutrient inflow in the autumn would be expected in boreal and temperate systems, because winter hardening involves significant augmentation of cellular components, in particular membrane phospholipids (Siminovitch et ah, 1975), which are remobilized to support the spring flush of aboveground growth. Environmental factors, in particular availability of soil moisture, may be superimposed on the inherent seasonality of ectomycorrhizal processes, but it is important to bear in mind, whether carrying out experiments under conditions of constant day length and irradiance in the laboratory, or collecting mycorrhizal roots in the field, that the results obtained at any one time may not reflect accurately the situation as it prevails in the field. There is scope for much more work on seasonality in function in mycorrhizal systems, although it appears likely that the main phase of activity is at the end rather than at the begirming of the growing season as surmised by Jones et ah (1991). Non-Nutritional Effects upon C Assimilation Ectomycorrhizal symbioses can influence the C balance of the plant via a number of normally interrelated processes, including net photosynthetic rate and mineral nutrition. However, there is an increasing awareness of the importance of sink strength in determining the rates of assimilation of C in the leaves (Herold, 1980; Gifford and Evans, 1981; Sonnewald et ah, 1994). Consequently, mycorrhizal fungi may, through their ability to maintain a net flux of C in their favour (Lewis and Harley, 1965a), and to increase the overall C demand of the root, exert direct nonnutritional impacts upon C assimilation by the plant. Several studies have shown that photosynthetic rates are enhanced in ectomycorrhizal plants relative to those grown in the non-mycorrhizal condition (Reid et ah, 1983; Nylund and Wallander, 1989) but the confounding influence of enhanced mineral nutrient concentration in the foliage of colonized plants has made it difficult to determine the relative importance of nutritional and non-nutritional effects. In order to discriminate between nutritional and sink effects, Rousseau and Reid (1990) grew seedlings of Finns taeda in the mycorrhizal and non-mycorrhizal condition, but provided the uncolonized plants with various additional amounts of P so that tissue P concentrations were the same as those of plants colonized by Pisolithus tinctorius. The photosynthetic rates of the mycorrhizal plants, some of which were lightly, some moderately and some heavily colonized by the fungus, were then compared with those of uncolonized plants from each of the P treatments, before harvests were taken to determine tissue P status. Relationships between mycorrhiza development, net photosynthetic rate and foliar P (Fig. 7.7) indicate that with low and medium colonization, assimilation of mycorrhizal seedlings responded to increasing foliar P concentration in the same way as uncolonized plants, suggesting that the rate was responding to increased P accumulation. However, at high (H) levels of colonization net rates of photosynthesis in mycorrhizal plants were significantly greater (up to 17%) than those seen in non-mycorrhizal plants of the same foliar P concentration. Clearly, this suggests an

Ectomycorrhizas

252

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Foliar Phosphorus (%)

Figure 7.7 Net rate of photosynthesis (mg CO2 g~' h~') versus foliar P concentration (percentage dry weight) for ectomycorrhizal seedlings. The different symbols indicate seedlings with different concentrations of glucosamine (mg g~') derived from the fungal symbiont associated with their roots: A , 0-7.99 mg; O, 8-15.99 mg; D , 16-24 mg. The dashed line shows the regression obtained from non-mycorrhizal seedlings. From Rousseau and Reid (1990), with permission.

effect of colonization upon photosynthesis not related to P status. The likelihood that the increased sink strength imposed by the highest level of colonization was responsible for the enhancement of assimilation was stressed by Rousseau and Reid (1990), although alternative explanations, including changes of hormone balance induced by the fungus, were also discussed. The possibility that nutrients other than P had become limiting for the highly colonized plants, despite the apparently adequate nutrient supplies, also deserves attention. A number of other studies have now shown that increased rates of photosynthesis in mycorrhizal plants can occur independently of nutrient enrichment. Dosskey et al. (1990) found that colonization of Pseudotsuga menziesii by Rhizopogon vinicolor led to significant increases of net photosynthesis whereas two other fungi, Hebeloma crustuliniforme and Laccaria laccata had no effect. Despite the increased net assimilation rate, Rhizopogon-colonized seedlings were smaller than the controls. The same type of result was observed in seedlings of Pinus pinaster when colonized by Hebeloma cylindrosporum (Conjeaud et a/., 1996). Despite significant increases of photosynthetic rate over non-mycorrhizal plants, with the same or even greater tissue N and P concentrations, there was an accompanying 35% reduction of growth. In these cases stimulation of photosynthetic rate arising from increases in sink strength are not sufficient to compensate for the costs of production of mycorrhizas and associated extraradical mycelium.

Growth and carbon economy in ectomycorrhizal plants

253

Community Level Patterns of C Allocation Rommell (1939b) provided an early estimate of the C demand of mycorrhizal fungi under field conditions, based upon fruit body production, and showed that the C required was 10% of that invested in annual timber production. The calculation assumed a fruit body yield of 180 kg ha~^ y~^, which some workers have suggested is a high value. Fogel and Trappe (1978), for example, quote values of dry weight of epigeous sporophores for a number of forest types in the northern hemisphere in the range of 3-180 kg ha"^ y~^. However, Vogt et al. (1982) recorded 380 kg ha~^ y~^ of hypogeous and 30 kg ha~^ y~^ of epigeous fruit bodies in a 180-year-old stand of Abies amabilis. In addition, Cenococcum sclerotia yield 2700 kg ha~^ y~^ in these forests. By combining all such values with estimated fungal biomass in sheaths and Hartig nets, Vogt et al. (1982) estimated that 15% of net primary production was allocated to the fungus, a value which still did not take account of vegetative mycelium in the soil, nor the possibility of increased C allocation due to turnover of the mycorrhizal rootlets. It is, of course, difficult to determine the biomass of ectomycorrhizal hyphae, growing as they do in mixed populations with other fungi in forest soil. However, some estimates are now available. On the basis of regression relationships between mycelial respiration and biomass, Finlay and Soderstrom (1989) estimated that in a Swedish pine forest soil dominated by the fungus Lactarius rufus there were 200 m mycorrhizal hyphae g~^soil. This figure is remarkably similar to that calculated by Read and Boyd (1986) for mycelial systems of Suillus bovinus growing in peat in observation chambers (see Chapter 6) and is not dissimilar to values of 100700 m g"^ presented earlier by Soderstrom (1979) for lengths of active hyphae (stained with fluoroscein diacetate) in the FH horizon of Swedish forests. The value of 200 m g~^ dry soil when converted to fungal biomass is equivalent to 3.5 kg of living mycelium ha~^ y~^. Assuming a turnover time of 1 week throughout a 5-month growing season, the hyphal production will be 70 kg ha~^ y~^. This is more than double the estimated dry matter yield of fruit bodies of L. rufus, which in Swedish forests is considered to be around 30 kg ha~^ y"^ (Richardson, 1970). Determination of total mycorrhizal biomass in the system also requires estimates of fine root production and turnover. Persson (1978) estimated that fine (<2 mm diameter) root biomass production in a forest of the kind studied by Finlay and Soderstrom (1989) was 2030 kg ha"^ y~\ Assuming that 90% of these roots are mycorrhizal and that 40% of the mycorrhizal root is fungal (Harley and McCready, 1952b; Vogt et al, 1982), 730 kg of fimgal sheath may be produced per year. Thus total fungal production (mycelium + sporophores + sheath) is 830 kg ha~^ y~^. If the C content of the fungal material is taken to be 40% and the efficiency is 60%, the C demand of the ectomycorrhizal fimgi in the forest studied by Finlay and Soderstrom (1989) is also 830 kg C ha~^ y~^. Photosynthetic production in the same forest has been estimated at 5800 kg C ha"^ y~^ (Linder and Axelsson, 1982), which means that ectomycorrhizal fungi use around 15% of assimilated C. This value, as Finlay and Soderstrom (1992) point out, is strikingly similar to those presented by Vogt et al (1982) and indeed by Rommell (1939b).

254

Ectomycorrhizas

Conclusions The construction of nutrient response curves has gone some way to providing an understanding of the influence of ectomycorrhizal colonization upon plant growth. When plants face a shortage of a mineral element such as P there is a threshold of availability below which growth will not occur. By enhancing access to the growthlimiting nutrient, colonization can significantly reduce the threshold. The extent of plant response varies with the fungus and its pattern has been shown to be distinctive at interspecific and even at intraspecific levels, both in the effectiveness with which the nutrient resource is exploited and in the C demands imposed upon the plant. It is now recognized that relationships between effectiveness of resource exploitation and extent of C demand can usefully be explored by means of costbenefit analysis. Costs and benefits of mycorrhizal associations are again shown to differ with fimgus and experimental circumstances. Considerable agreement exists as to the probable overall C costs of colonization by ectomycorrhizal fungi. Calculations based upon data collected from both field and laboratory experiments indicate that between 10% and 20% of current assimilate may be allocated to sustain the vegetative mycelium and fruit bodies of the heterotroph. Some further progress has been made towards elucidation of the biochemical mechanisms whereby plant-derived carbohydrates are converted to the compounds translocated in, and used by, the fungi but much remains to be learned. We now have a better understanding of the localization and control of invertase, but are still unsure about the feedback mechanisms which must influence its activity and role in the processes of transfer between symbionts. It must be recognized that although progress has been made in terms of understanding growth responses to mycorrhizal colonization in simple experimental systems, the conditions in these contrast markedly with those occurring in nature, where the individual plant is likely to be colonized at any time by a number of different fungal species and challenged by limitation in supply of more than one nutrient. For this reason, experiments of the kind described in this Chapter, while providing significant advances, must be seen to yield only the most basic insights. If we are to gain a realistic view of the function of ectomycorrhizas in nature, more elaborate experimental designs will be required. These will enable individual plants to be colonized by combinations of fungal species and challenged by nutritional conditions that reflect qualitatively, quantitatively, as well as temporally, regimens of the kind likely to be encoimtered in nature.

8 Nitrogen nutrition of ectomycorrhizal plants

Introduction Constraints upon plant growth imposed by low availability of N are a characteristic feature of many ecosystems dominated by ectomycorrhizal plants. Thus, in the extensive boreal (Tamm, 1991) and temperate (EUenberg, 1988) forests of the northem hemisphere N is the most important determinant of productivity. For this reason it is essential to establish the mechanisms and processes whereby the element is mobilized, assimilated and transported. Microbial activity in soil is of paramount importance in the interconversions of N in both inorganic and organic pools. Ammonium (NH4) is released from proteins and other organic N sources by ammonifying organisms with the rate of this process and the later conversion of NH4 to other forms being strongly dependent on soil conditions, particularly pH and aeration. When the soil is relatively acid, cold or poorly aerated, NH4 will remain the predominant form, but under conditions favouring nitrification, conversion to nitrate (NO^) will follow ammonification. In consequence, the form of inorganic N available to plants may be different in different habitats and at different stages of plant succession (Rice and Pancholy, 1973; Bowen and Smith, 1981; Kuiters, 1990). Thus the plants themselves, which are probably only able to utilize inorganic N sources, may be presented with different relative amounts of NH4 and NO^, depending on soil conditions. However, in many soils the rate of ammonification is relatively low and a high proportion of the total N is present in organic form as proteins or other N compounds of different accessibility to the microbial population and generally unavailable to plants, at least when they are non-mycorrhizal. Furthermore, availability of organic molecules including N compounds may be quite different in axenic or monoxenic culture, compared with soil, where processes of protection by chemical reactions or occlusion in spatially inaccessible pores may reduce their availability to organisms, including mycorrhizal fungi. The possibility of a direct involvement of ectomycorrhizal fungi in N acquisition by plants was first suggested by Frank (1894), but until recently more attention has been paid to their role in the capture of P. Harley (1989), recognizing this imbalance.

256

Ectomycorrhizas

pointed out that the ratio of N to P in most healthy plant tissues was of the order of 10:1 and that for this reason alone greater emphasis upon uptake and metabolism of N was necessary. Many researchers have responded to such pleas in recent times, with the result that a much clearer picture of the role of mycorrhizas in the N economy of plants and ecosystems is emerging. Studies have been carried out at two distinct levels; one involving analysis of the fungal partner in axenic culture, the other the mycorrhizal root or whole plant. Because the results obtained using the two approaches can be very different it is appropriate to consider them separately. Although the importance of organic N is now clearly recognized, investigations of the uptake and assimilation of inorganic forms are relevant for some mineral soils because they are likely to be utilized in preference to the organic forms when both are present. The competition between mycorrhizal and saprophytic fungi, both with respect to their growth in soil and their capacity to utilize inorganic N as soon as it is released by mineralization, is likely to be severe. Mycorrhizal fungi, with access to recent photosynthate from the plant, will not be limited by the availability of organic C in the soil. Furthermore, the investigations on the uptake and assimilation of inorganic N have provided useful insights into the enzymic pathways operating in mycorrhizal fungi and in intact mycorrhizal systems.

Use of N by Ectomycorrhizal Fungi in Pure Culture Inorganic N Sources The ectomycorrhizal fungi are similar to other fungi in the kinds of N compounds which they can use for growth in culture. The early workers (Norkrans, 1950; Rawald, 1963; Limdeberg, 1970) found a range of relative abilities to use NH4 and NO^ among the species and strains of mycorrhizal and saprophytic fungi that they studied (see Harley and Smith, 1983, for details). Nothing was discovered which distinguished mycorrhizal fungi as a group and more recent work has confirmed these observations (France and Reid, 1984; Genetet et al, 1984; Littke et al, 1984; Plassard et al., 1991). Most of them grow fastest on NH4 and some can use NO^ but others not. The experimental work on N absorption is beset with difficulties concerning pH change, which can be very significant in the unbuffered media usually employed. Absorption of NH4 results in a marked lowering of the pH, which may be followed by a sharp cessation of growth. The form of N present in the medium is strongly affected by pH. The pKa for protonation of ammonia (NH3) is 9.25, with the result that NH4 rather than NH3 will be the predominant form in most culture media and under most growth conditions. This has implications for the membrane transport processes likely to be involved in uptake. Absorption of NO^, as well as the release of NH4 from amides or other readily hydrolysed compounds, causes an increase in pH which is usually slower and may have a less marked effect on growth than the reductions in pH. Despite these problems, there is not much doubt about the broad results, although there is much variation between different species and strains of mycorrhizal fungi in requirements for inorganic N.

257

Nitrogen nutrition of ectomycorrhizal plants

While the earlier studies concentrated on the abilities of the fungi to grow on various media, more recent work has paid attention to the pathways by which inorganic N is assimilated in the fungal mycelium. Although work on inorganic N may be considered to be relatively unimportant, given the predominance of organic N in many soils colonized by ectomycorrhizal fungi, the details of these assimilatory pathways are interesting because they show what options may be available to the fungi during assimilation of all forms of N. In common with other fungi, most of the N from NH4 enters metabolism of ectomycorrhizal fungi either as the amide group of glutamine by the glutamine synthetase (GS) pathway, or as the amino group of glutamate through the glutamate dehydrogenase (GDH) pathway (Fig. 8.1). While both of these routes, together with that involving glutamate synthase

(a) COOH CH2 CH2

CONH2 CH2 CH2 CHNH2 COOH

r

c=o

COOH

^^GLUTAMINE

^ ^

^ \

.

GOGAT

1 GS

NAD(P)H FerredoxiHred

r

NH/A^

A T P - ^ ^ ^ " GLUTAMATE

A

^^^

a-KETOGLUTARATE

^^N^.^

GLUTAMATE

COOH CH2 CHNH2 COOH

COOH CH2 CHNH2 COOH

(b) a-KETOGLUTARATE

COOH CH2 CHj

c=o COOH

GLUTAMATE NAD(P)H

GDHl

V

NH/

GLUTAMINE

COOH ATP GS CH2 CH2 CHNH2 NH/ COOH

h

CONH2 CH2 -•CH2 CHNH2 COOH

Figure 8.1 Pathways of NH4 assimilation in plants and fungi, (a) The glutamine synthetaseglutamate synthase (GS-GOGAT) pathway, (b) The NADP-glutamate dehydrogenaseglutamine synthase (GDH-GS) pathway. Adapted from Martin and Botton (1993).

258

Ectomycorrhizas

(GS-GOGAT), can be present, the relative importance of each differs according to fungal species. Analysis of the major pathways of N metabolism in Cenococcum geophilum (Genetet et al, 1984; Martin, 1985; Martin et al, 1988a), Hebeloma cylindrosporum (Chalot et ah, 1991a) and Laccaria laccata (Bnm et ah, 1992) indicates that the GS pathway predominates in these fungi. Using ^^]S[H4 as tracer, up to 40% of assimilated ^^N was found in the amide group. Glutamine synthetase has been purified from L. laccata (Brun et al., 1992) and shown to have a very high affinity for NH4, suggesting that in this fungus at least GS is the main route of NH4 assimilation. This may also be the case in many ectomycorrhizal fungi and would be consistent with earlier work showing that in the presence of NH4 dark fixation of ^^C02 results in preferential incorporation of label into glutamine by excised Fagus mycorrhizas (Harley, 1964; Carrodus, 1967). The role of GDH should not, however, be overlooked. In Hebeloma crustuliniforme, for example, assimilation of NH4 appears to be mainly via the GDH pathway (Quoreshi et ah, 1995). Two forms of this enzyme are recognized: one NAD- and the other NADP-specific. Both are found in ectomycorrhizal fungi, but it appears that only the latter, which on the basis of K^ values probably operates in the direction of synthesis, has high activity (Dell et al, 1989; Ahmad et al,, 1990; Botton and Chalot, 1995). A considerable amount is known about this enzyme which has been isolated and purified from mycelia of C. geophilum (Martin et al., 1983a; Dell et al, 1989), L. hicolor (Ahmad and Hellebust, 1991) and L. laccata (Brun et al, 1992). Its properties are similar to those reported for NADP-GDH enzymes of Neurospora crassa and yeasts (Stewart et al, 1980). When GS is inhibited with methionine sulphoximine (MSX), glutamate, alanine and aspartate accumulate in mycelium of ectomycorrhizal fungi, confirming the presence and operation of the GDH pathway. These results suggest that NH4 assimilation, in the three mycorrhizal fungi is achieved by parallel action of GDH and GS (see also Chalot et al, 1994a,b). The need for examination of other fimgi is highlighted by the observation that in Pisolithus tinctorius GS activity is low (Ahmad et al, 1990) and that MSX blocked the synthesis of other amino acids, suggesting the operation of the GS-GOGAT pathway. An NADH-dependent GOGAT has been detected in L. hicolor (Vezina et al, 1989), but the instability of this enzyme renders characterization difficult and its status in ectomycorrhizal fungi is uncertain. There have been relatively few studies of the role of mycorrhizal fungi in the NO^ nutrition of plants. The greater mobility of the NO3 ion in soil, together with the inhibition of nitrification often seen in acidic soils occupied by ectomycorrhizal roots may mean that, as for vesicular-arbuscular (VA) mycorrhizas (see Chapter 5) the symbiosis is relatively unimportant in acquisition of this ion. However, there are clearly some situations in which nitrification does occur in soils supporting ectomycorrhizal plants. At one site for example NH4 and NO^ were present in the soil solution, both at 100 |XM (Stewart et al, 1993). Consequently some consideration of NO^ as a potential N source for fungus and plant is necessary. Some ectomycorrhizal fungi in culture use NO3 in preference to NH4. Growth of a strain of Hebeloma crustuliniforme, for example, was shown to be 10 times greater on NO^ than on NH4 supplied at the same N concentration. The nitrate reductase (NR) activity of this fungus is similar to that seen in herbaceous angiosperms. While induction of NR appears not to be dependent upon the presence of NO3",

Nitrogen nutrition of ectomycorrhizal plants

259

activity of the enzyme was depressed in the presence of NH4 (Scheromm ei ah, 1990a). There is, however, a striking variability both between (France and Reid, 1984; Plassard et ah, 1986) and within (Ho and Trappe, 1987) fungal species with respect to their abilities to use NO3. This should be borne in mind when attempting to interpret results of experiments using only one strain or species of a fungal symbiont. Organic N Sources The ability of some ectomycorrhizal fungi to use organic N sources has been appreciated for some time (see Harley and Smith, 1983). Lundeberg (1970), in his cultural studies of a number species and strains, identified several members of the genus Suillus that grew better on asparagine and glycine than they did on inorganic N. There is clearly a good deal of variability between different species and even between strains of the same species. Laiho (1970) published a study oi Paxillus involutus, in which he examined its cultural characteristics and ecology using many strains. All were able to use casein hydrolysate, peptone and a mixture of amino acids, and all isolates grew well on both glutamate and arginine. Ability to grow on other amino acids as sole N source was low and variable. There was no case of an organic N source being very readily used by one strain and being totally useless to another. To test the ability of mycorrhizal fungi to use orgaruc N compounds in a more realistic way, Lundeberg (1970) prepared humus agar in which the concentrations of inorganic N compounds were reduced and the organic N was labelled with ^^N. He allowed fungi to grow from an inoculum which straddled the interface between the N-free glucose agar and the humus agar. None of the mycorrhizal fungi absorbed organic N from the humus in significant quantities, although five other fungi which produced some or all of the hydrolytic enzymes - cellulase, pectinase, proteinase and laccase - were able to do so. These results agree with the findings of Mosca and Fontana (1975), on the use of protein N by Boletus luteus and together such data have been taken to support the tentative conclusion that mycorrhizal fungi may not be very effective at acquiring organic N and must compete with other organisms for inorganic N mineralized by other members of the soil microflora. Howevej, awareness of the possible importance of amino acids as primary sources of N for ectomycorrhizal fungi and thus for the plants which they colonize, has been heightened by the recognition that these can constitute a significant pool, particularly in acid organic soils (Nemeth et al, 1987; Abuarghub and Read, 1988), leading to re-examination of the role of ectomycorrhizal fungi in their mobilization. Some amino acids and amides, including glutamine, glutamate and alanine which appear to predominate in soil solution, are readily assimilated by ectomycorrhizal fungi in pure culture, and at equivalent concentrations of N and C they can support yields as large as those obtained on NH4 as sole N source (Abuzinadah and Read, 1988; Finlay et al, 1992; Table 8.1). Using Paxillus involutus, Chalot et al. (1994a,b) demonstrated by means of tracer and enzyme inhibition techruques that glutamate, glutamine and alanine were absorbed intact and incorporated, respectively, by GS, GOGAT and alanine aminotransferase (AIAT) into the various assimilation pathways. Here, the N sources

Ectomycorrhizas

260

Table 8.1 Yields of three ectomycorrhizal fungi, Suillus bovinus, Amanita muscaria and Hebeloma crustuliniforme, when grown with a range of mineral or amino N sources at a concentration of 60 mg N r ' and at the same C : N ratio N source

Weight of N source added

Weight of glucose added

(g)

(g)

Mineral N Ca(N03)24H20 (NH4)2S04

0.504 0.284

Acidic amino acids L-Aspartic acid L-Glutamic acid

Dry weight yields (mg) after 30 days S. bovinus

A. muscaria

H. crustuliniforme

3.004 3.004

17.7 ^3 1.5 30.7 ^^ 1.2

4.7 ± 0.3 26.0 ^ 1.2

12.6 ^3 1.2 25.3 ^ 1.4

0.572 0.632

2.372 2.216

31.3 ^ 2.6 32.0 ^ 1.0

34.3 ^3 2.0 36.3 + 1.5

23.0 + 0.9 24.9 + 1.3

Basic amino acids L-Arginine L-Lyslne L-Hlstidine

0.224 0.392 0.300

2.764 2.528 2.688

33.6 -^2 1.8 17.0 •J2 0.6 5.0 ^ 0.0

34.0 + 2.5 18.0 ± 0.1 14.7 ± 1.2

15.6 ± 0.7 5.9 + 0.5 4.2 ^ 0.4

Neutral Amino Acids L-Alanine L-Asparaglne L-Cysteine L-Cystine L-Glutamine L-Methionine Glycine L-Phenylalanlne L-Hydroxy-L-proline L-lsoleucine L-Leucine L-Proline L-Serine L-Threonine L-Tryptophane L-Tyrosine L-Vallne

0.380 0.284 0.520 0.516 0.312 0.640 0.324 0.708 0.560 0.564 0.564 0.492 0.452 0.512 0.436 0.776 0.300

2.528 2.688 2.528 2.528 2.608 2.216 2.688 1.588 2.528 2.216 2.216 2.216 2.528 2.372 2.136 1.588 2.220

31.6 ± 29.0 ^3 7.7 + 6.0 ^^ 29.3 ^^ 7.7 ± 5.7 ± 11.3 ^ 3.7 ^3 22.3 H^ 23.0 + 4.0 ^ 27.7 ± 4.7 -h 2.0 ^ 1.7 ± 17.0 ^^

21.7 23.3 3.3 19.7 36.3 2.3 22.7 4.7 4.7 7.3 7.3 6.3 18.7 6.7 2.7 5.3 11.3

Without nitrogen

--

3.004

0.3 1.5 0.3 0.6 1.4 0.3 0.3 0.7 0.3 2.3 3.2 0.6 0.9 0.3 0.0 0.3 1.2

7.3 ^^ 0.7

± ± ± ^ ^ + + ^ ^ ^^ ^ ^3

^ + ^ ^^ ^

1.2 1.9 0.8 0.7 0.3 0.3 1.4 0.7 0.3 0.9 0.3 0.9 0.7 0.3 0.7 0.9 0.9

4.3 ± 0.3

20.4 19.0 6.0 5.6 18.1 3.5 9.9 4.3 4.2 11.7 8.1 4.4 16.3 7.6 7.0 8.2 18.9

+ 1.3 0.7 + 0.5 ± 0.2 ± 0.7 ^ 0.2 ^^ 0.4 ± 0.7 ± 0.5 ± 0.3 ^ 0.4 + 0.3 ± 1.2 H^ 0.3 + 0.1 ^ 1.0 ^ 3.1 H^

6.2 + 0.2

Dry weight yields are means ± standard error. Data from Abuzinadah and Read (1988).

would supplement the free amino acid pools, which represent important sinks for C in mycelia fed with mineral sources of N. It has been shown, for example in Cenococcum geophilum and Sphaerosporella brunnea, that 16-40% of C, fed as [l-^'^C] glucose to the mycelium, entered the amino acid pools (Martin and Canet, 1986: Martin et al, 1988b). Exogenous supply of amino acids and amides would therefore be expected to supplement the organic C, as well as the N economy of the mycorrhizal fungus.

Nitrogen nutrition of ectomycorrhizal plants

261

In tracer kinetic studies of assimilation of glutamine, glutamate and alanine carried out over a range of pHs, with and without the presence of NH4, NO3 and glucose, Chalot et al. (1995) showed that amino acid absorption had a distinctly acid p H optimum (Fig. 8.2) and that neither NH4, at concentrations 0.05-0.5 mM, nor glucose, had an impact upon uptake. Over a period of 5-6 weeks, uptake of amino acids decreased by a factor of 4-10 as the mycelium aged, while the size of endogenous pools progressively increased. Extrapolation of information on pool turnover in pure cultures to symbiotic systems must be done with caution, because of the absence of a sink for assimilated N compounds which in mycorrhizas would be provided either by ongoing growth of the fungus in soil or by the associated plant. Alanine is a major metabolic sink for N, following glutamine assimilation (Martin et al, 1988a), but its metabolic fate is uncertain. It can be readily absorbed from exogenous sources and autoradiographic studies indicate that it (or label from it) is

Q

S

M -«—•

o H

Figure 8.2 Effect of external pH on the uptake ( • ) and respiration (O) of (a) L-glutamate and (b) L-glutannine by the ectomycorrhizal fungus Paxillus involutus. Data are means of three replicate determinations ± standard errors of the means. From Chalot et al. (1995), with permission.

Ectomycorrhizas

262

translocated through mycorrhizal mycelia to the colonized roots (Read et al, 1989; see below). While some of the amino acids present in the soil solution are undoubtedly derived jfrom the free pools of these molecules in living plant and microbial tissues, others are likely to be a product of the cleavage of polymeric peptides and proteins associated with decomposition processes in soil. Since these polymers constitute the bulk of the N in many ectomycorrhizal forest soils (see Chapter 15) it is desirable to determine whether they, too, might be accessible to ectomycorrhizal fungi. Abuzinadah and Read (1986a) have shown that Suillus bovinus (Fig. 8-3b), Rhizopogon roseolus and Pisolithus tinctorius readily use a series of alanine peptides of increasing chain length from the di- to the pentapeptide. Hexa-alanine was also used but more slowly. Laccaria laccata, in contrast, had poor growth on all these substrates (Fig. 8.3a). That proteins themselves can be used as N sources by mycorrhizal fungi has been known for some time (Melin, 1925; Lundeberg, 1970) but the extent of fimgal proteolytic capability has not been fully appreciated. Using soluble proteins of animal and plant origin, Abuzinadah and Read (1986a,b) screened eight ectomycorrhizal fungi over a wide range of pH conditions for the ability to exploit these polymers as sole sources of N. On both bovine serum albumin (BSA: MW 67000;

I

14 4-5

^^^

21

L//—^ 14

28 —

S ^ : * -

" * ^ ^

1 21

L 28

-

•'"<:::—-

3-5 r2-5 14

21 Days of growth

28

|~ iv—

""^^^^^* 1

14

1

21 Days of growth

1

28

Figure 8.3 Dry weight of mycelium, residual N and pH of the culture medium of (a) Laccaria laccata and (b) Suillus bovinus grown in liquid medium containing: , NH4; A, alanine; a range of alanine peptides of chain length 2-6 (A, A2; O, A3; # , A4; • , A5; • , A6); or , lacking N. Data are means of three replicates ± standard errors of the means. From Abuzinadah and Read (1986a), with permission.

263

Nitrogen nutrition of ectomycorrhizal plants

N content 16%) and gliadin (MW c. 30000; N content 14%) some ectomycorrhizal fungi produced yields as large as, or even larger than, those obtained with NH4 as sole N source. These, including Amanita muscaria, Cenococcum geophilum, Paxillus involutus, Rhizopogon roseolus, Suillus bovinus and Hebeloma crustuliniforme, were referred to as 'protein fungi'. In contrast, Laccaria laccata and Lactarius rufus, the so-called 'non-protein' fungi, produced only small yields on these organic N substrates. Pisolithus tinctorius was in an intermediate category. Proteolytic capability was strongly influenced by pH/ with an optimum for growth in the acidic range between p H 3 and 5. El-Badaoui and Botton (1989) isolated a protein-rich fraction from forest litter and observed that it induced greater proteolytic activity in A. rubescens, C. geophilum and H. crustuliniforme than did BSA or gelatin. There is obviously variation in the availability of commercial protein preparations for fungal growth. For example, Hutchison (1990) screened a very large number of ectomycorrhizal fungi for their ability to liquefy gelatin to its constituent amino acids, but failed to detect activity even in species such as H. crustuliniforme and P. involutus which are known to be able to hydrolyse BSA or gliadin. The extracellular acid proteinase of H. crustuliniforme has been purified and characterized (Zhu et ah, 1990). The enzyme was most stable and had greatest activity over the p H range 2-5. Protein and some individual amino acids, notably glycine (Table 8.2), induced activity when they were applied as sole N sources. Enzyme production was not affected by the addition of 1>IH4 at 3.2 roM, but was repressed at higher concentrations. Production required the presence of a simple C source and was not repressed by glucose between 0.5 and 2.0% (Zhu et al, 1994). Consequently it appears that, as in the case of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Leake and Read, 1991; see Chapter 12), the acid proteinase of Hebeloma crustuliniforme is largely regulated by induction. It may be repressed by some forms of N, but catabolite repression does not seem to play an important part in regulation. The observation that single amino acids can act as inducers is of interest because they Table 8.2 Effect of various N sources on growth and proteinase production of Hebtloma crustuliniforme after 20 days of growth* N sourcest

NHj Asparagine Glycine Glutamine Casein hydrolysate Casein Gelatin Gliadin BSA

Concentration

3.2 mM 10.0 mM 10.0 mM 10.0 mM 0.04% 0.04% 0.04% 0.04% 0.04%

Dry weight mg plate"'

21.3 36.1 10.4 89.2 37.4 36.1 44.7 25.7 28.6

± ± ± ± ± ± ± ± ±

1.5 3.4 1.6 5.6 2.7 3.6 3.2 2.1 2.4

Proteinase activity Units mg dry wt~'

% of control

32.4 16.7 74.7 8.6 9.6 18.4 60.1 52.9 70.8

Control 52 230 27 30 57 185 163 219

± ± ± ± ± ± ± ± ±

2.1 1.2 3.4 0.9 2.1 2.7 3.2 4.6 5.2

Final pH

2.8 5.4 5.3 6.5 6.4 4.9 4.8 4.1 4.5

* Data are presented as mean ± standard deviation of five replicates. t N sources were added to the basal medium containing 1% glucose. BSA, Bovine serum albumin. Data from Zhu etal. (1994).

Ectomycorrhizas

264

are increasingly reported to be present in solutions extracted from acid soils of the kind that are occupied by ectomycorrhizal roots (Abuarghub and Read, 1988a,b; Kielland 1994). Hebeloma. crustuliniforme, when grown on protein as sole N source, shows a prolonged lag phase followed by a phase of more rapid growth during which proteolysis results in accumulation of amino acids in the medium (Read et al., 1989; Fig. 8.4). These are subsequently assimilated, but only after the protein concentration has been markedly reduced. At no stage during growth is there evidence of NH4 release, indeed, NH4 ions are detectable in the medium only when the amino compounds have themselves been virtually exhausted. By this stage, C starvation of the fungus is likely to have led to deamination of the residual compounds and release and utilization of the C skeletons. Such starvation, and the release of NH4 associated with it, may both be artefacts of the pure culture environment, and not representative of the mycorrhizal condition in which C supply from the colonized plant is assured. Furthermore, the failure of NH4 to appear in the medium at earlier stages of growth could reflect the relative rates of deamination of protein (if that occurs), and uptake of NH4 which would be likely to be rapid in actively growing mycelium. The availability of proteins in soil depends on their structure and solubility and on their interactions with other soil components. Thus the relatively insoluble gelatin is less available in culture than is the soluble BSA, but in soil all proteins might be rendered unavailable by tanning reactions with phenolic compounds, by ionic reactions with soil organic matter or clays, or by physical occlusion in the soil 2.2-1

2.0 H 1.8H

iF

1.6H

c

1.4-j

JO c

1.2 J

0

8 1.0-]

0 0

c

^ Q-

0.8-^ 0.6-] 0.4 H 0.2-J 0.0-1 Time (days)

Figure 8.4 The relationship between protein utilization, mycelial blomass production and amino acid release when Hebeloma crustuliniforme is grown in liquid culture with protein as the sole N source. Note that NH4 does not appear in the medium until all the protein has been used and the fungus therefore starved of C. # , Protein concentration; O, amino acid concentration; • , mycelial dry weight. Data are standard errors of means. From Read et al. (1989), with permission.

Nitrogen nutrition of ectomycorrhizal plants

265

pores. The success of mycorrhizal fungi in obtaining these less available resources might very well be increased, compared with saprophytic fungi, because they are not dependent on soil organic C. For this reason, if for no other, it is clearly essential to extend studies of the N-assimilating abilities of the fungi to intact symbiotic systems in which the normal pathways of organic C supply would be operating.

Use of N by Ectomycorrhizal Roots and Intact Plants Inorganic N Sources The fate of ^^N from N O ^ and NH4 fed to external mycelium in intact mycorrhizal systems has been studied by Finlay et al. (1988,1989) and more recently by Ek et al. (1994). Glutamine has been identified as a major sink for absorbed N, with alanine, arginine and aspartate-asparagine also important (Finlay, et al. 1992; Martin and Botton, 1993; Botton and Chalot, 1995). The contribution of arginine to the pool of amino acids and amides differs with the fungal species involved and the suggestion has been made that this may relate to its accumulation in vacuoles, where arginine might play a role in stabilizing polyphosphate (Finlay et al, 1992). This point will be addressed again in Chapter 14. The data obtained with ^^N on intact mycorrhizal systems is consistent with earlier findings using ^^C with excised roots, confirming that glutamine is most likely to be the major form in which N is translocated in the mycelium and transferred across the symbiotic interface to the plant (Harley, 1964; Carrodus, 1967; Reid and Lewis, in Lewis, 1976). There is no doubt that transfer occurs very rapidly in the intact systems (Finlay et al, 1988, 1989). ^^N-labelling experiments have shown that NH4 is preferentially absorbed from NH4NO3 by Paxillus involutus in association with Betula pendula and also that NH4 inhibited ^^NO^ assimilation in the external hyphae (Ek et al, 1994). Again, the NH4 was assimilated into glutamine at the uptake site, providing evidence for the importance of this amide in translocation. The pathways of assimilation in the fungi have also been investigated. France and Reid (1983) provided a conceptual model of the mechanisms thought to be important for assimilation of NH4 in ectomycorrhizal roots, in which the pathways involving GS, GOGAT and GDH were all present. Subsequent studies (Martin and Botton, 1993; Botton and Chalot, 1995) have revealed a diversity of pathways in which the plant exercises considerable influence over the activities of the fungus in the symbiotic condition, as well as important differences between plant-fungus combinations (Fig. 8.5). Whereas the GDH-GS pathway appears to predominate in the fungi, NH4 assimilation in higher plants is known to occur primarily via GSGOGAT (Miflin and Lea, 1976; Robinson et al, 1991; Oaks, 1994). If fungal and higher plant cells have distinctive pathways of inorganic N assimilation, it is important to determine how these are controlled in the mycorrhizal condition. In some cases the fungal enzymes are down-regulated in proximity to plant tissues, a feature that has given rise to the concept of metabolic zonation (Fig. 8.6), while in others activity is relatively unaffected. Thus in Picea mycorrhizas formed by Hebeloma, NADP-GDH activity was greatest in the extraradical hyphae

266

Ectomycorrhizas

GS y / " " ^

(a)

GLUTAMINE

W

^

GLUTAMINE

- ^

y ^ - oc-KETOGLUTARATE

A

t

GOGAT Y

NKA GLUTAMATE M

B

GLUTAMINE

(b)

GLUTAMATE

Mr

^ ^

HEXOSES GLUTAMATE

GLUTAMINE

ASPARTATE

ASPARAGINE

NK-^ATP ^ ^GDH GLUTAMATE

-4-

GLUTAMATE

ALANINE

—

ALANINE

t

NH;-^ «-KETOGLUTARATE

V. (C)

GS

GLUTAMINE _^ ^^-

J

a^<ETOGLUTARATE

GOGAT J

NH:ATP>.,*Av^

^ ^

GLUTAMATE

HEXOSES

HEXOSES

HEXOSES

HEXOSES

\^ GLUTAMATE

GLUTAMATE •ROOT

FUNGUS INTERFACE

Figure 8.5 Possible localization of enzymes of NH4 assimilation in different types of ectomycorrhizas. (a) ?Qgus\ glutamine synthetase (GS) in the fungus and gtutamate synthase (GOGAT) in the root, (b) ?\C^Q\ NADP-glutamate dehydrogenase (GDH) and GS in the fungus and asparagine synthetase in the root, (c) Pisolithus mycorrhizas: GS and GOGAT in the fungus. Modified from Martin and Botton (1993).

and mantle, but reduced in the Hartig net (Dell et al, 1989). In line with this, the quantity of GDH polypeptide, revealed by immunogold labelling, decreased progressively from the peripheral cells of the mantle to the Hartig net (Chalot et al., 1990a). In Fagus mycorrhizas, on the other hand, both the activity (Dell et al, 1989) and the amount (Chalot et ah, 1990a) of GDH were strongly suppressed throughout the mantle whether it was formed by Hebeloma or by Cenococcum geophilum or Paxillus involutus. A similar picture is revealed in the c^se of Fagus roots with Lactarius-type mycorrhizas, there being successive incorporation of ^^NH4 into the amino N of glutamine, glutamate and alanine, consistent with GS activity (Martin et ah, 1986). The importance of this pathway was confirmed by incubation of roots in MSX which inhibited ^^N incorporation into glutamine and glutamate by 90%. Supporting evidence for the role of GS-GOGAT was obtained by use of the GOGAT inhibitor azaserine, which completely blocked glutamate synthesis from NH^. Such observations are suggestive of a complete down-regulation of fungal GDH in some mycorrhizal types, although there remains the possibility of expression of some activity in the mycelium as it grows away from the root. In addition, N sources (NH4 vs. NO^) and concentration (N-rich vs. N-starved) regulate NADP-GDH biosynthesis and activity, through the alteration of GDH mRNA

Nitrogen nutrition of ectomycorrhizal plants

267

Mantle

•

Extramatrical

Figure 8.6 Metabolic zonatlon of expression of enzymes of N assimilation in the extraradical mycelium of ectomycorrhlzas. Expression of both glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the fungus is down-regulated close to the root. From Martin et al. (1992), with permission.

in L. laccata S238 (Lorillou and Martin, 1996; Lorillou et al, 1996), suggesting the potential for regulation by N availability in soil. Further evidence for the role of NH4 concentration in controlling pathways of NH4 assimilation in the fungi (albeit from studies in pure culture) comes from the use of nuclear magnetic resonance (NMR) spectroscopy to monitor ^^N labelling, after feeding L. hicolor with ^^NHJ (Martin et al, 1994). Rapidly growing mycelium assimilated ^^NH4 into glutamine via GS which was probably the main route, but when this pathway was inhibited by MSX, GDH activity became apparent. In stationary-phase growth with low concentrations of NH4 in the medium, both pathways operated. The pathways of NH4 assimilation and the distribution of the enzymes involved have important implications for the mechanisms of transport of N across the fungus-root interface. Martin and Botton (1993) recognized three basic patterns of N incorporation. In Fagus, GS is localized in the fungal sheath and GOGAT in the root (Fig. 8.5a). In Picea (Fig. 8.5b), both GDH and GS occur in extraradical mycelium and sheath, respectively, with asparagine synthetase (AS) in the root. A third pattern (Fig. 8.5c) is representative of mycorrhizas formed by Pisolithus tinctorius, in which the GS-GOGAT pathway appears to operate in the fungus.

268

Ectomycorrhizas

These models are later considered in relation to transport of N and C across the plant-fungus interface (Chapters 14 and 15). Here it should be noted that although Figure 8.5 shows transfer of hexoses from plant to fungus, this is an oversimplification because (as shown in Chapters 7 and 14) transfer of organic C probably involves sucrose efflux to the apoplast and its hydrolysis to hexoses before uptake by the fungus. The question of uptake and transport of solutes can best be addressed in systems in which the sources of nutrient supply (the soil) and the ultimate sinks (the transpiring shoots) are intact. Enhancement of NH4 uptake was demonstrated when intact seedlings of Tsuga heterophylla, Picea sitchensis and Pseudotsuga menziesii were colonized by H. crustuliniforme compared with non-mycorrhizal seedlings over a pH range of 3-7 (Rygiewicz et ah, 1984). Significantly greater rates of uptake were maintained in all three species when the plants were mycorrhizal. However, absolute amounts absorbed were always lower in both categories of plant imder acidic conditions. Finlay et aL (1988) fed ^^NH4 to the distal parts of mycelial systems of Rhizopogon roseolus, Suillus bovinus, Paxillus involutus and Pisolithus tinctorius growing across peat from colonized roots of Pinus sylvestris. Measurements of the distribution of ^^N after 71 hours showed high labelling in glutamateglutamine, aspartate-asparagine and alanine in all plant-fungus combinations, except that involving P. involutus which showed no labelling in aspartateasparagine. Within this time period, 5-50% of the amino acids in the shoot were also labelled, indicating the very rapid operation of the transport and transfer processes. Within the plant, despite large differences in the size of the different amino acid pools, the levels of ^^N enrichment were similar, indicating that equilibrium between the pools, dependent upon activities of amino transferases, was rapidly achieved. Studies of the impact of mycorrhizal coloruzation upon NO^ assimilation and growth of the plant have again made clear the differences between patterns seen in the axenic and symbiotic systems. Scheromm et al, (1990a), using the isolate of H. crustuliniforme shown in pure culture preferentially to use NO^, could find no direct effect of the fungus upon either uptake or reduction of NO^ by mycorrhizal Pinus pinaster. Using the same fungus, Rygiewicz et al. (1984) observed no differences in NO^ uptake between colonized and uncolonized plants of Picea sitchensis or Tsuga heterophylla. Increases in uptake of the ion were, however, observed in colonized Pseudotsuga seedlings. pH was the major factor determining uptake of NO3 in both categories of plant, it being increased as pH was increased. Finlay et al. (1989) fed ^^NO^ to the extraradical myceliimi of Paxillus involutus associated with Fagus sylvatica and compared the patterns of uptake, assimilation and transport of the label with those obtained in systems fed with ^^NH4. ^^N was taken up from both sources, incorporated into a range of free amino acids and transported to the shoots (Table 8.3). However, the amounts of enrichment of most free and protein-boimd amino acids were usually greater in the systems fed with ^^NH4 , than in those fed with ^^NOaT. N assimilated from NO3 was only 62% of that obtained from NH4 (Table 8.4). Interesting though these results are, the inm\ense genetic variability, even within races of P. involutus itself (Laiho, 1970), with respect to NO^ utilization must be borne in mind. The possibility remains that where roots of trees such as Fagus grow in nitrifying environments, selection may favour colonization by fungi which preferentially use NO^.

269

Nitrogen nutrition of ectomycorrhizal plants

Table 8.3 Levels of '^N enrichment (atom % excess) in free amino acids in ectomycorrhizal systems of Fagus sylvatica infected with the fungus Paxillus involutus and supplied with '^N-labelled NH^ or NOg^ N source

Mycelium

Mycorrhical tips

Shoots

Roots

NO3"

NH;

NO3"

NH;

NO3"

NH;

NOB"

NH;

Alanine Glycine Threonine Serine Leucine Isoleucine Gaba Pipecolinic acid Asx Glx Lysine Tyrosine Arginine No. 1

30.8 11.5 2.5 8.6 0.0 3.5 30.8 0.0 30.0 37.7 0.9 9.9 1.2 38.1

57.9 24.7 8.0 26.6 6.7 5.1 61.5 0.0 53.9 68.5 5.8 10.7 0.0 69.6

25.9 4.7 I.I 3.1 0.0 0.9 22.0 11.9 14.0 25.3 1.2 2.6 6.8 28.5

38.6 7.9 1.6 41.3 3.7 6.6 29.8 1.3 17.7 51.1 3.5 5.3 II.1 56.1

6.4 0.0 0.8 0.9 2.7 5.5 5.1 0.0 4.6 6.4 1.3 1.0 0.4 6.6

13.0 1.9 2.4 6.5 7.1 7.8 16.2 0.3 16.9 21.0 3.1 13.2 4.1 23.3

0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 1.4 0.0 0.0 0.0 0.0 0.0

0.0 0.4 0.0 2.0 0.5 0.0 1.0 0.0 1.3 2.1 0.0 9.5 0.0 1.4

Mean

14.7

28.5

10.6

19.7

2.9

9.8

0.1

2.1

Gaba: gamma amino butyric acid Asx: aspartic acid + asparagine Glx: glutamic acid + glutamine No. I represents an unidentified amino acid. Data from Finlay et al. (1989).

Organic N Sources Since Melin and Nilsson (1953a) demonstrated uptake and transfer of ^^Nglutamate by Suillus granulatus in mycorrhizal association with Pinus, there have been surprisingly few studies of amino acid utilization by intact mycorrhizal systems. Alexander (1983) fed aspartic acid and serine at concentrations of 0.5 mM to mycorrhizal and non-mycorrhizal Picea sitchensis. While both depressed growth of non-mycorrhizal seedlings, the inhibitory effect of aspartic acid was removed by mycorrhizal colonization and that of serine was reduced. Clearly, at high concentrations toxicity of amino acids can be a problem for both fungus and plant. Accumulation of the amino acids alanine, arginine and aspartic acid in mycorrhizal roots of Pseudotsuga menziesii and Tsuga heterophylla has been demonstrated in short-term uptake studies using colonized and uncolonized plants (Sangwanit and Bledsoe, 1987). When grown with alanine or its peptides as sole N sources over a longer period of time, Betula showed striking responses to mycorrhizal colonization (Abuzinadah and Read, 1989a). In the absence of colonization, birch seedlings appeared to have no ability to use alanine and were N deficient. In contrast, when colonized by Hebeloma crustuliniforme, Amanita muscaria or Paxillus involutus the plants grew vigorously and their tissues showed N

Ectomycorrhizas

270

Table 8.4 Levels of '^N enrichment (atom % excess) in protein-incorporated amino acids in ectomycorrhizal systems of Fagus sylvatica infected with the fungus Paxillus involutus and supplied with '^N-labelled N H ^ or N O r . N source

Mycelium

Mycorrhizal tips

Roots

Shoots

NO3"

NH;

NOa'

NH;

NO3"

NH;

NO3"

NHj

Alanine Glycine Valine Serine Leucine isoleucine Proline Asx Phenylalanine Glx Lysine Tyrosine Arginine

I.I 0.2 1.2 1.3 1.8 I.I 0.6 2.1 1.0 3.5 1.3 1.0 3.2

4.7 1.6 2.8 1.9 4.6 3.6 3.3 5.4 2.8 9.3 I.I 0.1 1.4

3.8 3.1 2.6 2.9 4.4 3.7 1.9 5.0 3.2 6.0 1.4 0.8 2.9

6.8 3.4 3.4 2.9 5.3 4.8 2.8 6.2 3.7 8.3 1.5 1.5 1.3

1.6 1.5 1.5 1.6 1.8 1.7 1.2 2.0 2.3 2.2 0.7 0.5 1.2

4.1 3.0 2.4 2.3 3.2 3.1 1.9 4.5 3.5 4.4 I.I 1.6 3.4

0.3 0.2 0.0 0.2 0.1 0.0 0.0 0.4 0.1 0.2 0.3 0.5 1.3

0.3 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.4

Mean

1.6

2.7

2.8

3.4

1.4

2.6

0.3

0.2

For abbreviations see Table 8.3. Data from Finlay et al. (1989).

concentrations of the kind seen in healthy plants. When the plants were supplied writh peptides of alanine of chain lengths from two to six units, again, no growth was observed without colonization, whereas growth and N concentration were greatly increased in mycorrhizal plants. There were some differences in the effectiveness of the three fungi: H. crustuliniforme gave higher yields and N contents than A. muscaria which was, in turn, more effective than P. involutus. Yields and N contents were generally higher on the peptides than on the amino acids and in plants colonized by the two most effective fungi; the values were obtained with the largest peptide units (Fig. 8.7). Plants colonized by those mycorrhizal fungi which can hydrolyse BSA and gliadin, show increases in growth and N content when grown with these proteins as sole N source. Thus yields of Pinus contorta colonized by R. roseolus or S. bovinus were significantly higher than those of non-mycorrhizal plants supplied with BSA, and were similar to those grown with NH4 at the same N concentration. Colonization by P. tinctorius, shown in pure culture to have lower proteolytic ability, gave little access to the substrate, and the plants grew less well (Abuzinadah et al,, 1986; Fig. 8.8). The 'protein fungus' H. crustuliniforme gave similar responses in Betula pendula, Picea sitchensis and Pinus contorta, while none of these species had access to protein N without colonization (Abuzinadah and Read, 1986b). Mycorrhizal colonization had a major impact upon N utilization by Eucalyptus seedlings (TumbuU et ah, 1996). Both E. grandis and E. maculata used amino acids and protein when they were mycorrhizal, an ability lacking in non-mycorrhizal seedlings. Both species grew more than non-mycorrhizal plants on protein as well as on the amino acids

N i t r o g e n n u t r i t i o n of ectomycorrhizal plants (a) 8

T

6

n Root S Shoot H Whole plant

L-alanine (0.05)

(0.05)

271 (b) 8

Di-alanine (0.05) (0.05)

J (0.05) I

(0.05)T T

LSDJ I [

5Dl i i

R Sh PI § 4

41

^

^2]

^ 2

A.^Jm 8i N

(0.001)

N

(0.01) ] D.05) -T (0.05)

(0.001)

LSD J R Sh PI

'ill

c
(0.001)

(0.01)

LSD I R Sh PI

6

CNP"

I 4

NM

HC

f

AM

Penta-alanine

8 z 2i

EI

NM (d)

HC

AM

J

Hexa-alanine

8 (0.05)

(0.05)

(0.05)

-P 6 i LSD

III

41 5

c c d (0.01) (0.05)

cr6 f

(0.01)

4

z 2 c c b

HC

NM F i g u r e 8.7 G r o w t h (G) and N contents ( N ) of • Betula pendula involutus N

c o l o n i z e d b y Hebeloma

crustuliniforme

roots, ^

AM

s h o o t s a n d [ffl] w h o l e p l a n t s o f

( H C ) , Amanita

muscaria

(AM),

Paxillus

(PI), o r n o n - c o l o n i z e d ( N M ) g r o w n w i t h d i f f e r e n t a m i n o a c i d s a n d p e p t i d e s as s o l e

s o u r c e f o r 7 5 days. V e r t i c a l b a r s r e p r e s e n t least s i g n i f i c a n t d i g i t ( L S D ) f o r r o o t s ( R ) ,

s h o o t s (Sh) a n d w h o l e p l a n t s (PI), (a) A l a n i n e ; ( b ) d i - a l a n i n e ; (c) p e n t a - a l a n i n e ; ( d ) h e x a a l a n i n e . F r o m A b u z i n a d a h a n d Read ( 1 9 8 6 b ) , w i t h p e r m i s s i o n .

272

Ectomycorrhizas

12

(a)

I

(b)

I i

n 1I ******

8 6

1

1 20

10

1 I

1

(c)

'b ^1 -•o a;

www

2'

I

(d)

J. I '2| "c

o

^ 10

i

F i g u r e 8.8 D r y weights (a and b) and N contents (c and d) of Pinus contorta colonized by Suillus bovinus (Sb), Rhizopogon roseolus (Rr) o r Pisolithus tinctorius (Pt), o r non-mycorrhizal ( N M ) after 40 (a and c) o r 80 (b and d) days* g r o w t h on N H 4 (open bars) o r BSA (crosshatched bars) as sole N source. Vertical bars represent least significant difference (LSD) and asterisks indicate significant differences between mycorrhizal and non-mycorrhizal plants w i t h i n each t r e a t m e n t . * P<0.05; * * P
arginine, asparagine and histidine, when colonized by an Elaphomyces sp. (Fig. 8.9a). Although both species used protein less readily when colonized by a Pisolithus sp. (Fig. 8.9b), the mycorrhizal plants were still significantly larger that their non-colonized counterparts. When Betula was grown monoxenically with selected mycorrhizal or saprotrophic fungi, colonization by A. muscaria and H. crustuliniforme gave highest yields and N content on protein N, while P. involutus was somewhat less effective as a symbiont (Abuzinadah and Read, 1989b). In the presence of a seed-borne saprophyte, Ulocladium hotrytis, or an ericoid mycorrhizal fungus, Hymenoscyphus ericae, no growth responses or net increases of N content of the Betula were observed. In contrast, inoculation with the saprotroph Oidiodendron griseum, while giving lower plant yields and total N contents than A. muscaria and H. crustuliniforme, did facilitate accumulation of N in the tissues of Betula, presumably because N became available via mineralization (Abuzinadah and Read, 1989b). These results highlight one of the difficulties involved in extrapolating from experiments carried out under monoxenic conditions. In this case a non-mycorrhizal saprotroph presumably converted protein to a form which could be absorbed

273

Nitrogen nutrition of ectomycorrhizal plants

150-,

Egrandis

I

T 100-^ I

JL

I

^

PiPnPnPiPr^rlrj 200

E, maculata

OS

I

150

I

100-1

\

O £*0

z

CO

< o o

Nitrogen Source

50

s ^ 2

0 200-1

S

150-J 100-^

WW

50

I

100 ^

SO A

Q

£". grandis

150

CO X

50 H

<

(0 CQ

4

1 44

JUH-XSHLJBLCSH

E, maculata

I I

I WIAM

>- (0 < o X o - J X «^ < o o Nitrogen Source

Figure 8.9 Growth of seedlings of Eucalyptus grandis and £ maculata in agar culture on a range of inorganic and organic N sources, either non-mycorrhizal ( Q ) or ectomycorrhizal ( • ) with: (a) Baphomyces sp. or (b) Pisolithus sp. Data are means of 12 replicate plants, ± standard errors of means. Data from Turnbull et al. (1995), with permission.

and assimilated by the plant. In a mixed population of heterotrophs such products are less likely to accumulate. Clearly, the extent of benefit accruing to the plant from its fungal associates will depend upon a combination of factors which include plant-fungus compatibility, the ability of the symbiont to compete with other organisms for a resource and, having gained access to a substrate, its capacity to mobilize the nutrient elements which it contains. These factors can only be realistically evaluated in the presence of mixed natural populations of symbionts and saprotrophs although under these conditions the mechanisms operating will be hard to unravel. Dighton et al. (1987) approached this problem by examining the breakdown of the N-containing substrates powdered animal hide and chitin by mycorrhizal plants of P. contorta grown in the presence or absence of the basidiomycete decomposer Mycena galopus. When roots of the plant were colonized by the mycorrhizal fungi H. crustuliniforme or S. luteus, significantly greater degradation of these substrates occurred, together with enhanced plant growth. In the presence of the saprotroph the effectiveness of the mycorrhizal fungi in acquiring N was much reduced. However, interpretation of these results is complicated by the addition of readily available C to the medium, thus increasing the C:N ratio and probably enhancing, to an unrealistic extent, the ability of the saprotroph to compete for N in the organic substrates. Indeed, one of the advantages which all mycorrhizal fungi may have is that (in symbiosis) they are not dependent for C on orgaruc substrates

274

Ectomycorrhizas

in soil. In consequence, the C:N (or C:P, etc.) ratio of the substrates will be considerably less important for them than for saprotrophs. Ultimately, evaluation of the role of mycorrhizal fungi in the mobilization of N under natural conditions can be achieved only by supplying colonized plants with the substrates which they would normally encounter in the field, in the presence of a natural population of soil microorganisms. Bending and Read (1995a) grew plants of Pinus sylvestris colonized by Suillus bovinus or Thelephora terrestris on non-sterile peat in transparent observation chambers. Organic matter freshly collected from the FH horizon of a pine forest was supplied in weighed aliquots on plastic trays. On reaching these introduced organic materials the mycelium of the mycorrhizal fungi proliferated intensively to form dense 'patches' over the substrates (see Fig. 6.23). 'Control' trays of organic matter were placed in chambers containing peat but no plants and incubated under the same conditions. After a standardized period of occupation of the organic matter, the total N content of the material was measured and the quantities of N released during the incubation period determined. There was no significant loss of N in the controls, whereas in the organic matter colonized by both fungi there was a significant (23%) depletion of N in material colonized by S. bovinus and 13% in that colonized by T. terrestris (Fig. 8.10). These values are similar to those obtained at by Entry et al. (1991), who measured decline of N concentration in litter of Pseudotsuga menziesii colonized by mycelium of the matforming mycorrhizal fungus Hysterangium setchelli in the field. Over one year they observed losses of 32% of initial N from the litter. Colonization of natural substrates by mycorrhizal fungi both in the field (Griffiths and Caldwell, 1992) and in microcosms (Bending and Read, 1995b) leads to significant increases of proteolytic activity. These observations, coupled with those I5r

o

m

X

rh

12h

T3 O)

E L

9h

6h

m

Uncolonized

H h T. terrestris S. bovinus

F i g u r e 8.10 Utilization of N from litter collected from the FH horizon of forest soil. Hatched bar shows N content at the start of the experiment, and open bars show N content after incubation, uncolonized or colonized by mycelium of Thelephora terrestris or Suillus bovir)us, growing in symbiosis with Pir)us sylvestris. F H O M , fermentation horizon organic matter. Treatments with different letters are significantly different at P
Nitrogen nutrition of ectomycorrhizal plants

275

showing proteolytic capability of these fungi in axenic culture and on colonized plants under aseptic conditions, are strongly suggestive of a direct role of mycorrhizal associations in mobilizing N from natural substrates, but do not provide definitive evidence of this. An alternative explanation is that the presence of mycorrhizal mycelium in the substrates somehow facilitates the activities of saprotrophs. What is evident is that the mycorrhizal fungi, by intensive colonization of the substrate and provision of organized mycelial aggregates connecting resource deposits to roots, are extremely effective as scavengers for, and transporters of, nitrogenous materials. Conclusions The ease with which many ectomycorrhizal fungi can be grown in axenic culture has enabled extensive screening of their abilities to use different forms of N. Most species readily use NH4, NO^ and some simple organic N compoimds, although there are differences at both the inter- and intra-specific levels. Much new information has been gained concerning the biochemistry of N assimilation, in particular in relation to the enzymes involved in assimilation of NH4, which in many ectomycorrhizal fungi appears to be the preferred source of inorganic N. These studies have been extended to allow analysis of N assimilation by the fungi when grown in the mycorrhizal condition and, importantly, to investigate the influence of the plant upon the pattern of events. Increasingly, it has been recognized that much of the N contained in the superficial layer of soil occupied by ectomycorrhizal roots is in organic combination and that some ectomycorrhizal fungi have access to these more complex N sources. Emphasis is thus moving away from studies of the use of simple inorganic N sources towards investigation of more realistic questions of the extent of exploitation of the predominant forms of N in soil. These questions are themselves being addressed in laboratory studies of the biochemistry of proteolysis of pure substrates, as well as in experiments where the exploitation of naturally occurring substrates is being tested. It must be recognized that ectomycorrhizal fungi forage for nutrients only as components of complex microbial communities in soil, and that their abilities to mobilize N from organic residues will depend on competition and collaboration, as well as on biochemical attributes. Such attributes are likely to be best developed when the fungi are growing symbiotically and there is a clear need for more extensive investigations of their relative abilities to exploit N resources under circumstances where they coexist with members of the decomposer commuruty.

Phosphorus nutrition of ectomycorrhizal plants

Introduction The early view, effectively expounded by Frank (1894), that ectomycorrhizas were especially important in absorption of N, held sway for much of the early part of the twentieth century. Its prominence was weakened by the seminal paper of Hatch (1937), who demonstrated that mycorrhizal colonization in Pinus strobus led to increased concentrations of P and K, as well as of N (Table 9.1). Hatch was of the opinion that the importance of mycorrhizas lay in their ability to increase the uptake of any nutrient in short supply. This broad view of ectomycorrhizal fimction was also espoused by others (Mitchell et ah, 1937; Finn, 1942). However, some contemporary studies (e.g. McComb, 1938; McComb and Griffith, 1946; Stone, 1950), indicated that absorption of P was enhanced more than that of other nutrients. The publication of these findings was followed within a few years by the first commercial production of ^^P, which greatly improved the precision with which movement of P could be traced in biological systems. Its availability led to a significant shift of emphasis towards study of P nutrition of mycorrhizal plants. Table 9.1 Growth and specific nutrient uptake of N, P and K by P'mus strobus seedlings K

P

Degree of mycorrhizal infection

Dry weight (mg)

Root weight (mg)

T

SA

T

SA

T

SA

Mycorrhizal

448 361

180 170

5.39 4.62

0.030 0.027

0.849 0.729

0.0047 0.0042

3.47 2.57

0.019 0.015

Non-mycorrhizal 300 361 30!

174 182 152

3.16 2.5! 2.40

0.013 0.018 0.016

0.229 0.268 0.211

0.0013 0.0015 0.0014

1.04 1.94 1.17

0.006 0.0 II 0.008

N

T, Total absorbed (mg); SA, specific absorption (mg mg ' root dry wt). Data from Hatch (1937) and see Harley and Smith (1983).

Phosphorus nutrition of ectomycorrhizal plants

277

and studies of ^^P uptake were carried out by Kramer and Wilbur (1949), Harley and McCready (1950) and Melin and Nilsson (1950). These studies set a trend of work on P uptake and nutrition of mycorrhizas, which was performed ahnost to the exclusion of work on uptake of the other elements until the 1980s (see Harley and Smith, 1983). As Jack Harley was wont to point out, the obsession with P became analogous to that with the three-legged rabbit 'Alphonse', which the gamekeeper produces from a bag as a target for those hunters whose vision is inadequate to enable them to hit a healthy animal: 'We all shoot Alphonse' he would say. While these studies laid the foimdation for much elegant research on the uptake of P by roots, which will be examined in this chapter, the broader view of mycorrhizal function espoused by Hatch (1937) and by Harley and Smith (1983) is the one which now more closely reflects the cumulative results of research. There are, as discussed in Chapter 15, many circumstances in nature where P deficiency is clearly the primary limitation on productivity of plants and in which, therefore, ectomycorrhizal colonization may be of particular importance for P nutrition. These are the systems in which further work on P nutrition is particularly necessary. In those even more widespread areas where elements other than P limit plant growth (see Chapters 8 and 15) work on these nutrients (N, K and so on) should be emphasized and the apparent significance of mycorrhizas to P nutrition of plants should be assessed with circumspection.

Uptake of Phosphorus by Excised Ectomycorrhizal and NonMycorrhizal Roots Much of the detailed experimental work on the mechanism of uptake of nutrients, particularly P, by ectomycorrhizas has been done with excised roots, in which both the external mycelial system in the soil and the throughput of water are eliminated. The overwhelming reason for using excised mycorrhizas for investigating certain aspects of mycorrhizal physiology is that uniform samples can be obtained for studying specific aspects of the uptake processes. Whole root systems are composed of mycorrhizas, uncolonized primary roots and secondarily thickened axes in different proportions so that it is almost impossible to conclude anything about detailed mechanisms in mycorrhizal roots from them. It is easy to obtain large numbers of similar mycorrhizal roots from the surface layers of forest soil, wash and prepare them with no great effort. Of course, excised mycorrhizas and nonmycorrhizal roots can also be obtained from aseptically grown plants in the laboratory, but the labour of providing them in sufficient quantity for experimental work on a large scale is daunting. The criticisms levelled at the use of excised mycorrhizas are those applicable to all experiments with excised roots, i.e. that the transpiration stream is eliminated and the tissue may become starved of organic C during the experiment. In the case of mycorrhizas there is the additional problem that the root is detached from what is increasingly seen to be the critical absorbing system, the extraradical mycelium. Setting these problems aside for the moment, the factors which affect the rate of absorption of nutrients by excised mycorrhizas are similar to those which affect the rate of absorption by most plant material, including roots. This was an important

278

Ectomycorrhizas

contribution from the work of Harley's group using Fagus roots most probably colonized by Lactarius subdulcis (e.g. Harley and McCready, 1952a,b; Harley et al, 1953, 1954, 1956; Harley and Jennings, 1958; Harley and Wilson, 1959; Carrodus, 1966; and see Harley and Smith, 1983; see Plate 3), and served to focus attention on the fact that nutrient absorption by mycorrhizas was more relevant to the natural situation than work with non-colonized roots. In the ensuing account the aim will be to consider the manner in which the absorption physiology of ectomycorrhizas differs from that of non-colonized roots. The work with excised mycorrhizas was predicated on the idea that the sheath tissue which covers the root resembled the extraradical mycelium and that uptake characteristics of the sheath could be extrapolated in developing ideas of nutrient uptake by hyphae from soil. As we have seen from discussions of the changes in enzyme activities in mycelium and sheaths (see Chapter 8), this assimiption must now be viewed very much as a first approximation. The development of the fungal sheath, as well as the activity of the extraradical myceliimi, have important impacts on the duration of active absorption by a root system. The most active non-colonized root apices differ from mycorrhizas in that they are dividing and growing. It is well known that the rate of uptake in the tip region of a growing root is much greater than that in the region behind it. This is not generally true of mycorrhizal apices. McCready (unpublished) found that the uptake of P by Fagus mycorrhizas did not change greatly over distances of 12 cm. However, the rate of absorption of mycorrhizal and non-mycorrhizal roots may be very different as shown in Table 9.2 (Harley and McCready, 1950; Bowen and Theodorou, 1967). The differences were also emphasized by the autoradiographs of Kramer and Wilbur (1949) and also of Harley and McCready (1950), which show intense P accumulation in the mycorrhizas and in the extreme apices of noncolonized roots, but not elsewhere. Bowen (1968) scanned the long roots of P. radiata with a Geiger counter and showed that whereas the most active region of accumulation in uncolonized roots was at the apex and in the positions of the apices of developing short roots, that of a long root bearing mycorrhizas was at its apex and more particularly at the positions of the mycorrhizal rootlets.

T a b l e 9.2 Comparative uptake rates of phosphate by excised mycorrhizas and uninfected roots Authors and host

Fully colonized

Uncolonized

Sheath poorly developed

Harley and McCready (1950) Fagus sylvatica

5.18 6.68 1.97 2.72 1.69

0.88 0.75 0.42 0.61 0.72

4.76 1.20 0.62

7.5 15.5 15.0

3.5

Bowen and Theodorou (1967) Pinus radiata

The values for each host are relative to one another.

5.5

— 2.13

5.5

Phosphorus nutrition of ectomycorrhizal plants

279

Harley and McCready (1952a) showed, using ^^P, that the exposure of excised roots to a bathing solution resulted in a great accumulation of phosphate in the fungal sheath. By dissecting the fungal layer from the plant tissue (the core; see Fig. 7.2c) they were able to estimate the relative quantities accumulated in the two symbionts. This method was later used for more detailed analyses, in particular of C (see Chapter 7) as well as P fractions and other nutrients. In the case of P, about 90% of that absorbed was found in the sheath after uptake from low concentrations. This observation has since then often been confirmed. Harley and McCready (1952a) verified that the accumulation was not dependent on, or influenced by, excision. They compared the distribution of P between the sheath and the root tissue (the core) in mycorrhizas attached to adult trees in the forest and those detached. Comparisons were made on three occasions: when the trees were leafless, developing their leaves and in full leaf. On all occasions there was a great accumulation of P in the sheath (Table 9.3) although the extent to which P was absorbed within rather than adsorbed onto the surface of the sheath was not determined. These observations of Harley and McCready (1952a) extended only up to July and suggested little seasonal variation in rate of removal of P from solution. It has been shown since that the ectomycorrhizal roots of Abies balsamea (Langlois and Fortin, 1984) and Picea sitchensis (McDonald et al., 1991) show a distinct seasonality in their ability to absorb P, maximum rates being achieved in late summer or autumn after completion of extension growth above ground. The results of Harley and McCready (1952a,b) were obtained by dissecting roots following a period of exposure to PO^ solution. Two features must be noted. First, as external solution is applied to the sheath accumulation can appear to take place as P is adsorbed onto its surface or as it passes through the fungal tissue to the core within. Second, there may also be a real accumulation depending upon the species of the fungus and its activity. Garrec and Gay (1978) analysed the mycorrhizas of Pinus halepensis using an electron probe and concluded that P is mainly accumulated in the fungal sheath and Hartig net region and is lower in the plant tissue. Table 9.3 Estimates of the proportion of P which accunnulates in the fungal sheath of Fagus mycorrhizas when attached to the parent root system or when excised Condition of mycorrhizas Attached

Detached

31 March

II May

23 July

31 March

II May

mMKH2^^P04

0.074

0.32

0.16

Mean percentage in sheath Range

88 74-96

88 83-94

23 July

1.6

0.074

0.32

0.16

1.6

90

87

91

89

85

91

89-94

86-93

85-96

79-93

83-89

91-93

Experiments In Bagley Wood with roots of adult trees at three seasons. Mycorrhizas In aerated phosphate solution pH 5.5 at ambient temperature. Data from Harley and McCready (1952a).

280

Ectomycorrhizas

With the methods of specimen preparation used at that time it appeared that Ca^"^ was located with the P, in solid granules. This is artefactual, and it now seems certain that polyphosphate is present in a soluble form of relatively short chain lengths associated with K^ (see Chapter 14). Since the sheath, whether it absorbs nutients, accumulates them or acts only as a temporary store, separates the plant, tissue from the soil, the mechanism of the passage of substances to and through it and factors affecting their rate of transfer to the plant, require investigation. In this context the extent of impermeability and hydrophobicity of the sheath surface in different fimgi (Ashford et al., 1988; Unestam, 1991) as well as their ability to produce external mycelial systems (see Chapters 6, 14, 15) will all influence the processes of P capture and transfer. The absorption of soluble inorganic phosphorus (Pi) into excised mycorrhizas of ¥agus results in its immediate incorporation into nucleotides and sugars (Harley and Loughman, 1963). Both separated sheath and plant core exhibit rapid incorporation of applied ^^P, but in intact mycorrhizas the core tissue receives less P and appears more sluggish in incorporating Pi into other compounds, with only about 10-20% of that absorbed passing steadily into the core tissues through the sheath. By studying the time course of esterification of the P entering the root core, Harley and Loughman were able to show that the labelling of inorganic orthophosphate represented 100% of the radioactivity in that tissue initially and that the proportion fell as nucleotides, sugar phosphates and other fractions became labelled. Since both the sheath and the plant tissues showed the same labelling pattern of soluble P compounds if allowed separately to absorb P from radioactive solutions, it was concluded that in the intact excised mycorrhizas orthophosphate was the form which passed from the sheath to the plant when low concentrations were applied externally. Harley and McCready (1952b) and Harley et al. (1958) studied the possible routes by which P might pass through the sheath from the external solution to the plant. They showed first that the sheath prevented the plant from absorbing P at its maximum possible rate, except when very high concentrations were present in the solution. They concluded that from low P concentrations, such as might be expected in the soil, diffusive movement through the sheath did not take place at a significant rate. It might, however, occur at concentrations above about 1 mM, which are totally unrealistic ecologically. The P passing through the sheath to the plant root did not equilibrate with a large part of the P in the fungus. If it did so, as Harley et al. (1954) showed, a lag phase in the arrival of P in the core tissue would be expected, and the quantity of P in the pathway to the core would be related to the length of the lag phase. Using low external P concentrations the lag phase was exceedingly short, so that the quantity of P in the sheath with which the passing Pi equilibrated was very small - of the order of 0.017 ^ig P per 100 mg dry wt of mycorrhizas. This is extremely low, compared with the amount of P present in the sheath and it was concluded that Pi is incorporated first into the metabolic pools of the fungal symplast in the sheath and that these constitute a small proportion of the total P, as they do in other roots (Crossett and Loughman, 1966). In mycorrhizas, some of the P in the sheath is present as poly-phosphate. This was pointed out by Ashford et al. (1975) using Eucalyptus and subsequently shown to be true of Pinus radiata, of the arbutoid mycorrhizas of Arbutus unedo and of the vesicular-arbuscular (VA) mycorrhizas of Liquidambar styraciflua

Phosphorus nutrition of ectomycorrhizal plants

281

(Ling-Lee et al, 1975). The presence of polyphosphate is certainly important, but almost all the work on the dynamics of formation and utilization must be viewed with some caution in the light of the findings that the 'granules' that have been much studied are likely to be artefacts (see Chapter 14). The exceptions are the recent work of Orlovich and Ashford (1993) and the nuclear magnetic resonance (NMR) studies, particularly those of Martin et al. (1983b, 1985). Using the same cytochemical methods as Ling-Lee et al. (1975), Chilvers and Harley (1980) described particles believed to be polyphosphate in the sheath of Fagus mycorrhizas. The number and size of particles increased during P absorption at rates similar to the rate of absorption and similar factors affected their formation as affected P uptake. The formation of polyphosphate in Fagus mycorrhizas was further examined by Harley and McCready (1981) using the method of extraction and precipitation described by Aitchison and Butt (1973). Assuming that there was little hydrolysis extraction and that the precipitation with BaCl2 was complete, a large amount of the P which is accumulated in the sheath tissue is polyphosphate. More recent work has confirmed the location of the polyphosphate to be in the fungal vacuoles where it is stabilized by K"^ (Ashford and Orlovich, 1994). This seems to be the way in which a great part of the P in the sheath is separated from the mobile P which can move to the plant tissue. Phosphate Absorption in Intact Plants Following uptake, the slow transfer in excised roots is consistent with data from experiments on whole plants of Pinus radiata (Morrison, 1957a, 1962), in which seedlings were grown at two levels of P in pots in the greenhouse for 17 weeks. With high P, the non-mycorrhizal plants grew levels better than the mycorrhizal ones, but the reverse was true with low P levels. Plants of each kind were then grown with fresh supplies of P, labelled with ^^P. In all experiments the movement of ^^P to the shoot tip of the non-mycorrhizal plants was rapid at first, but later the rate decreased and almost ceased, but could be increased again by further addition of P to the soil. Movement to the shoot tips of mycorrhizal plants, although much slower than the initial rate in non-mycorrhizal plants, continued steadily for weeks and was little affected by further additions of labelled P to the soil. The accumulated '^^P in the shoot tips of mycorrhizal plants eventually exceeded that of the non-mycorrhizal controls. If mycorrhizal and non-mycorrhizal plants were deprived of P after a period of uptake of "^^P, radioactivity continued to pass to the shoots of mycorrhizal plants for three weeks, but ceased to move to those of non-mycorrhizal plants after only a short period. This behaviour is explicable in terms of the accumulation of P in the fungal sheath, coupled with a steady rate of transfer to the plant when P is available. When P supplies are deficient, movement from the stored P in the sheath occurs. This type of study is instructive in terms of distribution of P in the plant, but tells us little about the processes of P capture from soil by the extraradical mycelium. Stone (1950) compared two samples of seedlings of Pinus radiata with very different development of extraradical hyphae. Those with the more extensive system absorbed ^^P from the soil faster and translocated a greater quantity to their needles. Similarly, Melin and Nilsson (1950) showed that ^^P orthophosphate fed to

282

Ectomycorrhizas

the extraradical mycelium of P. sylvestris, was translocated to the root by the hyphae and thence through the plant to the needles. Skinner and Bowen (1974a,b), using P. radiata and Rhizopogon luteolus, confirmed the transport of P in mycorrhizal rhizomorphs. P absorption by rhizomorphs was inhibited by cyanide and was temperature dependent. Subsequent translocation occurred over distances of up to 12 cm. However, there were large differences in extent of mycelial growth in the soil between strains of fungus and between samples of the same fungal isolate in different soil conditions (Skinner and Bowen, 1974b). Experiments such as these emphasize the need for the extent of production of extraradical hyphae and rhizomorphs to be fully described in experiments on the efficacy of different combinations of fungal strain and plant genotype. Finlay and Read (1986b) used autoradiography to examine the uptake of "^^P by the extraradical mycelium of Suillus bovinus and its transport to seedlings of Pinus spp. which were interlinked by the fungus (Fig. 9.1). A seedling of P. sylvestris colonized by the fungus was first introduced to an observation chamber containing non-sterile peat, and the system was incubated for a period sufficient to enable the mycelium of S. bovinus to colonize both the peat and a series of previously uncolonized seedlings of P. contorta. [^^P]orthophosphate fed at about 30 cm from the seedling roots was absorbed by the fimgus over a period of 72 hours and was translocated throughout the peat and to the mycorrhizal roots of all the plants interlinked by the fungus. '^^P accumulated in mycorrhizal roots, in a pattern that would be predicted from the studies of excised roots described above. In some seedlings onward transfer of '^^P to the shoots took place in the same period. Distribution of "^^P in the peat itself was irregular. It was clear that the rhizomorphs provided the main pathways for long-distance translocation, but there was also directional transport towards the actively growing hyphae at the advancing mycelial front and into patches of dense mycelium. The extraradical component of ectomycorrhizas is extremely important in colonizing the soil and may play a role similar to that described for VA mycorrhizal hyphae (see Chapter 5). However, both the extent of development of the ectomycorrhizal mycelium and the abilities of some of the fungi to utilize sparingly soluble organic phosphorus (Po) sources may be generally greater in ecto- than in VA mycorrhizal symbiosis (Marschner, 1995; George and Marschner, 1996). The hyphae extend far beyond any zone depleted of nutrients near the surface of the sheath, and selectively proliferate in resource-rich microsites (Bending and Read, 1995a; and see above). The possible physiological and ecological significance of mycelial links between plants and of selective exploration of some reserves is discussed in Chapters 14 and 15. Rousseau et al. (1994) quantified the difference in potential absorbing surface area between seedlings of P. taeda colonized by the fungi Pisolithus tinctorius and Cenococcum geophilum and those that grew in the non-mycorrhizal condition. They also examined the differential effect of the two fimgi on P uptake by the plants (Table 9.4). Whereas P. tinctorius stimulated some increase in branching of root tips and hence surface area of fine roots, C. geophilum did not. The major impact of the fungi on the area available for absorption was provided by the extraradical hyphae which led to an increase of approximately 40-fold in the case of P. tinctorius and 25-fold for C. geophilum, in relation to the non-colonized controls. The greater lengths, and consequently surface areas, of the mycelium were associated with significant increases in P uptake by both fungi and with greater shoot

Phosphorus nutrition of ectomycorrhizal plants

283

Figure 9.1 Transport of ^^P through the extraradical mycelium of Suillus bovinus, linked to seedlings of Pinus sylvesths and P. contorta, (a) Root observation chamber showing the mycelial connections between the plants and the site of feeding with ^^P in half-strength Melin-Norkrans medium (arrowed), (b) Autoradiograph of the same chamber showing the distribution of ^^P after 82 hours. Label has accumulated In the rhizomorphs (A), mycorrhizal roots (B) and the shoots (C). There is also some accumulation in the advancing mycelial front (D). From Finlay and Read (1986b), with permission.

weight in the case of P. tinctorius. While there appears to be a correlation here between hyphal development and P uptake, studies of this kind do not conclusively demonstrate a causal relationship between the two. To determine such a relationship, P uptake must be measured in terms of inflow (uptake per unit length of root per unit time) or specific uptake rate (uptake per unit weight per unit time). That the effects of the fungus can be large has been appreciated for some time and as Bowen (1973) pointed out, estimates of the specific uptake rate can be calculated from the data of Hatch and others (see Table 9.1). The results demonstrate the greater uptake that follows from the improved exploitation of the soil with respect to immobile P, even though the values must be underestimates because the contents of nutrients in the seed are not known. It is surprising that few studies have followed up the work of Harley and McCready (1950), who demonstrated ectomycorrhizal increases in inflow of the order of four- to five-fold, whereas similar increases have been shown repeatedly in VA mycorrhizal plants (see Chapter 5). Two studies, one of Eucalyptus pilularis colonized by a fungus of uncertain identity but probably Cenococcum geophilum (Heinrich and Patrick, 1986) and another of Salix viminalis colonized by Thelephora terrestris (Jones et al, 1991) have now shown unambiguously that

284

Ectomycorrhizas

Table 9.4 A comparison oiP plant and fungal parameters for Pinus taeda seedling colonized by Pisolithus tinctohus (Pt), Cenococcum geophilum (Cg) orleft uncolonized (control)

Mycorrhizal infection (%) Shoot weight (g) Foliar P cone, (g) Shoot P content (mg) Fine root diameter (mm) Root:tip ratio Area fine root (mm^) Area (mm^ g~' soil) Hyphae Rhizomorphs Total Length (m g~' soil) Hyphae Rhizomorphs Total Dry weight (|ig g~' soil) Hyphae Rhizomorphs Total Hyphal diameter (}im)

Pt

Cg

Control

P*

69.5 a 1.09 a 0.066 a 0.669 a 0.477 b 3.72 b 4.02 b

66.5 a 0.830 b 0.043 b 0.340 b 0.573 c 1.39 a 1.49 a

O.Ob 0.710 b 0.034 b 0.238 c 0.299 a 1.55 a 1.30 a

< 0.0001 0.015 < 0.000! < 0.0001 < 0.000! < 0.000! < 0.000!

33.8 a 13.6 a 47.4 a

28.1 a 0.00 b 28.1 b

1.50 b 0.00 b 1.50 c

< 0.000! 0.0012 < 0.000!

2.80 b 0.00 b 2.80 b

0.28 c 0.00 b 0.28 c

< 0.0001 0.00!! < 0.000!

7.85 0.00 7.85 3.18

0.22 c 0.00 c 0.22 c

< < < <

6.42 a 0.36 a 6.78 a 4.98 a 14.3 a 19.3 a 1.60 a

b b b b

—

0.000! 0.0001 0.000! 0.0001

* P, Probability values from one-way ANOVA between inoculation treatments. Values within a row having the same letter are not statistically different (Duncan's multiple range test, P < 0.05). Data from Rousseau et al. (1994).

mycorrhizal colonization of intact root systems increases P inflow significantly. Heinrich and Patrick (1986) established relationships between numbers of Cenococcum-iype ectomycorrhizas and both seedling dry weight (Fig. 9.2a) and total seedling P content (Fig. 9.2b). Significant correlations were observed for both relationships. The inflow of P for colonized roots was calculated to be increased by 7.7 X 10~^^ mol P m~^ s~^, compared with non-colonized roots. Mycorrhizal colonization of Salix gave a twofold increase in growth of the plants and a P inflow of 3.2 X 10"^^ mol m~^ s~\ which was three times greater than that of nonmycorrhizal roots. In both cases the increased inflows must have been due to the mycelial contribution to P acquisition. There are, however, studies indicating that hyphal development in soil can be a poor indicator of mycorrhizal effectiveness. Thomson et al (1994) found that while those fungi which were most effective in increasing the uptake of P and growth in Eucalyptus globulus were also those that colonized the roots most extensively, P uptake correlated poorly with hyphal length. Thus the fungus most effective in increasing plant growth, Descocolea maculata, formed the smallest amount of external hyphae per metre of colonized root while isolates of Laccaria laccata developed more external hyphae per metre of root than other fungi, without any apparent

285

Phosphorus nutrition of ectomycorrhizal plants

additional benefit to the plant. Observations such as these indicate the need for caution in generalizing about the role of hyphal length in P uptake. Clearly other factors, amongst which the viability and physiological characteristics of the mycelium and the compatibility between fungus and plant, may be important and should be taken into account. A number of other studies of responses of colonized and uncolonized plants to changing doses of P, have now been published. That of Bougher et al. (1990) on Eucalyptus has already been described (Chapter 7). Pinus taeda responded positively to inoculation with four fungi (Pisolithus tinctorius, Rhizopogon roseolus, Scleroderma aurantium and Thelephora terrestris) when growing in Piedmont forest soil, over a gradient of available P, in the greenhouse, at all P concentrations, relative to uninoculated controls after 10 months (Ford et al, 1985). Responses to S. aurantium were much greater than to the other fungi. This was a pot study which, if considered in isolation, could be taken to indicate that similar responses to P and inoculation might be expected in the field. However, the authors point out that attempts to improve growth of P. iaeda in plantations established on these soils, by application of P fertilizers, had failed because N was in fact the limiting nutrient in the field. The growth responses to P achieved in pots occurred because N limitation had been removed by N fertilization at the start of the experiment. The experiment was thus instructive in terms of the potential of mycorrhizal colonization to improve growth, but tells us little about what role the fungi might play in nature.

l O r (a:

0 Number of black ectomycorrhizas (cm root length"^ x10^)

1 2 3 4 Number of black ectomycorrhizas (cm root length"^ x10^)

Figure 9.2 Growth and P uptake of Eucalyptus pilularis colonized by Cenococcum geophWum 176 days after sowing, (a) Relationship between number of black ectomycorrhizas and total seedling dry weight, (b) Relationship between number of black ectomycorrhizas and phosphate content of the seedlings. From Heinrlch and Patrick (1986), with permission.

286

Ectomycorrhizas

Sources of Phosphorus in Soil Some discussion of the nature and availability of P sources in soil has been provided in Chapter 5. The possibility that poorly soluble Pi might be absorbed from the soil by ectomycorrhizal plants was examined by Stone (1950) and by Bov^en and Theodorou (1967). The latter authors showed that in culture four mycorrhizal fungi, Suillus granulatus, S. luteus, Rhizopogon roseolus and Cenococcum geophilum, could bring P into solution from rock phosphate (RP). They ascribed this to secretion of acid and expressed doubt whether it could occur in conditions prevalent in soil. Like Stone, they observed that mycorrhizal plants of Pinus radiata, absorbed more P from RP than non-colonized plants when grown in soil. They raised the possibility that contaminating microorganisms on the mycorrhizal surface might have contributed to the effect. The matter clearly needs further investigation in view of the likelihood that mycorrhizas might excrete organic acids capable of chelating metals and solubilizing P from sparingly soluble forms such as Fe and Al phosphates. There is increasing evidence for such activity. The ectomycorrhizal fungus Hysterangium crassum exudes large amounts of calcium oxalate into the mycelial mats which it produces under stands of Pseudotsuga menziesii (Cromack et al., 1979). Soil oxalate concentration was significantly higher and p H significantly lower on mat than on non-mat soils. Scanning electron microscopy showed intense chemical weathering attributable to oxalate attack in the immediate vicinity of hyphae. Working in similar systems Griffiths et al. (1994) have subsequently shown that concentrations of dissolved organic carbon (DOC), oxalate and soluble Pi are considerably higher in the mat than non-mat soils and that there are significant statistical correlations between DOC, oxalate and Pi, indicating that the organic acids are involved in weathering and release of Pi in ectomycorrhizal mats. In sand culture Pisolithus tinctorius can effectively extract P from insoluble aluminium phosphate and thereby improve the P nutrition of Pinus rigida (Cumming and Weinstein, 1990). Measurements of the pH of the solution indicated that H"*^ production may have been important in solubilizing the aluminium phosphates, but the possibility that organic anions such as citrate were also important was not excluded. A large part of the P contained in the surface horizons of forest soils, where most ectomycorrhizas are localized, is present in organic forms (Dalai, 1977; Harrison, 1983). These can occur as phosphon\onoesters such as inositol hexaphosphate, often referred to as 'phytate', or as phosphodiesters, amongst which nucleic acids and phospholipids are likely to be important. Some of the monoesters, although important constituents of living cells, may have only a short life span in soil because endogenous phosphomonoesterases will attack them in the course of cell breakdown (Beever and Bums, 1980). Others are clearly more resistant to breakdown since a significant proportion of the Po in acidic soils is in the form of the phytates inositol penta- and hexa-phosphates (Cosgrove, 1967; McKercher and Anderson, 1968). The phosphomonoesterases are easily studied and have been detected in most ectomycorrhizal fungi, but in future it will be important to investigate other (hitherto neglected) enzymes that can degrade Po. Evidence for the ability of ectomycorrhizal fungi to produce phosphomonoesterase has taken two forms: one indirect, involving studies of their growth on phytate

Phosphorus nutrition of ectomycorrhizal plants

287

supplied as sole P source, and the other direct and dependent upon measurement of phosphatase activity using paranitrophenyl phosphate (PNPP) as the substrate. Growth of Suillus granulatus, S. luteus, Cenococcum geophilum and Rhizopogon roseolus took place with phytates of Ca and P as sole sources of P (Theodorou, 1968) although it was shown that iron phytate, likely to be of greater quantitative significance in acid organic soil, was little used. The latter fungus produces two types of phytase (Theodorou, 1971). Bartlett and Lewis (1973) examined the surface phosphatase activity of mycorrhizas of Fagus. They also showed that there was more than one phosphatase present, because the activity had a double pH optimum and hydrolysed a range of P compounds including inorganic pyrophosphates and organic compounds, especially inositol phosphates. They emphasized that phosphatase of such an activity on the surface of the fungal component of ectomycorrhizas might result in the immediate recycling of the phosphates present in the fallen litter back into the mycorrhizal system. Williamson and Alexander (1970) also examined Fagus mycorrhizas. They found that acid phosphatase was present throughout the fungal tissue and was not associated with contaminating microflora to any significant extent. They agreed with Bartlett and Lewis that more than one phosphatase enzyme was present, and that each had different characteristics. Alexander and Hardy (1981) also showed that mycorrhizas of Finns sitchensis possessed surface phosphatase activity that was inversely correlated with the concentration of extractable Pi in the soil. In this respect the work is extremely reminiscent of that of Calleja et ah (1980), who showed that the phosphatase activities of four species of ectomycorrhizal fungi were more strongly developed in the absence of soluble P in the culture medium. The effects of such environmental variables as pH, temperature and substrate concentration on the activities of acid phosphatase have now been examined in a number of ectomycorrhizal fungi (Antibus et aL, 1986) and it has again been shown (Antibus et aL, 1992) that enzyme activity and phytate utilization are greatest at low concentrations of Pi. Dinkelaker and Marschner (1992) demonstrated that phosphomonoesterase activity was greater in the mycorrhizal roots of spruce and in the rhizomorphs of Thelephora terrestris than in non-mycorrhizal roots. The probable importance of the extraradical mycelial system in the production of phosphomonoesterases is suggested by the detection (Fig. 9.3) of a positive correlation between mycelial length and phosphatase activity in spruce forest organic matter (Haussling and Marschner, 1989). The surface activity on the fungal hyphae and sheath means that substrates would have to be located close to them for hydrolysis to occur. The extraradical mycelium, whether in hyphal mats in the field (Griffiths and Caldwell, 1992) or in mycelial 'patches' in microcosms (Bending and Read, 1995b; see Fig. 6.27), proliferates locally in the vicinity of substrates enriched in P or N and is associated with elevated activities of phosphomonoesterase, thus achieving the necessary localization. To date, studies of what are likely to be the more important phosphodiesterase activities have been few. Griffiths and Caldwell (1992) found that the mat-forming ectomycorrhizal fungi Gautieria monticola and Hysterangium gardneri, together with an unidentified Chondrogaster species were capable of hydrolysing the major phosphodiester RNA, and it has been shown by Leake (unpublished) that Suillus

288

Ectomycorrhizas

14 — 12 >

10

CO

a. §

2h J400

J800

-L

J_ 1200

1600

Hyphal length (m mP^ soil)

Figure 9.3 The relationship between length of external hyphae and phosphatase activity (in enzyme units, eu) in the humus layer soil in which mycorrhizal plants of Picea abies were growing. From Haussling and Marschner (1989), with permission.

hovinus, one of the fungi known to produce dense mycelial patches (see Fig. 6.29) can use DNA as sole source of P. There is, of course, the likelihood that both phosphomono- and diesters contained in senescent organic residues will be sequestered, along with Nous components, in more complex aromatic and aliphatic macromolecules. Evidence is emerging that some ectomycorrhizal fungi can produce enzymes capable of hydrolysing these 'protected' substrates (see Chapter 15).

Conclusions Early work emphasized the role of the fungal sheath in the processes of absorption, storage and transfer of P and laid the basis for our understanding of the physiology of nutrient transfer between fungus and plant in the ectomycorrhizal symbiosis. Most of these studies were carried out using excised roots, but emphasis has moved more recently towards consideration of intact systems in which the effectiveness of different fungi in captures transport and transfer of P to the plant has been examined in soil. The relationship between P acquisition and growth is now much more clearly understood and a number of studies have provided estimates of P inflow in intact ectomycorrhizal systems. These are of the same order of magnitude as those seen in VA mycorrhizal systems. The role of the extraradical mycelium in exploring the soil and facilitating the mobilization of P from complex sources, both inorganic and organic, has been emphasized. The success of combined laboratory and field-based studies in elucidating the mechanisms and significance of increased P capture is clear. Both production of phosphatases which release P from organic sources, and production of protons and organic anions which can accelerate processes of chemical weathering, appear to be important. There is now a need to characterize more precisely the chemical nature of the main sources of P used by ectomycorrhizal plants in nature and to investigate the relative

Phosphorus nutrition of ectomycorrhizal plants

289

effectiveness of different species and races of fungal symbionts in providing access to them. Considerations of P uptake cannot be made in isolation. In many natural environments P and N occur together in organic substrates and their release requires a complex suite of enzyme activities, some of them involving prior breakdown of polymeric C sources. We know little of the relative abilities of mycorrhizal and non-mycorrhizal microbial communities to achieve this breakdown, or of any competitive interactions that may take place between them.

Plate 2. Effects of mycorrhizal inoculation of a range of crop plants in fumigate soil. Right-hand block, inoculated with VA mycorrhizal fungi. Left-hand block, not inoculated. Crops (front to back): Allium, Catalpa, Pisum, Vicia, Zea. Non-host plants trimmed. Photograph courtesy of V. Gianinazzi-Pearson.

Plate 3. Lactahus subdulcis mycorrhiza, formed between L subdulcis and Fagus sylvotica. (a) Irregular pyramidally branched mycorrhizal system (X5.5). (b) Irregular pyramidally branchged mycorrhizal system ( X I 0.6). (c) Tip of mycorrhizal axis (X44). (d) Rhizomorph ( X 4 4 ) . From Brand (1987), with permission.

10 Ectendomycorrhizas

Introduction Mycorrhizas with many of the characteristics of ectomycorrhizas, but also exhibiting a high degree of intracellular penetration, have been described at various times in the last century in various species of tree. These ectendomycorrhizal structures appear to be quite distinct from ectomycorrhizas where a few cells only are penetrated by the fungus, or where the senescent cortex becomes fully colonized by hyphae in the late Hartig net zone (see Chapter 6). Ectendomycorrhizas are also distinct from 'pseudomycorrhizas' described in Pinus by Melin (1917, and later) as forms of colonization by septate fungi which did not form sheath and Hartig net. The term 'ectendomycorrhiza' should be used as a purely descriptive name for those mycorrhizal roots which exhibit some of the structural characteristics of both ectomycorrhizas and endomycorrhizas, and it implies no functional significance. The symbioses described here, occurring mainly in conifers, are distinct from ageing ectomycorrhizas and from mycorrhizas in some members of the Ericales in which a considerable degree or a specialized kind of intracellular penetration occurs.

Occurrence and Structure Laiho and Mikola (1964) examined the initiation of mycorrhizal colonization in Pinus sylvestris and Picea abies seedlings. Picea developed normal ectomycorrhizas, but on Pinus a kind of mycorrhiza having a coarse Hartig net, intracellular colonization and a thin or absent fungal sheath was formed. TTiese ectendomycorrhizas were initially very abundant and invariably present on Pinus in nursery beds of soil of agricultural origin, but were very infrequent indeed on Picea. The earliest sign of colonization was the formation of a Hartig net which followed closely behind the apical meristem as the root grew. Behind this, intracellular penetration increased in intensity towards the older part of the root, so that the cells became almost filled with coils of septate hyphae which were up to 15 jiim thick. These mycorrhizal roots persisted for at least a year, with few signs of hyphal degeneration. As Mikola (1965) wrote: 'Intracellular hyphae do not injure the cortical cells; both plant cells

Ectendomycorrhizas

291

and intracellular hyphae were observed to live at least one year after commencement of colonization; even the nuclei of such heavily colonized cortical cells were clearly visible in stained preparations'. Scales and Peterson (1991a) made detailed examination of the structure and development of the ectendomycorrhiza of Pinus banksiana formed by Wilkoxina mikolae var. mikolae. Emergent short roots become covered with hyphae which appear to be embedded in a matrix material (Fig. 10.1a,b). The hyphae in this matrix are highly branched (Fig. 10.1b). The sheath forms first behind the apex (Fig. 10.1c), where it develops between protruding root hairs. At this stage only a few hyphae of narrow diameter traverse the root apex (Fig. 10.1c). In the mature structure (Figs lO.ld; 10.2) the apex is completely ensheathed, except in those cases where the lateral roots grow very rapidly. As shown in Figure 10.2a, a uniseriate Hartig net begins to form just behind the apex. This penetrates between the epidermal and outer cortical cells initially but eventually extends to the inner cortex adjacent to the endodermis. The Hartig net has a labyrinthine structure (Fig 10.2b) typical of that seen in ectomycorrhizas. Intracellular penetration occurs in one or two cells distal to those in which the earliest Hartig net formation is observed (Fig. 10.2a). The hyphae, having penetrated the cell, branch repeatedly (Fig. 10.3). All aspects of the structure and development of this ectendomycorrhiza, with the exception of the intracellular penetration, appear to be similar to those seen in an ectomycorrhiza. When the same fungus was used to inoculate Picea mariana and Betula alleghaniensis (Scales and Peterson, 1991b) ectomycorrhizas typical of those formed by other fungi were produced. A well developed Hartig net with labyrinthine growth occurred in both types, but neither showed any evidence of intracellular penetration. These descriptions show that the contention sometimes made that the cortical cells are dead and devoid of cytoplasm in ectendomycorrhizas is generally not true. Neither Mikola (1965) nor Scales and Peterson (1991a) saw any signs of hyphal 'digestion'. Mikola concluded that the kind of ectendomycorrhizas formed by E-type fungi (see below) were confined to young one- to three-year-old Pinus in nursery soil over a wide range of soil fertility, acidity and humus content, and that their intensity of formation was not greatly dependent on light intensity. The longevity of this kind of mycorrhiza equalled that of typical ectomycorrhizas and was many times longer than non-mycorrhizal roots of similar position in the root system. Laiho (1965) made a wide survey of ectendomycorrhizas in nurseries and forests in Europe and America and experimentally synthesized mycorrhizas on a number of species of tree, using the E-strain fungi isolated from them. He encountered one form of ectendomycorrhiza only, in which the roots were inhabited by coarse septate mycelium which formed a strong Hartig net and intracellular colonization. He concluded that E-strain fungi were present in all ectendomycorrhizas that he examined in detail, but that there must be other causative fungi, for Etype fungi would only colonize species of Pinus and Larix, whereas some ectendomycorrhizas undoubtedly occurred on species of Picea and on other genera. He stressed, however, that there was a possibility of error because senescent mycorrhizas often had intracellular colonization and he quoted Mikola's (1948) observation that Cenococcum could penetrate the cortical cell walls of plants growing in

292

•ftin^yg

F i g u r e 10.1 (Caption opposite)

Ectomycorrhizas

Ectendomycorrhizas

293

Figure 10.1 Scanning electron microscopy of short roots of Pinus banksiana-Wilcoxina mikolae var. mikolae mycorrhizas. (a) Young emergent short root covered with hyphae (arrowheads), that appear to be partly embedded in matrix material (*). (b) Subapical region of a mycorrhizal short root, showing highly branched hyphae (arrowheads) embedded in matrix material (double arrowheads) on the surface of the root, (c) Short root showing apical root hairs (large arrows) and hyphae (arrowheads) contacting the root surface in the colonization zone. Hyphae of small diameter (double arrowheads) traverse the root surface, (d) Apical region of a monopodial mycorrhizal roots, showing the mantle (*). Bars, iOOjLim. From Scales and Peterson (1991a), with permission.

unsuitable conditions. This point is even more strongly reinforced by the observation that it is quite usual for senescent cortical cells to be invaded (Harley, 1936; Atkinson, 1975; Nylund, 1981). Laiho was impressed by the absence of a deleterious effect of ectendomycorrhizal colonization on Pinus seedlings and concluded that it was 'balanced symbiosis' and that it gave place to ectomycorrhizas as the seedling aged, especially in woodland conditions. Wilcox (1968b, 1971) described similar ectendomycorrhizas on young plants of Pinus resinosa in which coarse hyphae form the Hartig net in both long and short roots. These hyphae tend to spiral around in the cell wall following the angle of the cellulose fibrils. They penetrate the cell wall through pit areas and also by means of rather complicated appressoria and grow into the lumen of the cell. The hyphae seem to be stimulated to spread intercellularly behind the apex of the long roots and around developing laterals, but do not invade the meristems. They may be active around the lateral initials and colonize the young roots as they pass through the cortex. Although ectendomycorrhizas are often present on most of the short roots of young seedlings in nursery soils, they are later replaced by ectomycorrhizas formed by slender hyphae, probably from another fungus.

Ectendomycorrhizal Fungi The fungi involved in the formation of ectendomycorrhizas were first examined in detail by Mikola (1965) and Laiho (1965) who isolated more than 150 strains from

294

Ectomycorrhizas

Figure 10.2 Light microscopy of dichotomous short roots of Pinus banksiana-WilcoxIna mikolae van mikolae mycorrhizas. (a) Longitudinal section showing Hartig net (arrowheads), sheath (*) and intracellular hyphae (double arrowheads). Bar, 100 |Lim. (b) Higher magnification of an area of the main axis of the root seen in (a). Hyphae in both Hartig net (arrowheads) and intracellular hyphae (double arrowheads) are quite vacuolate. Bar, 50 |Lim. From Scales and Peterson (1991a), with permission.

Pinus seedlings growing in Finnish nurseries. On grounds of gross morphology of the mycelia growing on nutrient agar, Mikola considered that all of these strains belonged to the same species which he called the 'E-strain' fungus. These mycelia typically have a brown coloration and consist of two h5^hal forms: one dark with verrucose walls and one thinner, smooth walled and hyaline. The diameter of the hyphae range from 2 to 12 |xm. The taxonomic position of the fungus was unclear because no fruiting structures were found. Wilcox et al. (1974) isolated a similar fungus from seedlings of Pinus resinosa growing in nursery soils of the USA and observed the production of chlamydospores in culture. The chlamydospores of Estrain fungi have a distinctive crenulate surface and dimensions in the range 100200 jxm. A spore of this kind isolated from soil in a pine plantation in Iowa was named Complexipes moniliformis by Walker (1979) and placed in the Endogonaceae. However, this was later challenged (Mosse et al., 1981; Danielson, 1982; Thomas

Ectendomycorrhizas

295

Figure 10.3 Scanning electron microscopy of a single cortical cell of a mycorrhiza formed between Pinus banksiana and Wilcoxina m/ko/ae var. mikolae. It can be seen that the intracellular hyphae (arrowheads) are branched and that the Hartig net (black arrowheads) is unisereate. Bar, 100 |im. From Scales and Peterson (1991a), with permission.

and Jackson, 1982; see Chapter 1). The septal pores of vegetative hyphae of fungi producing these spores are of the simple type (Thomas and Jackson, 1982) and the fungus is resistant to benomyl (Danielson, 1982), both being features of fungi with ascomycetous affinities. Danielson (1982) postulated that E-strain fungi may belong to the order Pezizales. This was confirmed when ascocarps of an operculate discomycete in the genus Tricharina, then called T. mikolae, were produced on a pot culture of soil supporting ectendomycorrhizal seedlings of Finns resinosa (Yang and Wilcox, 1984). The soil had been inoculated with Complexipes-type chlamydospores collected in an Oregon Douglas fir nursery. Later, Yang and Korf (1985) delineated the new genus Wilcoxina for 'E' strain fungi, T. mikolae being subsumed into Wilcoxina, as W. mikolae.

Analyses of restriction fragment length polymorphisms (RFLP) in the nuclear (Egger and Fortin, 1990) and mitochondrial (Egger et al, 1991) genomes of E-strain isolates have confirmed that most can be referred to Wilcoxina, and that the majority of strains can be assigned to two taxa: W. mikolae and W. rehmii, each of which has a distinctive habitat preference. W. mikolae is the chlamydospore-producing fungus, which is found predominantly in disturbed mineral soils such as those of nurseries, while W. rehmii occurs in peaty soils and does not produce chlamydospores.

296

Ectomycorrhizas

The Taxonomic and Functional Status of Mycelium radicis atrovirens Another group of largely sterile fungi commonly inhabiting tree roots, in this case producing cultures on agar primarily of grey or grey-black coloration, was first described by Melin (1923) as Mycelium radicis atrovirens. This fungus penetrated the cortical cells of the fine roots of Pinus and Picea without forming either Hartig net or mantle. Melin contrasted what he believed were the harmful effects of this type of colonization, which he designated pseudomycorrhizal (see Chapter 6) with those of ectomycorrhizal fungi which were considered to be both beneficial and essential for the development of trees such as Pinus and Picea. A pathogenic role of fungi of the M. r. atrovirens type has since been observed in herbaceous plants such as strawberry (Wilhelm et al., 1969), as well as in short roots of Picea mariana (Richards and Fortin, 1973). Gams (1963) isolated numerous strains of fungi with the cultural characterics of M. r. atrovirens from roots and soil particles and, on the basis of the failure of many paired cultures to form anastomoses, concluded that M. r. atrovirens was a genetically heterogeneous group. Some of Gams' cultures produced conidia of the Phialocephala type, and were identified as P. dimorphospora (Fig. 10.4a). Of the 41 strains of M. r. atrovirens obtained from short roots of P. mariana by Richards and Fortin (1973), 15 were subsequently shown to produce conidiophores, phialides and conidia of the P. dimorphospora type. Understanding of the taxonomic status of the M. r. atrovirens group was advanced by Wang and Wilcox (1985), who isolated numerous dark, sterile fungi resembling M. r. atrovirens from conifer roots, some of which produced ectendomycorrhizal and others pseudomycorrhizal associations with Pinus resinosa. By

Figure 10.4 Conidiophores of: (a) Ptiialoceptiala dimorptiosporo Kendrick; and (b) Phialocephala fortinii Wang and Wilcox. Bars, 10 |im. Photographs courtesy of J. Wang.

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297

incubating cultures of these normally sterile fungi for periods of six months to a year at low temperatures (5°C) on conventional nutrient media, such as modified Melin-Norkrans (MMN) medium, they were able to induce sporulation in a few isolates. These cultures were shown to represent three different genera of hyphomycete, namely Phialophora finlandii, Chloridium paucisporum and Phialocephala fortinii. The first two of these produced ectendomycorrhizas on P. resinosa, while P. fortinii was pseudomycorrhizal. The relationship between P. dimorphospora and P. fortinii remains unclear. While resemblances between the two are evident, the conidiogenous apparatus of the two species show some structural differences: the conidiophores are verrucose in P. fortinii, but smooth in P. dimorphospora (compare Fig. 10.4a and b). They yield chains of up to nine conidia aggregated together in a dense slimy head in the latter species whereas in P. fortinii chains are shorter with only three to four conidia, on sparse heads lacking slime. To date, only a very small proportion of cultures of the M. r. atrovirens type have been seen to sporulate and none has produced teleomorphs; consequently, the taxonomic status as well as the functional aspects of their association with roots remain obscure. The group is clearly a heterogeneous complex including, for example, P. finlandii and C. paucisporum which form ectendomycorrhizas with Hartig net and mantle, that are likely to be of benefit to the plant. The main question concerns the status of the P. fortinii group. Interest in this topic has recently been heightened by the realization that fungi of this type are included amongst the so-called dark sterile forms which become prominent as colonists of roots in soils of high altitude and latitude (see Chapter 15). Assessment of the function of this group of fungi will require experiments of careful design. Wilcox and Wang (1987b) concluded, on the basis of studies carried out under conditions of high exogenous C concentration, that P. fortinii was a pathogen of pine. This agrees with circumstantial evidence obtained from many field observations made from the time of Melin's (1923) original description of this group of fungi to the present. However, since it is now known that availability of free organic C can enhance aggressiveness and change compatibility of mycorrhizal fungi (Duddridge, 1986a,b), indications of pathogenicity obtained under these conditions must be viewed with caution. In the absence of experiments in which the relationships between plants and fungi of the M. r. atrovirens type are examined in natural soils, some (e.g. Danielson and Visser, 1989; Visser, 1995) have chosen to include associations of this kind in the ectomycorrhizal category. Replacement of E-strain fungi by those of the M. r. atrovirens type was observed to occur on individual roots of Pinus banksiana growing on tailings (Danielson and Visser, 1989) but, at the level of the community on P. banksiana, Visser (1995) found that quantities of M. r. atrovirens mycorrhizas were similar in 6-year-old and 121-year-old stands. There is clearly a need for evaluation of the status of these fungi under realistic soil conditions, bearing in mind the possibility that the status may change according to vigour and age of the individual root. It will be important to determine whether colonization of short roots by M. r. atrovirens is a cause of accelerated senescence or merely a symptom of their declining vigour. Bearing in mind that the life span of a healthy ectomycorrhizal root of spruce or pine is probably only 3-4 months (Alexander and Fairley, 1983; Downes et al., 1992), replacement of ecto-

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mycorrhizal fungi by other types would be expected to occur as a normal part of succession associated with ageing, and it would not be surprising if the fungi of M. r. atrovirens type occupied a stage in this process of decline and turnover of fine roots in soil. Conclusions It seems clear that ectendomycorrhizas in conifers are not 'aberrant' or minor parasitic infections which occur in the absence of ectomycorrhizal fungi. They are symbiotic associations which may be mutualistic and are frequently the early colonization state of seedlings. Perhaps comparative estimates of the drain of C from the plant caused by them and by ectomycorrhizal fungi should be made to see if the least carbohydrate-demanding fungi are selected by seedlings before full photosynthetic potential is reached. One very interesting question raised by Wilcox concerns the consistent formation of ectendomycorrhizas on Pinus and ectomycorrhizas on Picea by the same strains of E-type fungus. This is not an observation unique to these fungi; it is met again in arbutoid mycorrhizas, where one and the same strain colonizes species of Arbutus or Arctostaphylos with extensive cell penetration to form arbutoid mycorrhizas, and forms typical ectomycorrhizas with conifers (see Chapter 13). Other examples of strong plant influence on the structure of mycorrhizas are the formation, by one fimgal species, of monotropoid mycorrhizas on Monotropa with intracellular haustoria, and typical ectomycorrhizas on forest trees such as Picea or Fagus (see Chapter 11). Similarly Paris- or Arum-type mycorrhizas can be produced on different plant species by the same isolate of VA mycorrhizal fungus (Chapter 2).

II Arbutold and monotropoid mycorrhlzas

introduction Considerable increases in our knowledge of the structure and development of arbutoid and monotropoid mycorrhizas have been made possible by the use of electron microscopy. Careful studies over the last 15 years of the ultrastructure of arbutoid mycorrhizas in Archostaphylos (Scannerini and Bonfante-Fasolo, 1983) Arbutus (Massicotte et aL, 1993) and Fyrola (Robertson and Robertson, 1985), and of the monotropoid mycorrhizas of Sarcodes, Pterospora (Robertson and Robertson, 1982) and Monotropa (Duddridge and Read, 1982a) have done much to clarify aspects of structure and development that had previously only been sketchily described by light microscopy. Molecular techniques have also thrown light on the possible phylogenetic relationships between plant taxa forming arbutoid and monotropoid mycorrhizas, and as on the likely taxonomic positions of the fungal associates of the latter. The closest relatives of the achlorophyllous monotropoid group are probably chlorophyllous members of the genus Arctostaphylos, which themselves form arbutoid mycorrhizas. This suggests that the two mycorrhizal types may have arisen from a common ancestral form and that structural differences between them are related to the different methods of acquisition of organic C and the way in which C is transferred between the symbionts. Polymerase chain reaction (PCR)-based analysis of the taxonomic positions of the fungi forming monotropoid mycorrhizas (CuUings et aL, 1996) supports the view, expressed some time ago (Bjorkman, 1960; Castellano and Trappe, 1985) that the suilloid fungi (Boletaceae), which are frequently associated with roots of Pinus spp. in the forest floor under monotropoid plants, are important symbionts of both types of hosts, forming ecto- and monotropoid mycorrhizas, respectively. However, some Monotropa spp., notably M. uniflora, appear to form mycorrhizas, preferentially with members of the Russulaceae, namely Lactarius and Russula spp. Sadly, progress in understanding the physiology of the relationships between achlorophyllous monotropoid plants, their fungal associates and other autotrophic plant partners has not kept pace with structural and phylogenetic information.

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Speculation about possible processes involved in C and nutrient transfer abound, but there is an overriding need for these ideas to be tested in rigorous experiments.

Arbutoid Mycorrhizas Mycorrhizas in Arbutus and Arctostaphylos The fungi of Arbutus and other arbutoid mycorrhizas were long believed to be basidiomycetes because of the structural similarities between ecto- and arbutoid mycorrhizas. This has been confirmed both by synthesis experiments and by the descriptions of dolipore septa in fungi associated with mycorrhizas of Arctostaphylos (Duddridge, 1980; Read, 1983; Scannerini and Bonfante-Fasolo, 1983). The work of Zak (1973, 1974, 1976a,b), who traced mycelium and performed synthesis experiments, showed that mycorrhizas in Arbutus menziesii and Arctostaphylos uva-ursi are formed by fungi which also form ectomycorrhizas. The fungi involved included Hebeloma crustuliniforme, Laccaria laccata, Lactarius sangufluus, Porta terrestris var. subluteus, Rhizopogon vinicolor, Pisolithus tinctorius, Poria terrestris, Thelephora terrestris, Piloderma bicolor and Cenococcum geophilum. Similarly, Molina and Trappe (1982a) tested the ability of 28 ectomycorrhizal fimgi to form mycorrhizas with Arbutus menziesii and Arctostaphylos uva-ursi in pure culture. All but three produced arbutoid mycorrhizas with both species. The conclusion here must be that the plant plays an important part in regulating the development of mycorrhizas, with the consequence that different structures are produced in different plant taxa. There is no evidence that will allow comparison of function in arbutoid and ectomycorrhizas, but the assumption is that they operate similarly. The plants are all woody and photosynthetic and since mycorrhizas are the common form of absorbing organ of members of the Arbutoideae, an important and ecologically significant group of species, the symbiosis must be assumed to be of selective advantage. This is even more likely because the sheath on the roots, as in ectomycorrhizas, may not only have a storage function, but also separates the plant from the soil. Hence the fungus calls the time in absorption by the short roots, and everything absorbed by them must pass through it. It seems extremely likely that the mycelium and rhizomorphs in soil are important in nutrient scavenging. Massicotte et al (1993) carried out a detailed analysis of the structure and histochemistry of arbutoid mycorrhizas, synthesized in growth pouches between Arbutus menziesii and the basidiomycetes Pisolithus tinctorius and Piloderma bicolor. The morphology of the mycorrhizas was strongly influenced by the identity of the mycobiont. In the case of plants colonized by P. tinctorius, repeated pinnate branching of first- and second-order lateral roots produced a compound structure (Fig. 11.1), similar to that originally described by Rivett (1924) in Arbutus unedo. This pattern of branching, which has been observed in associations between Arbutus spp. and a range of other fimgi (Molina and Trappe, 1982a; Giovannetti and Lioi, 1990) appears to arise from precocious initiation of individual lateral roots, rather than by dichotomy of the apical meristem of the root which is typically seen in ectomycorrhizas of Pinus (Piche et ah, 1982; and see Chapter 6). Each of the rootlets colonized by P. tinctorius is ensheathed in a well developed mantle, from the outer

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Figure I I.I Scanning electron microscopy of mycorrhizal rootlets formed between Arbutus menziesii and Pisolithus tinaorius, showing three-dimensional pinnate branching. Bar, 500 ^ m . From Massicotte et al. (1993), with permission.

layer of which an extensive system of rhizomorphs develops. This is a feature previously recorded in associations between Arbutus and several other species of ectomycorrhizal hmgi (Zak, 1976b; Molina and Trappe, 1982a). Mycorrhizas formed by P. bicolor, in contrast, were largely unbranched and had a thm or no mantle, with sparse surface hyphae being embedded in mucilage in a manner smiilar to that seen in ectendomycorrhizas formed in Pinus by Wilcoxina spp. (Piche et al, 1986; Scales and Peterson, 1991a; and see Chapter 10). A longitudinal section of the compound type of mycorrhiza formed by P. tmctonus (Fig. 11.2) reveals a thick mantle, intercellular development of mycelium to produce a Hartig net and penetration of some epidermal cells by fungal hyphae which proliferate to form dense hyphal complexes (Massicotte et al, 1993). The combined presence of mantle, Hartig net and intraceUular proliferation are diagnostic features of arbutoid mycorrhizas which can only be revealed by anatomical investigation. There are reports (e.g. Largent et al, 1980) that Arbutus spp. are ectomycorrhizal, but these are based only on superficial recognition of the presence of a mantle. Clearly, in the absence of more detailed structural analyses such reports must be regarded with suspicion. It was observed by Rivett (1924) and subsequently confirmed (Fusconi and Bonfante-Fasolo, 1984; Miinzenberger, 1991; Massicotte et al, 1993) that the Hartig net m Arbutus is of the paraepidermal kind typically found in ectomycorrhizas in

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Figure 11.2 Longitudinal section (light microscopy) of a compound mycorrhiza formed by Pisolithus tinctorius on Arbutus menziesii. The individual roots have flattened apices (double arrowheads) and are covered by a thick mantle (*). Many of the epidermal cells are colonized by intracellular hyphae (arrows). From Massicotte et al. (1993), with permission.

the majority of angiosperms (Brundrett et al, 1990; and see Chapter 6). Massicotte et al. (1993) proposed that deeper penetration may be prevented by deposition of suberin lamellae and a Casparian strip in radial walls of the outer tier of cortical cells, so forming a hypodermis. The epidermal cell walls contain phenolic substances but no suberin and clearly do not inhibit fungal penetration. The physiological activity and potential storage role of the fungal tissue are indicated by the presence of glycogen rosettes and of polyphosphate (Ling-Lee et al, 1975). Mycorrhizas in Pyrola The seed germination and early growth of Pyrola have been studied because some species have been described as difficult to germinate. The work of Lihnell (1942) is of interest because in aseptic culture, seed of P. rotundifolia and P. secunda germinated to form colourless branched axes with root-like structures. These plant bodies continued to develop for 3.5 years without further differentiation. Lihnell

Arbutoid and monotropoid mycorrhizas

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isolated four fungi from the mycorrhizas of P. rotundifolia and one from P. secunda, all of which produced sterile septate mycelia, perhaps similar to the sterile forms isolated from other Ericales. Although they had some effect on seed germination if present in the medium, the fungi did not form any association with the 'procaulomes' produced by germination, in the conditions used. It is interesting that Liick (1940) had isolated a clamp-bearing basidiomycete from Pyrola, so here (as with ericoid mycorrhizas; see Chapter 12) there may be several potentially symbiotic fungi. Precise identifications have not been obtained for any of the mycorrhizal associates of Fyrola, and again there is considerable scope for investigation using molecular techniques to identify the fungi, as well as to obtain clues on possible links between Fyrola spp. and other, normally ectomycorrhizal, plants with which they are associated in nature. In adult plants, all species of Fyrola, with the exception of P. uniflora, produce an extensive system of white subterranean rhizomes. Adventitious roots arise in the axils of scales along the rhizome. They are normally of relatively small diameter and, when not mycorrhizal, sparingly branched. Colonization of these roots leads to increases in their diameter and the amount of branching. Young mycorrhizal roots are frequently translucent at their tips, and become light or dark brown, or even black with age. Robertson and Robertson (1985) describe the distinctive situation in P. uniflora where the rhizome is replaced by a horizontal root, which then bears secondary roots along its length. In this case fungal colonization does not noticeably affect the diameter of the root or stimulate branching, but produces lightbrown patches interspersed among the normally translucent regions of the root. Careful ultrastructural analysis by Robertson and Robertson (1985) of development of the arbutoid mycorrhizas of a range of Fyrola species has enabled us to visualize the sequence of events involved in the formation and subsequent decline of the arbutoid association at the cellular level. The mycorrhiza is initiated a few millimetres behind the root tip, where hyphae from the surface weft (Fig. 11.3) penetrate between the radial walls of epidermal cells to form a Hartig net. In Fyrola this, again, is of the paraepidermal type (Fig. 11.4) associated with labyrinthine developments of the hyphal walls (Fig. 11.5). The walls of these hyphae are more electron-dense than those that penetrate the cells (Figs 11.5 and 11.6). As hyphae enter the epidermal cells from the Hartig net they are surrounded by the invaginated plasma membrane of the plant cell and by a matrix of material that is continuous with the plant cell wall (Fig. 11.7). This matrix is considerably thicker (around lOOnm) than the wall of the hypha itself, which attains a thickness of only 25-35nm (Fig. 11.8). Tannin-like deposits occur along the plant membrane surrounding the hyphae and on the tonoplast (Fig. 11.9). Despite this apparent resistance response, the fungus grows extensively within the cells, forming complexes which, in transverse section, appear as hyphal profiles (Figs 11.4 and 11.7). At the mature stage, the plant cytoplasm in colonized cells is packed with organelles including mitochondria, endoplasmic reticulum, ribosomes, dictyosomes and plastids of various kinds (Figs 11.8 and 11.9). The cytoplasm of the intracellular hyphae is even more dense and, in addition to abundant mitochondria and nuclei, can have membranous sheets and multi-vesicular bodies (Fig. 11.8). Work of Robertson and Robertson (1985) also revealed intraspecific differences in the structural organization of the interfacial matrix in Fyrola species. In P. minor the matrix is a homogeneous granular layer between fungal wall and plant plasma

306

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Figure 11.3 Mycorrhiza formation in ?yro\Q secunda, A surface weft of fungal hyphae covers the root a short distance behind the root tip, but there is no organized sheath. Bar, 50)im. From Robertson and Robertson (1985), with permission.

Figure 11.4 Mycorrhiza formation by Hysterangium separabile on Pyrola secunda. Section shows sparse development of mycelium on the surface of the root (*), paraepidermal Hartig net (arrowed, HN) and extensive intracellular colonization of the epidermal cells, but no penetration into the root cortex. IH intracellular hypha. Bar, 50|im. From Robertson and Robertson (1985), with permission.

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Figure 11.5 Mycorrhiza formation in Pyrola secunda. Surface view of Hartig net (HN) formation, showing anastomoses and labyrinthine developments in the hyphal walls. E, Epidermal cells. Bar, lOjLim. From Robertson and Robertson (1985), with permission.

Figure i 1.6 Mycorrhiza formation in Pyrola secunda. The Hartig net (HN) surrounds the epidermal cells (E). Note the differences in wall thickness between the hyphae of the Hartig net (arrowhead) and those colonizing the epidermal cells (arrow). IH, intracellular hypha. Bar, I.OjLim. From Robertson and Robertson (1985), with permission.

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Mycorrhizas in the Ericales

Figure 11.7 Intracellular hyphal development in an epidermal cell of Pyrola secunda, showing penetration by a hypha from the Hartig net (HN) and invagination of the plasma membrane of the plant cell (arrowed). Bar, I.OjLim. Inset: Light micrograph of epidermal cell showing hyphal invasion from the Hartig g ^ and extensive intracellular colonization. From Robertson and Robertson (1985), with permission.

membrane (Fig. 11.8), whereas in P. secunda it has a characteristic honeycomb appearance (Fig. 11.10a,b). The process of senescence of the association is similar to that observed in the ericoid mycorrhizas of Rhododendron (Duddridge and Read, 1982b; and see Chapter 12). It begins in a localized region of a cell (Fig. 11.11), with degeneration of cytoplasm of the plant and loss of integrity of its organelles. Again, as in Rhododendron, at this stage the fungal hyphae and surrounding matrix materials have a normal appearance. Degeneration of the fungal hyphae routinely occurs only after disorganization of the plant cytoplasm (Figs 11.12 and 11.13), although Robertson and Robertson (1985) occasionally observed collapsed hyphae embedded in apparently healthy plant cytoplasm. It thus seems clear that in arbutoid, as in ericoid mycorrhizas, degeneration of the plant cell frequently precedes that of the fimgus, a situation which again contradicts earlier observations, based upon light microscopy, that 'digestion' of the fungus takes place as the association matures.

Monotropoid Mycorrhizas The family Monotropaceae consists of ten genera of entirely achlorophyllous plants (Wallace, 1975). It has been known since early in the last century (Rylands, 1842) that

Arbutoid and monotropoid mycorrhizas

309

Figure 11.8 Development of granular interfacial matrix (arrowed) surrounding the intracellular hyphae in Pyrola secunda. Note the relatively thin hyphal walls and extensive development of organelles in both fungal hypha and plant cells. IH intracellular hypha. Bar, I.Ojim. From Robertson and Robertson (1985), with permission.

Figure M.9 Intracellular fungal development in Pyrola minor, showing extensive tannin deposits on plant plasma membrane and tonoplast (arrowed). Note development of organelles in both symbionts. IH, intracellular hypha. Bar, I.Ojim. From Robertson and Robertson (1985), with permission.

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Mycorrhizas in the Ericales

Figure 11-10 (a) and (b) Honeycomb structure of the interfeclal matrix (arrowed) In fVrato secndo. IH mtracellular hypha. Bars. I.O^m. From Robertson and Robertson (1985) With permission. ^ ''

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^P'''^'"'""' " " ' °^ '^''°'°' '^°^'"'S paraepidermal Hartig net -ntracellular colonization. Localized degeneration of the plant

T d Roh . '"noo'c? " " ? ^^P'^'" '' '"^'^^'"^ ''y ^'•'•°^^- B^--- '0^^"^- From R o b e L n and Robertson (1985). with permission.

311

Arbutoid and monotropoid mycorrhizas

Figure 11.12 Advanced stage of senescence, showing degeneration of plant cytoplasm around living hyphae in Pyrola secunda. Bar, l.0|im. From Robertson and Robertson (1985), with permission.

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Figure 11.13 Collapsed intracellular fungal hyphae (CH) in the epidermis of Pyrola. Bar, 1.0 jxm. From Robertson and Robertson (1985), with permission.

312

Mycorrhizas in the Ericales

the roots of a widespread representative of the family, Monotropa hypopitys, were enveloped in fungal hyphae, and a later detailed study of this plant (Kamienski, 1881) produced some of the earliest clear descriptions of what Frank, in the same period, was first calling a 'mycorrhiza'.

The Symbionts and Development of Monotropoid Mycorrhizas Monotropa (Fig. 11.14) and its relatives such as Pterospora andromeda (Fig. 11.15) and Barcodes sanguinea (Fig. 11.16) frequently grow in forests of pine and other conifers, whose roots and mycorrhizas, as well as those of the monotropoid plants, are closely associated in tight complexes know as 'root-balls' (Fig. 11.17). The plants overwinter underground and from the 'root' system flowering scapes develop. Adventitious buds develop on the apices of some of the roots of first and second order, and the flowering shoots formed from them grow above the ground, mature and senesce over a period of months. The amount of organic C required for this development must be considerable. As with the arbutoid mycorrhizas it has been assumed that monotropoid mycorrhizal fungi are ectomycorrhizal on other types of plant. Some evidence for this comes from labelling experiments which demonstrate functional links between the plants (see below). Synthesis experiments have not been carried out because of the difficulties of germinating seed of Monotropa and its allies under controlled conditions. Recently, molecular methods have been applied to this problem of identification, with illuminating results. CuUings et ah (1996) used PCR-based methods to obtain sequence information both from mitochondrial large subunit genes and ITS regions of rDNA, and thus identified the fungi forming mycorrhizas with members of three genera of monotropoid plants: P. andromeda, M. hypopitys, M. uniflora and S. sanguinea. Samples were obtained from a range of geographically separate habitats in California, Wyoming and Oregon. The results showed that a variety of fungal associates were involved in the symbioses, and that the specificity of the associations, although quite variable, was higher than expected from the ectomycorrhizal host ranges of the fungi (see Chapter 15). S. sanguinea was apparently relatively unspecific in its relationships, forming mycorrhizas with three unrelated families of fungi. M. hypopitys associated only with suilloid fungi (including Suillus and Rhizopogon) and M. uniflora only with members of the Russulaceae. The most specific relationship was found in P. andromeda, of which all 31 individuals sampled were associated with Rhizopogon subcaerulescens. The variations in specificity appear to be controlled by interactions between the symbionts and not caused by such factors as fortuitous occurrence of the fungi at different sites. For example, in the five locations where P. andromeda occurred with M. hypopitys or S. sanguinea, the differences in fungal associates were maintained. The cellular and physiological basis for the observed specificity is still a matter for speculation. From an ecological standpoint it is clear that the success of these mycoheterotrophic plants depends on the persistence of particular species of fungi. Kamienski (1881) described the presence of a sheath and Hartig net similar to that seen in Pagus and made the observation that materials entering the plant from the soil must pass through the sheath of the fungus. He also expressed the view that Monotropa plants might be dependent upon the neighbouring trees for nourishment

313

Arbutoid and monotropoid mycorrhizas

Figure 11.14 Mature scapes oi Monotropa hypopitys. Photo, G. Woods, with permission.

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Figure 11.15 Flowering scape of Pterospora andromeda. From Robertson and Robertson (1982), with permission.

314

Mycorrhizas in the Ericales

Figure 11.16 Emerging scapes of Sarcodes sanguinea. From Robertson and Robertson (1982), with permission.

Figure 11.17 Excavated plant of Pterospora andromedOy showing the highly developed Voot-bair (arrowed). From Robertson and Robertson (1982), with permission.

Arbutold and monotropoid mycorrhizas

315

because he thought that they were associated with a common mycelium. The distinctive nature of the anatomy of mycorrhizas in Monotropaceae was first recognized by MacDougal (1899) who described the occurrence of peculiar peglike structures which he called haustoria in the outer cells of the Monotropa root. This structure has subsequently been recognized as the characteristic feature of Monotropa mycorrhizas (Francke, 1934). It was envisaged that the 'haustorium' arose as a result of encapsulation of the invading fungal hypha by the cell wall of the plant. Such a process by itself, however, would not be unique since a similar phenomenon is commonly observed in ericoid mycorrhizas (Burgeff, 1961). The distinctive nature of the peg in Monotropaceae has been revealed by ultrastructural analysis (Lutz and Sjolund, 1973; Duddridge and Read, 1982a; Robertson and Robertson, 1982), which demonstrates that the intrusive structure is not a true haustorium. The peg is, however, of sufficiently elaborate and specialized construction to justify a differentiation between this type of mycorrhiza for which the term 'monotropoid' was proposed by Duddridge and Read (1982a), and the arbutoid type in which extensive internal proliferation of the fungus occurs. The fungal mantle surrounding monotropoid roots consists of a multilayered and compact sheath in which the boundaries between the layers are (or sometimes are) demarcated by tannin deposits (Fig. 11.18). In Monotropa (Duddridge and Read, 1982a) and Pterospora the sheath encloses the root apex while in Sarcodes sanguinea the apex remains free (Robertson and Robertson, 1982). In all three genera a Hartig net surrounds the outer epidermal layer of relatively small cells, but does not penetrate into the underlying cortex. There is no evidence of labyrinthine development in the Hartig net as seen in ectomycorrhizas (see Chapter 6). From the Hartig net single hyphae grow into the epidermal cells, the walls of which often appear to extend around the fungus, or at least to deposit a very substantial interfacial matrix between the two organisms. This composite structure is referred to as the fungal 'peg'. Elaborations in the form of ingrowths of the wall of the epidermal cell surrounding the hypha occur in the intracellular position. Robertson and Robertson (1982) point out that in P. andromeda and particularly in S. sanguinea, the intracellular penetration of the hyphae occurs in a precise manner. Fungi always enter the cell through that radial wall of the epidermal cell that is orientated towards the tip of the root. In Sarcodes the point of entry occurs near the base of the epidermal cell (Fig. 11.19). As intrusion takes place, ingrowths develop from the encapsulating wall of the peg into the plant cytoplasm and become progressively more elaborate, their appearance differing according to their position along its length. Near the base of the peg these ingrowths are sac-like and filled with granular contents (Fig. 11.20), whereas closer to the tip they are thinner, and in Pterospora (Fig. 11.21) and Sarcodes (Fig. 11.22) they have a long ribbon-like conformation. Both the basal and apical protuberances are extensions of the cell wall surrounding the peg but, since this wall is essentially fused with that of the fungus throughout its length, it is not possible, in the absence of cytochemical data, to be precise about the origin of the materials from which it is built. However, the effect of their proliferation is a massive increase of surface area within the epidermal cell to produce a structure that Duddridge and Read (1982a) recognized to be analogous to a 'transfer cell'. Cells of this type are widely

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Figure 11.18 Light microscopy of development of the sheath (S) in Sarcodes, showing tannin deposits (arrowed) and hyphal penetration between the epidermal cells (E) and large cortical cells (C) containing starch grains. Region marked (*) is shown in higher magnification in Fig. 11.19. From Robertson and Robertson (1982), with permission.

Figure 11.19 Detail of fungal colonization of the epidermal cells (E) of Sarcodes (refer to Fig. 11.18) showing the development of fungal pegs (arrow) in the epidermal cells. C, cortical cells. S, sheath. From Robertson and Robertson (1982), with permission. distributed in the plant kingdom and are particularly associated with tissues that are involved in nutrient exchange (see Gunning and Robards, 1976). The final stage of development of the monotropoid mycorrhizas involves the opening of the tip of the peg and what appears to be the release from it of the fungal contents. The fungal material enters the epidermal cell, but is contained by a membranous sac derived from the plant plasma membrane which extends, balloon-like.

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Figure 11.20 Development of the fungal peg (FP) in Pterospora, showing the numerous protruberances (arrows). CW, Cell wall. From Robertson and Robertson (1982), with permission.

Figure M .21 Development of the fungal peg (FP) in Pterospora, showing the long ribbonlike protruberances (arrowed). From Robertson and Robertson (1982), with permission.

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Figure 11.22 Epidermal cell of Sarcodes, showing fungal peg (FP) entering the wall. Note the membranous sac (MS) and mitochondria, Golgi bodies and endoplasmic reticulum in the plant cytoplasm. From Robertson and Robertson (1982), with permission.

into the cytoplasm of the epidermal cell (Fig. 11.23). These latter events were observed under the light microscope by Francke (1934) who considered them to be indicative of 'digestion' of the fungus. However, while the process is rapid and has been referred to as 'bursting' of the peg, it is clearly a controlled event during which fungal and plant structures retain their integrity. During the event, an osmiophyllic neck-band (Figs 11.23, 11.24) develops around the open tip of the peg. Duddridge and Read (1982a) suggested that this may prevent back-flow of nutrients through the wall of the peg. This type of ring is seen around the haustorial neck of some biotrophic pathogens such as Erysiphe (Gil and Gay, 1977) and Puccinia (Heath and Heath, 1975) and is believed to create an apoplastic seal at the plantfungus junction (Smith and Smith, 1990). It has been suggested (Duddridge and Read, 1982a) that 'bursting' of the fungal peg involves transfer of materials from fungus to plant, but as yet there is no evidence of this. If the membrane of the plant retains its integrity then 'normal' processes of membrane transport would be expected to be involved in any such transfer. In Pterospora and Sarcodes the sac appears to be a stable and well organized structure clearly delimited by the plasma membrane of the epidermal cell (Robertson and Robertson, 1982; Fig. 11.23). It contains linear arrays of fibrils which have the dimensions of microtubules (Fig. 11.24) and its membrane is, in places, greatly invaginated to produce what in section appear as small membrane-bounded vesicles in the cytoplasm. Duddridge and Read (1982a) attempted to throw light on the functional aspects

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Figure 11.23 Epidermal cell of Pterospora, showing a median section of the fungal peg (FP) the tip of which has opened to form a membranous sac (MS). Note the osmiophyllic ring at the tip of the peg (arrowed). From Robertson and Robertson (1982), with permission.

of the structures observed by relating differentiation of the fungal peg to the different stages of shoot development in Monotropa through the growing season. The maximum period of peg formation coincides with elongation of the flowering scape in June, while 'bursting' occurs as a late phenomenon at the time of seed-set from July onwards. They suggested that in the extension phase the epidermal proliferations act as transfer cells facilitating steady supply of nutrients to support the extension of the scape. There is clearly a possibility that the 'bursting' event provides a late surge of transfer of residual materials which could be used for seed production before the scape senesces. However, all such suppositions require experimental investigation. Function of Monotropoid Mycorrhizas The seed of Monotropa is small and has proved difficult to germinate, like the seeds of so many orchids which also have a period when they are dependent on an external supply of organic compounds. As in some of these, asymbiotic germination can be brought about by prolonged washing in water before the seed is set upon the germination medium (Francke, 1934). The embryo consists of a minute axis of very few cells set in an endosperm of about a dozen large cells. On germination it forms a small plant body, similar to the primary protocorm of orchids, which possesses an apical growing point and a broad base attached to the remainder of the endosperm. No further development occurred in Francke's cultures unless they were inoculated with a fungus which he had isolated from the roots of adult plants. This fungus, which he believed to be a species of Boletus,

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Figure 11.24 Epidermal cell of Sarcodes, showing median section of the fungal peg (FP). The membranous sac (MS) appears to be surrounded by the plant plasma membrane and the osmiophyllic ring at the tip of the peg is again evident. Inset: Higher magnification showing linear array of microtubule-like inclusions of the membranous sac. From Robertson and Robertson (1982), with permission.

formed a hyphal sheath around the protocorm and penetrated into a few cells of the outer layer in a manner similar to the colonization of the roots. Some further growth and cell division took place after colonization but soon ceased. Francke was unable to obtain further development to produce plants that were differentiated into root and shoot. Clearly, the embryo possesses limited ability to absorb nutrients in the absence of the fungus, but some factor or nutrient must be lacking because development comes to a halt. Under natural conditions the stimulus is possibly derived via the fimgus from a secondary host or from the soil (see below). As mentioned above, Kamienski (1881) thought that Monotropa possessed a common symbiotic fungus with the forest trees near which it grew. He also believed that it might be nourished, not saprophytically, but through the common mycelium from the neighbouring trees. Bjorkman (1960) tested this hypothesis. He first separated Monotropa plants from the tree roots by metal sheets, and observed that they grew poorly compared with attached plants. Later, ^^C-labelled glucose and ^^P-labelled orthophosphate were found to be translocated in 5 days from the Picea and Pinus trees into which they had been injected, to the tissues of Monotropa growing close by. The distance between the trees and Monotropa plants was 1-2 m and young developing plants became more radioactive than old n\ature plants. Bjorkman confirmed the view of Kamienski that the fungus infecting the tree roots and Monotropa was probably of the same mycelium. Other plants in the

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neighbourhood, such as Calluna vulgaris, Vaccinium vitis-idaea and V. myrtillus, did not become labelled from either [^^C]glucose or [^^P]orthophosphate injected into Picea and Pinus, and that was regarded as confirmation of the need for hyphal connections. In a similar experiment briefly reported by Furman (1966), Monotropa plants were injected with ^^P, which was transported to neighbouring Quercus and other trees which were ectomycorrhizal and (surprisingly) to an Acer which had vesiculararbuscular mycorrhizas. Attempts to confirm transfer of ^^C after feeding ^^C02 to Salix associated with M. hypopitys failed to confirm these findings (Duddridge, Stribley and Read, unpublished data), but raise the interesting possibility that the mycoheterotroph may be supported not by current photosynthate (which would be labelled during ^^C02 feeding), but from carbohydrate stored in the bud. More, longer-term experiments are obviously required. Bjorkman called the behaviour which he observed 'epiparasitism', but it is probably more appropriately referred to as a form of 'mycoheterotrophy', implying the dependence of an achlorophyllous plant on a fungus (Leake, 1994). In the so-called epiparasitic examples, the fungus obtains organic C for itself and its mycoheterotrophic dependant via a mycorrhizal symbiosis with an autotrophic partner. Mycoheterotrophs are found in many families of plants including members of the Orchidaceae (see Chapter 13) and Burmanniaceae (see Chapter 4), and also in the gametophyte stages of some bryophytes and pteridophytes. In some instances these myco-heterotrophic plants are supposed to be associated not with mycorrhizal, but with parasitic or saprophytic fungi. For example, Furman and Trappe (1971) and Harley (1973) noted the similarity of the behaviour of Monotropa with that of orchids, such as Gastrodia, in which the fungus Armillaria mellea is parasitic on the neighbouring trees and apparently mycorrhizal with the orchid. Campbell (1971) noted that in the USA Monotropa uniflora was also associated with Armillaria mellea in a similar fashion, although M. hypopitys seemed to be associated, as Bjorkman observed, with the mycorrhizal fungi of neighbouring trees. The relationship of the Armillaria with the tree roots was described as less 'harmonious' in the case of M. uniflora, as might indeed be expected from its parasitic behaviour. As it turns out, neither M. uniflora nor the Gastrodia spp. are always associated with Armillaria or other parasites, but in the adult stages form typical monotropoid or orchid mycorrhizas with basidiomycetes ectomycorrhizal on associated trees (Taylor and Bruns, personal communication; and see Chapter 13). This finding certainly vindicates the concern expressed by Harley and Smith (1983) who stated that: '. . . the supply of readily available carbon compounds even in the litter and humus layers of the soil of temperate forests does not seem to be quantitatively sufficient to support such large saprophytes as Monotropa spp. and an indirect source of carbon from photosynthesis or via a large pool in a parasitized plant seems necessary for achlorophyllous plants of any size'. With respect to monotropoid mycorrhizas, as well as other mycoheterotrophs, there is a wealth of exciting research to be done on the identity of the fungal symbionts, as well as on the cellular interactions leading to their apparently relatively specific relationships and their physiological interactions with the fungi and the autotrophic partners in the tripartite symbioses.

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Conclusions It is clear that striking advances have been made in our knowledge of the structural aspects of arbutoid and monotropoid mycorrhizas. However, our understanding of the functional attributes of many of the newly revealed features, particularly in the monotropoid symbiosis, remains lamentably poor. In the case of those plants with both types of mycorrhiza that produce minute seeds, there are, as in the Orchidaceae, outstanding general questions concerning the nutrition of the seedling in its early stage of growth. In the achlorophyllous Monotropaceae, which must be dependent upon exogenous sources of carbon through adulthood the question extends to the whole cycle. There is an urgent need to analyse quantitative and qualitative aspects of C supplies to these plants; such analysis must include examination both of the sources and periodicity of C supply. The challenge here will be to relate the patterns observed in the development and function of the structures revealed by microscopy. Amongst these, the roles of the peg and membranous sac in the monotropoid mycorrhiza are of perhaps the greatest interest. Despite the technical challenges involved, there will be no substitute for direct examination of transfer processes between plants of this type grown from seeds with their fungal symbionts and those autotrophic partners with which they are associated in the field.

12 Ericoid mycorrhizas

Introduction The order Ericales has long been recognized as a natural group of closely related families (Engler and Prantl, 1897), with members of worldwide distribution (Table 12.1). Some, notably the Ericaceae, have representatives which occur as dominant plants over vast areas of the northern hemisphere, forming distinctive heathland ecosystems (see Chapter 15). In the southern hemisphere the Epacridaceae is an important family. The Ericaceae and Epacridaceae are the largest families in the order. Their members, with the exception of a small number of ericaceous genera notably Arbutus and Arctostaphylos, which have arbutoid mycorrhizas (see Chapter 11), have, together with members of the Empetraceae, a distinctive form of the symbiosis referred to as 'ericoid' mycorrhiza. Much controversy surrounded the early studies of ericoid mycorrhizas, especially with respect to the identity of the fungal partner and, in particular, its distribution within the tissues of the plant. These arguments distracted attention from more important questions about the role of mycorrhizas in the growth, nutrition and ecology of the plants. The controversies have been discussed elsewhere (Harley, 1969; Read, 1983) and will only be briefly referred to here. While doubts remain about the taxonomic position of some of the fungi isolated from ericoid mycorrhizal roots, there is now much better understanding of the processes involved in formation of ericpid mycorrhizas and some of the major functions of the symbiosis. This chapter will place emphasis upon these advances and aspects of their possible ecological significance are reviewed in Chapter 15.

Structure and Development The ericoid mycorrhizal root is a delicate structure, the anatomy of which shows considerable uruformity (Burgeff, 1961; Nieuwdorp, 1969; Peterson et al, 1980; AUaway and Ashford, 1996; Ashford et al, 1996; Read, 1996). The apical meristem, a small group of undifferentiated cells, is protected distally by a root cap which itself is invested in mucilage (Fig. 12.1). The mucilage extends backwards from the apex as a thin sheath over the outer surface of the root. The finest hair

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T a b l e 12.1 Some taxonomic, geographic and mycorrhizal relationships w i t h i n the Ericales Order

Family

Important genera

Distribution

Mycorrhizal status

Ericales

Clethraceae

Clethra

Not known

Grubbiaceae

Grubbia

Cyrillaceae

Cyrilla, Cliftonia,

Restricted to central America and SE Asia Restricted to southern Africa Restricted to Central America Worldwide

Purdiaea

Ericaceae

Arbutus, Arctostaphylos, Cassiope, Calluna,

Not known Not known Mycorrhizal, some 'arbutoid' but mostly 'ericoid'

Erica, Gaultheria, Kalmio, Ledurr), Rhododendror), Vaccinium

Epacridaceae

Dracophyllum, Epochs, Richea, Styphelia

Empetraceae

Ceratiola, Corema, Empetrurr)

Pyrolaceae

Chimaphila, Moneses, Pyrola, Orthilia

Monotropaceae

Monotropa, Pterospora

Restricted to southern South America, Indonesia, Australasia some Pacific Islands including Hawaii Throughout northern hemisphere and in southern South America Boreal and subarctic distribution in northern hemisphere

Mycorrhizal, ericoid

Mycorrhizal, ericoid

Mycorrhizal mostly arbutoid Mycorrhizal monotropoid

F i g u r e 12.1 Light micrograph of the apical region of a hair r o o t of CoWuna vulgaris, showing the r o o t cap (*) and the commencement of mycorrhiza formation in cells distal t o the r o o t apex (arrowed). Photograph courtesy of D.J. Read.

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roots have a stele consisting only of one or two tracheids, a sieve element and companion cell. Around the stele is a cortex typically consisting of only two layers, an outer hypodermis and an inner endodermis (Fig 12.2). In the Epacridaceae the cells of the hypodermis have a suberized lamella around their walls, while those of the inner layer carry a distinct Casparian strip on the radial walls of each cell (Allaway and Ashford, 1996). Surrounding the hypodermis and forming a single layer is the epidermis, consisting of a small number of longitudinal rows of swollen cells extending back from the apex. This layer forms the outer surface of the root in the region which, in a conventional plant root, would be the piliferous zone. However, in the ericoid root the epidermal cells do not produce root hairs. Instead, many of them are colonized by mycorrhizal fungi which proliferate in the intracellular position (Fig. 12.2) to produce the dense hyphal complexes that are diagnostic of what Harley (1969) first called 'ericoid' mycorrhizas. As a result of the general simplification of anatomy, the diameter of the ericoid mycorrhizal root is normally less than 100 |Lim. Beijerinck (1940) referred to this structure as a 'hair root'. The epidermal layer of the hair root is an ephemeral structure, older roots losing their epidermes. Suberized and thickened cells derived from the two cortical layers then come to form the outer surfaces of the root (Peterson et al, 1980; Allaway and Ashford, 1996). Since mycorrhizal colonization is restricted to expanded epidermal cells, these maturation processes define the 'window of opportunity' for formation of the symbiosis in both space and time. Secondary thickening provides for longevity in the more mature part of the roots, whereas the unthickened hair roots

I

—I

F i g u r e 12.2 Transverse section of a mycorrhizal r o o t of Calluna vulgaris^ showing the cortex of one cell layer fully colonized by fungal hyphae. Bar, I m m . Photograph courtesy of

D.J. Read.

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appear to be completely shed each season. In drought-prone heaths of the southern hemisphere this shedding occurs in the dry season (Read, 1978; Bell et ah, 1994), whereas in wet heaths it is more likely to occur during the winter when high water tables produce anoxia at or near the surface. Ashford et al. (1996) described an interesting situation in the epacridaceous species Lysinema ciliatum, where apparently specialized thick-walled cells of the epidermis, which are readily detached from the root surface, become preferentially colonized by mycorrhizal fungi. It is suggested that these cells may act as resistant propagules, able to survive dry conditions and colonize new hair roots as they emerge after rain.

The Colonization Process The hyphae of ericoid mycorrhizal fungi form a loose network over the zone of the hair root which contains a mature epidermis (Fig. 12.3). As the root apex extends, new epidermal cells are differentiated and the hyphae at the leading edge of the network advance to colonize them. Slowing of root growth, for whatever reason, can enable the hyphal network to reach the root apex, but normally there is a zone of differentiating cells immediately behind the meristem which is free of fungal hyphae. The molecular basis of the processes of recognition between fungi forming this

F i g u r e 12.3 Scanning electron micrograph of a r o o t of Rhododendron colonized by loose wefts of fungal hyphae. Bar, 10 jiim. From Duddrldge and Read (1982b), w i t h permission.

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network over ericoid roots is so far unknown, but ultrastructural and cytochemical observations, most of them using strains of the ascomycete Hymenoscyphus ericae, are beginning to provide insights. Some strains of H. ericae typically produce an extracellular fibrillar sheath which is rich in polysaccharides (Bonfante-Fasolo and Gianinazzi-Pearson, 1982). This sheath is more strongly developed in infective (Fig. 12.4a) than in non-infective (Fig. 12.4b) strains (Gianinazzi-Pearson and BonfanteFasolo, 1986), and it has been suggested (Gianinazzi-Pearson et ah, 1986) that it may anchor the fungus to the plant as the first step in mycorrhiza formation (Fig. 12.5). Using the gold-labelled lectin Concanavalin A (Con A) as a cytochemical marker for mannose or glucose residues, Bonfante-Fasolo et ah (1987b) observed an abundance of these compounds when an infective strain was in association with a plant root. While the fibrillar sheath may assist in attachment, which is clearly a prerequisite for further colonization, doubt remains about the extent to which it is involved in recognition of host as distinct from non-host plants because strains with a typical fibrillar sheath formed attachments to and penetrated roots of the nonhost Trifolium pratense (Bonfante-Fasolo et ah, 1984). Differences between the responses of host and non-host roots to challenge by the fungus were seen only in the intracellular situation where, in the case of one ericoid plant, the fibrillar material was lost as the hyphae became invested by plant plasma-membrane while in non-host roots it was retained over a period during which organization of the invaded cell broke down.

Penetration of the Plant Wall From the hyphae of the surface network branches emerge at right angles to penetrate the epidermal cells (Figs 12.3, 12.5, 12.6). Typically, there is a single penetration point per cell but multiple entry points are sometimes observed (Read and Stribley, 1975). Each entry may be preceded by the production of an appressorium, but as this structure is not always found it is clearly not a prerequisite for successful colonization.

Figure 12.4 Development of surface fibrils on hyphae of Hymenoscyphus ericae in the presence of roots of Calluna vulgaris, (a) Highly infective isolate, producing many fibrils, (b) Sparse production of fibrils by poorly infective isolate. Diameter of hyphae approximately 3 jiim. Photographs courtesy of V. Gianinazzi-Pearson.

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r€^

Figure 12.5 Transmission electron microscopy of a hypha of Hymenoscyphus ericae (FH) attached to the surface of a root of Rhododendron poniticum. Note the presence of (SL) fibrils; (EC) root epidermal cell; (HCW) cell wall; and (EIA) electronlucent zone, at the site of penetration. (Duddridge and Read 1982b), with permission.

The mechanism of penetration is not fully understood. There is evidence from the use of the periodic acid-thiocarboxydrazide-silver proteinate (PATAG) test for localization of polysaccharide that the carbohydrate-rich fibrils which ensheath the hyphae, although present at the point of attachment to the surface of the epidermal cells, disappear as the hyphal tip penetrates the outer wall. An electronlucent zone becomes evident where the fungus enters the inner wall layer of the plant (Bonfante-Fasolo and Gianinazzi-Pearson, 1982; Duddridge and Read, 1982b; see Fig. 12.5). This might represent dissolution of the plant cell walls by fungal enzymes. When grown in pure culture, H. ericae has the ability to use a range of plant cell wall-related mono-, di-, and poly-saccharides including carboxymethyl cellulose (CMC), as sole sources of C (Pearson and Read, 1975; Varma and Bonfante-Fasolo, 1994). Two polygalacturonases were identified when the fungus was grown with pectin as sole C source (Perotto et al, 1993) and activity of both (3 (1,4) and P(l,3)-glucanase was detected in cultures supplied with CMC or sterile root segments (Varma and Bonfante-Fasolo, 1994). Because these enzymes are involved in penetration of cell walls by a number of plant pathogenic fungi (Hahn et al, 1989) their production by H. ericae, combined with the ultrastructural evidence indicating weakening of the plant cell wall in advance of the penetrating fungal hypha, strongly suggests the involvement of hydrolase enzymes in the penetration process. There is also the likelihood, discussed later, that these enzymes might be

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Figure 12.6 Newly colonized cell of a Rhododendron seedling, showing point of penetration (EP) by a hypha of Hymenoscyphus ericae. Invagination of the plasma membrane of the plant cell is shown (HPL). HC, Plant cytoplasm; HCW, plant cell wall; EH, intracellular fungal hypha. Inset: Higher magnification of fungal penetration through the plant cell wall, showing a simple septum in the hypha (SSP) and Woronin body (WB). From Duddridge and Read (1982b), with permission.

deployed in soil organic matter, resulting in release of the nutrients contained within it.

Features of Intracellular Colonization Electron microscopy of mature infection units (Nieuwdorp, 1969; Bonfante-Fasolo and Gianinazzi-Pearson, 1979; Peterson et ah, 1980) have shown that colonizing hyphae retain a discrete structural integrity within the plant cell. In addition, there is some deterioration in the appearance of plant cytoplasm when there is no loss of integrity in the fungus. In a detailed analysis of the sequence of events from colonization of the plant cell through to collapse of the association it has now been confirmed that breakdown begins with deterioration of the plant rather than the fungal tissue (Duddridge and Read, 1982b). Seedlings of Rhododendron ponticum were grown either in soil partially sterilized by y-irradiation and then inoculated with the endophyte, or in soil freshly collected from underneath R/zo-

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dodendron bushes. Surface colonization of roots occurred in irradiated soil after 3 weeks and in natural soil after 4 weeks, with penetration of the cortical cells following immediately (Figs 12.5, 12.6). Once within the cell the fungal hyphae proliferate extensively. The plant plasma membrane invaginates to envelop each branch of the invading fungus, but is separated from the fungal cell wall by a thin electron-lucent layer, the so-called 'interfacial matrix' (Fig. 12.8). This contains flocculent, electron-dense pectic material (Duddridge, 1980). The scanning electron microscope (SEM) and transmission electron microscope (TEM) studies have confirmed that colonization occurs through the outer wall of the epidermal cell, so that each cell is an individual infection unit. Consequently, even adjacent cells may have fungal complexes which are of different ages (Fig. 12.7). When each complex is mature the volume of the plant cell is almost completely occupied by fungal hyphae and little or no vacuolar volume is apparent. Outside the electron-lucent zone the hyphae are ensheathed by plant cytoplasm which is packed with rough endoplasmic reticulum and mitochondria, suggesting that at this stage the unit is the site of considerable physiological activity (Fig. 12.8).

Figure 12.7 Transmission electron microscopy of a root of Rhododendron ponticun), showing three adjacent cells in longitudinal section and intracellular colonization of different ages. Cell (a) Healthy, mature colonization by hyphae that fill the cell. Cell (b) Plant cytoplasm is degenerating, but the intracellular hyphae appear healthy. Cell (c) Cytoplasmic degeneration in both plant and fungus. DjHC, degenerating plant cyloplasm; PPG, phosphate granule; LI, lipid droplet; HCW, plant cell wall; INF, intracellular fungal hypha. From Duddridge and Read (1982b), with permission.

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Figure 12.8 Early stage of degeneration of mycorrhizal colonization in roots of Rhododendron ponticun). The plant cells contains abundant rough endoplasmic reticulum (RER). M, Mitochondria; IM, interfacial matrix; HPI, plant plasma membrane; InF, intracellularhypha; W M , wall material. Bar, I |xm. From Duddridge and Read (1982b), with permission.

The first sign of breakdown observed by Duddridge and Read (1982b) was a loss of structural integrity of organelles in the plant cell, particularly the mitochondria. Plant cytoplasm then degenerates and the electron-lucent area between the plant plasma membrane and fungal cell wall becomes progressively wider. The integrity of the plant plasma membrane is finally lost and most of the plant organelles degenerate before fungal deterioration occurs (Fig. 12.9). Evidence of deterioration in the fungal hyphae is seen as an increase in the size of the vacuoles, only in the later stages of plant degeneration. Final breakdown of the fungus is not complete until after the plant cell loses it integrity. At the end of the breakdown process, therefore, the cell is empty, except for the debris of earlier fungal occupation. In the studies of Duddridge and Read (1982b) the first indication of breakdown was observed 8 weeks after inoculation and hence about 4 weeks after penetration in inoculated soil and about 11 weeks after planting into natural soil. The differences of timing are probably attributable to differences in the vigour of the partners in the two systems, both fungus and plant being more active in the irradiated soil. The events revealed by TEM are thus distinct from those previously described by light microscopy. Since breakdown occurs first in plant cytoplasm, nutrient transfer either from fungus to plant or in the reverse direction must take place during the few weeks after colonization, when both partners have full structural and hence presumably physiological integrity. This means that the active life span of the individual colonized cells is not more than 5 or 6 weeks. In addition, the notion of digestion or lysis of the fungus by the plant is not tenable. Ultrastructural analysis also provides information concerning the taxonomic position of the fungal endophytes. Bonfante-Fasolo and Gianinazzi-Pearson

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Figure 12.9 Late stage in the degeneration of mycorrhizal colonization in roots of Rhododendron ponticum. The plant cytoplasm (DHC) has almost completely degenerated, whereas the fungal hyphae (InF) are apparently still alive. Lipid droplets (Li) and polyphosphate granules (PPG) are apparent. Bar, I |im. From Duddridge and Read (1982b), with permission.

(1979), showed that the intracellular hyphae in Calluna contained simple septal pores indicting ascomycete affinities (Fig. 12.6). In the course of examination of material from a wide range of ericaceous species, including some from southern Africa, Duddridge (1980) also found only hyphae with simple septal pores in an intracellular position, although dolipore septa were occasionally seen in rhizosphere hyphae. Both Bonfante-Fasolo (1980) and Peterson et ah (1980) have observed hyphae with dolipore septa within ericoid cortical cells. Careful analysis of the status of such colonizations is necessary since presence inside the cell is alone not evidence of a mycorrhizal or even a biotrophic association.

The Fungi Forming Ericoid Mycorrhizas The earliest attempts to determine the identity of the fimgi involved in the formation of ericoid mycorrhizas were surrounded by controversy. There were claims (Rayner, 1915,1927) that roots were colonized by a species of Phoma, the mycelium of which extended from the roots, through the shoots and into the floral organs

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where it reached the seed coat. This so-called 'systemic infection' was considered to enable the radicle of the germinating seed to become colonized as it passed through the seed coat. The view was expressed that subsequent development of the seedling was dependent upon this transfer of colonization (Rayner, 1915; Addoms and Mounce, 1931). At the time, some workers, notably Knudson (1929, 1933) in America and Christoph (1921) in Europe, took issue with this conclusion, showing that systemic infection was neither normal nor an essential prerequisite for seedling development, which could readily be obtained under aseptic conditions. If nothing else, these controversies exposed the need for rigour in isolation of fungi from roots and for re-inoculation to ensure that a typical mycorrhiza was produced. These simple prerequisites for verification of the status of microbial colonists had earlier been clearly expounded in the form of Koch's postulates (Koch, 1912), but while they were widely followed in plant pathology, their application to studies of mycorrhizal colonization especially of ericaceous plants, was largely overlooked by Rayner and her colleagues. Doak (1928) was the first to report isolation of an endophyte from an ericoid mycorrhizal root, in this case of Vaccinium, and to confirm its mycorrhizal status by back-inoculation. Bain (1937) repeated this type of observation in a number of genera of North American Ericaceae, as did Freisleben (1933, 1934, 1936) in Europe. These, the earliest of the systematic observations, revealed that when grown on nutrient agar the isolated fungi produced dark-coloured, slow-growing, sterile mycelia. These basic features have been repeatedly confirmed in studies of roots of Ericaceae (Burgeff, 1961; McNabb, 1961; Pearson and Read, 1973a; Singh, 1974) and Epacridaceae (Reed, 1987) and the range of plant genera and species from which such fungi have been isolated has been considerably extended (Read, 1996). The apparent absence of reproductive structures in most such isolates has hindered their classification. However, Pearson and Read (1973a) observed that a significant proportion of their isolates produced hyphae which segmented to form chains, in zigzag formation, of arthroconidia (Fig. 12.10), a feature of the hyphomycete genus Scytalidium. One isolate, from Vaccinium angustifolium, was subsequently identified as being Scytalidium vaccinii Dalpe, Litten and Sigler (Dalpe et ah, 1989). One of the segmenting isolates obtained by Pearson and Read (1973a) subsequently produced apothecia both in pure culture and in cultures in which the fungus was forming mycorrhizas with Calluna vulgaris. This fungus was named Pezizella ericae Read (Read, 1974). Later, Kernan and Finocchio (1983) described another small discomycete, Hymenoscyphus monotropae Kernan and Finocchio, growing on roots of a mycoheterotrophic member of the Ericales, Monotropa uniflora. On the basis of structural similarities between this fungus and that described by Read (1974), they proposed the transfer of Pezizella to the new combination Hymenoscyphus ericae (Read) Korf and Kernan. Apothecia of the fungus have not yet been observed in nature, nor have they been produced by isolates of ericoid mycorrhizas obtained in North America. However, because of the phenotypic similarities between Scytalidium vaccinii and H. ericae, Egger and Sigler (1993) investigated the possibility that S. vaccinii was the anamorph of H. ericae. They examined a number of North American isolates which had the cultural characteristics typical of H. ericae and observed that the propensity to produce arthroconidia which remained connected in zigzag chains was a unifying characteristic of all strains studied. Using polymerase chain reaction (PCR) techniques to obtain

334

Mycorrhizas in the Ericales

Figure 12.10 Hyphae of an isolate of Hymenoscyphus ehcaey showing segmentation into zigzag chains characteristic of Scytalidium, Photograph courtesy of D.J. Read.

nucleotide sequences of homologous regions of ribosomal RNA subunits and of the 5' internal transcribed spacer, Egger and Sigler (1993) showed that divergence between isolates identified as Scytalidium or H. ericae was extremely low, ranging from 1.2 to 3.5%. The morphological and molecular evidence, taken in combination, therefore suggests that S. vaccinii and H. ericae are indeed anamorph and teleomorph states of a single taxon. Amongst the other fungi which have been isolated from ericoid mycorrhizal roots, members of the genus Oidiodendron figure most strongly. Burgeff (1961) obtained O. griseum from the roots of C. vulgaris and Vaccinium spp. and Pearson and Read (1973a) observed an unidentified Oidiodendron sp. amongst their isolates. O. griseum can form mycorrhizas of the ericoid type (Couture et al, 1983; Dalpe, 1986; Xiao and Berch, 1992) and a number of Oidiodendron spp. have been shown to colonize the roots of aseptically grown seedlings. These include O. maius in Rhododendron (Douglas et al, 1989). Hutton et al. (1994) carried out a detailed study of the fungi forming ericoid mycorrhizas in the Epacridaceae of Western Australia. They obtained over 400 isolates from the roots of 14 species belonging to seven genera. In culture all of the isolates were dark-coloured, sterile and slow growing, resembling H. ericae at least superficially. Their hyphae had the simple septal pores characteristic of

335

Ericoid mycorrhizas

ascomycetes. Eight of the isolates, divided into three groups on the basis of small differences in appearance, were further investigated using pectic zymograms. European isolates of H. ericae and several Oidiodendron species were included for comparison (Fig. 12.11). The three cultural groups (Table 12.2) had distinct zymogram patterns. Whereas none of these coincided precisely with those of H. ericae or the Oidiodendron spp., similarities between profiles of isolates EC2 and E. ericae 100 are evident (Fig. 12.11). This study suggests that there may be considerable genetic diversity amongst isolates that are superficially similar in appearance. This diversity has been confirmed in European isolates of ericoid mycorrhizal fungi, using the PCR-RAPD (random amplification of polymorphic DNA) technique to amplify ribosomal DNA (Perotto et ah, 1995). Isolates were obtained from surface-sterilized roots from different plants of Calluna vulgaris and also from different cells of the same root. Each isolate was re-inoculated onto Calluna seedlings under axenic conditions to confirm its mycorrhizal status before DNA extraction. Very distinct amplification patterns were observed in isolates obtained from different plants and, more surprisingly, from those from the same plant. A parallel study of isolates of Oidiodendron maius showed that even within this species a wide range of amplification patterns was evident. Clearly, isolates from the epidermal cells of ericoid mycorrhizal roots are genetically diverse. Whether the differences between isolates are sufficient to justify the suggestion (Perotto et ah, 1995) that these are different

Western Australian isolates

R ericae

Oidiodendron species*

* 89 = Oidiodendron sp. * * gri = 0. griseum, mai = 0. maius, cer = 0, cerealis, cit 0. citrinum, scy = 0. scytaloides, fla = 0. flavum, rho = 0. rhodogenum, per = 0. periconioides. Figure 12.11 Pectic zymograms of fungi infective with members of the Epacridaceae (Western Australian isolates) and Ericaceae {Hymenoscyphus ericae and Oidiodendron sp.). Dark bands correspond to pectin depolymerase activity and white bands to pectin methyl esterase activity. From Hutton et al. (1994), with permission.

336

Mycorrhizas in the Ericales

Table 12.2 Morphological and cultural characteristics of fungi isolated from Western Australian members of the Epacridaceae grown on agar Characteristics

Colour Mycelium White margin (2-3 mm) Colony appearance Indents agar Exudes droplets Growth rate^ (mm wk~') PDA Malt agar 1 - 3 Oat agar

Isolate groups Lc 1 Asc Anc Ec 1

Lc2 Lc3

Ec2 Ec3

Smokey-grey Fluffy, appressed at margin Yes Dry Yes Yes

Black Appressed No Wet Yes Yes

Light brown Fluffy, appressed at margin No Dry No No

4-5 1-3 5-6

7 6-7 7-9

7 1-2 7-9

^ Plates incubated in the dark at 24°C. Data from Hutton et o/. (1994), colours based on Rayner (1970).

species is uncertain, particularly in view of the range of genotypes seen within O. mains. Perhaps of greater importance is the functional significance of the observed variability. Genetic diversity is increasingly recognized to be considerable in ectomycorrhizal as well as ericoid mycorrhizal fungi (see Chapter 6). Having established its occurrence, it is now necessary to determine whether such differences have any impact upon performance of plants grown under controlled conditions. Fruit bodies of Clavaria argillacea can often be produced in soil surrounding ericaceous plants in nature (Gimingham, 1960; Seviour et ah, 1973; MooreParkhurst and Englander, 1982), leading to the view that this fungus may be a mycorrhizal associate. Serological (Seviour et al, 1973), immunocytochemical (Mueller et al, 1986) and nutrient transfer techniques (Moore-Parkhurst and Englander, 1982) have demonstrated that this fungus can indeed form close associations with ericoid roots. When roots of Rhododendron were collected from soil underneath fruit bodies of C. argillacea, hyphae with dolipore septa, typical of basidiomycetes were observed in epidermal cells, together with those forming simple septa (Peterson et al, 1980). Since C. argillacea appears never to have been grown in culture, the nature of its relationship with the ericoid roots is unclear. The application of molecular methods of identification to the fungi colonizing the roots could be illuminating.

Functional Aspects of Ericoid Mycorrhizas The soils which support ericaceous vegetation are characteristically extremely poor in available nutrients and it is logical to expect nutritional benefits to arise from mycorrhizal colonization. Experimental analysis of mycorrhizal effects has, however, been made difficult by the fact that sterilization of these typically organic

337

Ericoid mycorrhizas

soils, which is essential if non-mycorrhizal plants are to be grown as controls, may release nutrients or toxins. Using autoclave-sterilized peat, Freisleben (1936) concluded that the main effect of mycorrhizal fungi was to detoxify the peat. Subsequent experiments also using autoclaved soil (Brook, 1952; Morrison, 1957b) provided some indication that mycorrhizal colonization might improve plant nutrition, but problems arising from toxicity and the uncertain nature of the inoculum made interpretations of their results difficult. These problems make it important to define clearly the nature of the substrate and the sterilization treatment being employed in experiments. Progress has been made by using a range of systems from those that are simple and readily defined such as hydroponic sand culture, to those like the heathland soil itself which are more realistic but correspondingly more difficult to handle. Use of irradiation to sterilize soil has helped because it does not release toxins, but there can be problems of excessive nutrient release, especially of ammonium (NHJ) (Stribley et ah, 1975). Using small quantities of irradiated heathland soil and mycorrhizal endophytes isolated by Pearson and Read (1973a), colonization of Calluna and Vaccinium consistently resulted in increases of yield, associated with increased concentrations not only of P but also, particularly, N in the plant tissues (Read and Stribley, 1973). These observations, which had parallels with the effects of mycorrhizas on ectomycorrhizal trees, led to a more intensive analysis of the nutritional role of ericoid mycorrhizal fungi. Perhaps the most striking feature to emerge has been the versatility which these organisms show in relation to nutrient use. This is well exemplified in the case of N. Of the mineral forms, H. ericae can readily use NH4 in liquid culture (Fig. 12.12). Assimilation of NH4 takes place by both the glutamate dehydrogenase (GDH) and glutamine synthetase (GS) pathways (St. John et ah, 1985), the latter being most

Mineral nitrogen

Amino acids (acidic)

Amino acids (basic)

50-

8

X

1^ 7

1

i

f

^

40-

1

30

6 -III

5 i

h

-• 20 0

Glutamic acid

•

Aspartic acid

10-

— Yield — pH 1

1.

1

J

10

20 Days

30

2'-x o Arginine • 2-Amino-n butyric acid — Yield — pH

10

20 Days

30

F i g u r e 12.12 Dry weight yields (solid lines) and changes in pH (dashed lines) of the medium after growth of Hymenoscyphus ericae on nitrate ( N O ^ ) , NH4 or a series of acidic and basic amino acids as sole sources of N . (a) Inorganic N or no N source, (b) Acidic amino acids, (c) Basic amino acids. From Bajwa and Read (1986), with permission.

338

Mycorrhjzas in the Ericales

important when external concentrations of NH4 are low (1 HIM). In sand, enhanced scavenging by the mycorrhizal fungus for NH4 can, at these concentrations, support increases of yield of colonized plants (Stribley and Read, 1976). The ability of H. ericae to use NO3 (Pearson and Read, 1975; Bajwa and Read, 1986) should not, however, be overlooked (Fig. 12.12), especially because plants in the Ericaceae and their counterparts in Epacridaceae appear to have very little NO^ reducing ability (Stewart et al, 1993). Facilitation of NO^ utilization may be of considerable ecological importance under some circumstances (see Chapter 15). The possibility that mycorrhizal colonization might provide the plant with access to organic sources of N emerged from a study using small quantities of irradiated heathland soil upon which Vaccinium plants were grown with and without colonization by H. ericae, and supplied with ^^NH4, which was then thought to be the only significant source of N in this soil (Stribley and Read, 1974b). Colonized plants showed a stimulation of growth and N concentration, but a lower ^^N enrichment than their non-mycorrhizal counterparts (Table 12.3). Since little of the ^^NH4 had been incorporated into the organic constituents, dilution of label in the colonized plants was attributed to them having access to N from the soil organic matter. This experiment led to others which were designed to determine the possible sources of this organic N. Amino acids were obvious candidates, although only later (see Chapters 8 and 15) was it found that potentially significant pools of these were present in heathland soil. Mycorrhizal and non-mycorrhizal plants were grown in sand culture to which the amino acids alanine, aspartic acid, glutamic acid, glutamine or glycine were added individually as sole N sources. Yields of colonized plants were comparable with those obtained on NH4, but most of the amino compounds were little used by non-mycorrhizal plants whether they were grown in sterile or non-sterile conditions (Stribley and Read, 1980). It seems that both amino acids and their amides are equally well assimilated by H. ericae (Fig. 12.12), and the products transfered to mycorrhizal plants. There are two major sources of amino acids in soil. On one hand, they may leak from living roots and microTabl6 12.3 N content and yield and '^N excess of shoots of mycorrhizal, non-mycorrhizal and nonmycorrhizal saprophyte-inoculated plants of Vaccinium macrocarpon after six months' growth on '^N-labelled soil Growth stage

N content (% oven -dry wt)

Sterile seedlings Plants 6 months after inoculation Mycorrhizal (M) Non-mycorrhizal (NM) Inoculated with saprophytes: Trichoderma sp. Aspergillus sp.

0.94

4.23

1.20 0.98

30.32+ 20.97+

0.36+ 0.21 +

15.38+ 20.03+

0.94 0.82

18.80 16.20

0.18 0.18

ND ND

Yield (mg oven-dry wt)

Total N (mg plant" ')

0.04

'^N excess (atom%)

0

Each figure represents a mean of 14 plants, except for sterile seedlings, when 30 plants were analysed. +, Figures significantly different within the growth at /) < 0.001; ND, not determined. Data from Stribley and Read (1974b).

Ericoid mycorrhizas

339

organisms, largely as monomers. On the other hand, polymeric proteinaceous material may be rendered unavailable to various extents, by reaction with phenolic and other soil constituents as decomposition proceeds. The loss of availability to the microbial population causes this type of N source to accumulate in many organic heathland soils (see Chapter 15). The organic N will be present in a range of sizes from the smallest peptides to macromolecular proteins. The extent to which these complex sources of N are available to ericoid mycorrhizal fungi has been investigated using a combination of approaches, involving both axenic culture and mycorrhizal synthesis. When supplied as sole N sources to H. ericae, the tripeptide glutathione and peptides in the form of alanine units of 1 to 6 amino acid residues were all readily used (Bajwa and Read, 1985, 1986). Their assimilation was generally slower the longer the chain length, but ultimate yields were the same on all peptides, as well as being equivalent to those obtained on NH4. Mycorrhizal plants of V. corymbosum had higher N contents than their nonmycorrhizal counterparts in all cases, and higher yields on all but two of the peptide sources. Slower assimilation of the larger polypeptides was attributable to the requirement for peptidase activity and subsequent work using a range of polymeric N sources has confirmed that ericoid mycorrhizal fungi have very well developed peptidolytic and proteolytic potential. H. ericae can utilize protein as sole source of N both with (Spinner and Haselwandter, 1985) and without (Bajwa et al, 1985) the additional supply of C. In the latter study soluble animal protein in the form of bovine serum albumen (BSA) of molecular weight (MW) 67000, and the plant proteins gliadin (MW 27000) and zein (MW 40000) were used as sole N and C sources. These proteins were broken down preferentially at low pH, there being progressively lower yields as p H increased from 3.0 to 7.0. When V. macrocarpon was grown on cellulose filter paper platforms in the mycorrhizal and non-mycorrhizal condition with BSA as its only potential N supply, significantly greater dry matter yields (Fig. 12.13a) and N contents (Fig. 12.13b) and concentrations were obtained in the colonized plants. Expression of proteolytic activity in heathland soils which contain significant amounts of polymeric organic N could be of considerable importance for plants with ericoid mycorrhizas and much effort has been devoted, particularly by Dr J. R. Leake, to characterizing the factors which might determine the production and activity of proteinase enzymes of ericoid fungi. Enzyme production and activity is regulated by pH, with both being maximal between p H 2 and 5 and practically eliminated above pH 6.6 (Leake and Read, 1990a; see Fig. 12.14). The possible ecological significance of this attribute is highlighted in Chapter 15. Proteolytic activity can be induced by the presence of protein itself, or of hydrolysates of protein (Leake and Read, 1990b,c), and is increased by the presence of NHJ, at a concentration 10 mM (Leake and Read, 1990c). If pure protein was provided as the sole source of N for the fungus, both total and specific activities of the proteinase were strongly repressed by the presence of glucose. In contrast, no repression by glucose was observed if protein hydrolysate was the sole N source. Leake and Read (1990b,c) have suggested that H. ericae employs a 'Noah's Ark' strategy of enzyme regulation (Bums, 1986), in which an emissary extracellular proteinase is produced under conditions of de-repression. In the presence of appropriate substrates this leads to the release of reporter molecules in the form of products of protein hydrolysis that induce full enzyme production. The rapid

Mycorrhizas in the Ericales

340 (b)

(a)

(1.9)(1.9)(1.8)

140 ^120

NS

I• l rl r i NM

M

No nitrogen

NM

M

BSA

^

nooh

(i.i)(i.i)(i.2) 80 h(1.0)(1.0)(1.2) NS 60

NS

40 20

(i.2)(i.i)(i.3) P ; ^

M

IT

1

1*1 Pi NS Ki

0 NM

M

No nitrogen

NM

ii I

NS

M

BSA

F i g u r e 12.13 Partitioning of (a) dry weight, and (b) N content into ^ , w h o l e plant, ^ , roots and H , shoots of plants of mycorrhizal (M) and non-mycorrhizal ( N M ) Vaccinium macrocarpon g r o w n w i t h bovine serum albumin (BSA) o r w i t h no N f o r 30 days. * * * , Significant differences between M and N M plants w i t h i n the n u t r i e n t regimens at P <0.001; NS, no significant difference. Values are means and standard e r r o r s of means of 24 replicate plants. From Bajwa et al. (1985), w i t h permission.

release of proteinase by H. ericae when the fungus was exposed to substrate (approximately 80% of maximum activity of the enzyme being detected within 36 hours of inoculation of the medium and well before the major growth phase of the fungus) suggests that this strategy may be very effective. Whereas conditions of de-repression might prevail in the typical soil environment of the ericoid hair root, the relatively high pH and free sugar content of the intracellular environment would be expected, in combination, to ensure that little or no production of the acid proteinase would take place in the colonized cells (see Leake and Read, 1990c). While it is necessary to appreciate the potential role of proteolytic activity in mobilizing N from organic substrates, it has to be recognized that most of the organic N in soil will be present in more recalcitrant forms. Attempts to investigate the extent to which such N is accessible to the mycorrhizal plant have taken two forms. In one (Leake and Read, 1990d), protein was co-precipitated with tannin before being supplied as sole N source to mycorrhizal and non-mycorrhizal plants. The fungus gained access to N contained in the protein-phenol complexes. The second approach involved axenic production of substrates likely to be present in soil. These include mycelial products which, in acid pine-heath soils, constitute a significant pool of stored N (Baath and Soderstrom, 1979). The fungus certainly has some chitinolytic capability (Leake and Read, 1990b). Read and Kerley (1995) showed that dead mycelium or purified cell wall fraction of H. ericae could act as sources of N for mycorrhizal, but not non-mycorrhizal, plants of Vaccinium macrocarpon. N was released from both materials in sufficient quantity to sustain growth of the mycorrhizal plants whether it was supplied as entire mycelium (Fig. 12.15a) or as purified hyphal walls (Fig. 12.15b). Since separate assays of both substrates revealed that mineral N was scarcely detectable, it can be assumed that mobilization of organic N was achieved by a combination of proteolytic and

Ericoid mycorrhizas

341

'•°"^^EEr^^?^>'"" Figure 12.14 Proteinase activity (fluorescence units released in 3 h, corrected for residual protein) in culture filtrates of Hymenoscyphus ericae in relation to the pH of the medium and pH at which the assay was performed. Values are means of four replicates. Vertical bar, least significant difference; P = 0.05. From Leake and Read (1990a), with permission.

chitinolytic activity. The possible ecological significance of these observations is discussed in Chapter 15. Comparisons of P concentrations in tissues of ericaceous plants grown with or without mycorrhizal colonization indicate that the fungi can increase access to this element and also to N (Read and Stribley, 1973; Mitchell and Read, 1981). In many heathland soils the main sources of P will again be the organic residues in which the roots proliferate. Phytates have been identified as being quantitatively the most important sources of Po in soils (Cosgrove, 1967), although it is increasingly recognized that the phosphodiesters such as nucleic acids may be present in significant amounts and are more labile than the monoesters (Griffiths and Caldwell, 1992; and see Chapter 9). Under the acidic conditions prevailing in heathland soil, phosphomonoesters are likely to be complexed with Fe and Al to form, respectively, Fe- and Al-phytates. These have been shown to be accessible to ericoid endophytes isolated from Vaccinium and Rhododendron (Mitchell and Read, 1981) and considerable progress

Mycorrhizas in the Ericales

342 (a) 10

A M+IHE A M-IHE • NM+IHE NM -IHE

^«(^

E

r

6

^ ^ J5

24 36 Day of harvest

(b) i Orr A M +CWF

8h h

:E

A M -CWF • NM+CWF o NM-CWF

www

1

6

1 ^ \ ^

W WW

O)

^

4

ca Q-

2 [h

^

^

-T

_

% ^A-*-

[

I

12

1

• i .

24 36 Day of harvest

.

J

48

1 60

F i g u r e 12.15 Use of hyphal material as sole N source by Vbcc/n/um macrocarpon mycorrhizal w i t h Hymenoscyphus ericae (M) o r non-mycorrhizal ( N M ) . (a) G r o w t h of plants w i t h (+IHE) o r w i t h o u t ( - I H E ) necromass of H. ericae as sole N source, (b) G r o w t h of plants w i t h ( + C W F ) and w i t h o u t (-CWF) pure cell wall fraction of H. ericae as sole N source. Vertical bars represent least significant differences at P = < 0.05. * * * , Result of one-way A N O V A of M + substrate vs. N M + substrate: P = < 0.001. From Read and Kerley (1995), w i t h permission.

has been made towards characterization of the phosphomonoesterase enzymes involved in release of P from these sources. It was established some time ago that activity was greatest under conditions of low external P concentration (Pearson and Read, 1975). The production of two isoenzymes has subsequently been demonstrated (Straker and Mitchell, 1986; Straker et a/., 1989), the activities of both of which are stimulated in the presence of Fe^"^ at low concentration. The low molecular weight form of the enzyme that appears to be produced selectively

Ericoid mycorrhizas

343

under conditions of low P supply (Lemoine et ah, 1992) is known to be able to hydrolyse such compounds as ATP, ADP and AMP (Straker and Mitchell, 1986). In a comparative analysis of the extracellular acid phosphatase of endophytes isolated from a number of plant species, Straker and Mitchell (1986) found highest activity in a fungal isolate from the South African species Erica hispidula. Soluble Pi made available by mineralization could subsequently be absorbed by low- and high-affinity uptake systems, depending on the concentration (Straker and Mitchell, 1987). By ultrastructural localization using immunogold labelling, Straker et al. (1989) demonstrated that the high molecular weight phosphatase was exclusively associated with the walls and septa of living hyphae. There is some evidence that activity of this wall-bound enzyme is at its greatest in hyphae close to the root and that it is much reduced as distance from the plant increases (Gianinazzi-Pearson et al, 1986). This could be due to lower concentrations of P in the vicinity of the root surface or to control of enzyme activity being exerted by the plant. In any event, maximal expression of activity in the rhizosphere could, especially in view of the low specificity shown by acid phosphatases towards phosphomonoesters, provide the plant with access to P from a range of organic sources. There is increasing recognition of the quantitative importance of phosphodiesters in acid organic soils. Leake and Miles (1996) have investigated phosphodiesterase production by H. ericae, together with the ability of the fungus to use DNA as a sole source of P. The fungus grew well on this compound and achieved greater mycelial dry weight than on orthophosphate (Fig. 12.16). At least part of the enzyme activity was attributed to the exonuclease 5 -nucleotide diesterase. The p H optimum for the activity was between 4.0 and 5.5. When P was supplied to mycorrhizal and nonmycorrhizal plants of Vaccinium macrocarpon in the form of purified nuclei, only those colonized by H. ericae absorbed P in appreciable amounts (Myers and Leake, 1996; Fig. 12.17). Such results confirm the need to consider diesters as well as monoesters as possible sources of P for mycorrhizal plants in the field. In the experiments of Leake and Miles (1996) the fungus was supplied with mineral N in order that phosphatase activity alone could be examined. In view of the proteolytic capabilities of H. ericae, described earlier, nucleotides could provide N as well as P sources for plants with ericoid mycorrhizas. In addition to their involvement in the capture and transport of the major nutrients N and P, ericoid mycorrhizal fungi may play a role in acquisition of other elements by the plants. Their influence upon Fe nutrition has been studied most intensively. The chemical form, and hence availability, of this element can change drastically in heathland soils where redox conditions fluctuate with seasonal changes in water balance. Shaw et al. (1990) emphasized that under such circumstances regulation of supply of the element across a wide range of concentrations was likely to be more important than acquisition or exclusion as individual processes. H. ericae was able to sustain productivity over a range of external Fe concentrations typical of those to be expected in heathland soils. At the highest external Fe concentration (144 |ig ml"^) the concentration in the mycelium was raised to 6000 |Lig mg~^dry wt. This suggests that the fungus may retain the potential to regulate plant Fe consumption, even when exposed to soil solutions which are enriched in the element. Regulation appears to be achieved by processes which control the affinity of the fungus for Fe. Both in pure culture and in colonized roots of Vaccinium macrocarpon or Calluna vulgaris, H. ericae showed an extra-

344

Mycorrhizas in the Ericales

100 n o>

^

80

4-»

SI D) 0)

^ >»

60

1.

•D

E

40

3

0)

o >»

20

2

Time (days) Figure 12.16 Growth of mycelium of Hymenoscyphus ericae on DNA ( • ) or orthophosphate (5 mM P) ( • ) compared with growth with no P supply ( A ) . Vertical bars are standard errors of the mean and letter codes (a-c) indicate significant differences between means at each harvest derived from Tukey's tests. From Leake and Miles (1996), with permission. 0.25

NM M No Phosphorus

NM M Orthophosphate

NM M Nuclei

Figure 12.17 P content of roots (solid bars) and shoots (cross-hatched bars) of mycorrhizal (M) and non-mycorrhizal (NM) plants of Vaccinium macrocarpon after growth for 28 weeks on nutrient agar containing no added P, orthophosphate or salmon sperm nuclei. Different letters indicate significant differences P < 0.05 according to Tukey's test. From Myers and Leake (1996), with permission.

Ericoid mycorrhizas

345

ordinarily high affinity for Fe at low concentrations {c. 2.0 |ig ml"^), which was progressively reduced as the external concentration of Fe increased. The high affinity appears to be explained by the production of Fe-specific siderophores (Schuler and Haselwandter, 1988; Federspiel et al, 1991), some of which have now been chemically characterized. The predominant siderophore in H. ericae and O. griseum is ferricrocin, while in a related endophyte isolated from the calcicolous shrub Rhodothamnus chamaecistus it was fusigen (Haselwandter et al., 1992; Haselwandter, 1995). Such siderophores may be involved in facilitating the significant increases in the specific absorption rate of Fe seen in mycorrhizal plants of Calluna grown in the presence of Ca salts (Leake et a/., 1990), and may be particularly important in those plants such as R. chamaecistus which typically occur in relatively Ca-rich environments. The mycorrhizal fungus also appears to play a role in determining the response of colonized plants to the non-essential (and potentially toxic) element Al. It has a remarkable tolerance of the presence of this metal in solution, no inhibition of mycelial yield being observed in cultures containing 800 mg P^ of Al^"^ (Burt et ah, 1986). Non-mycorrhizal plants of Vaccinium when exposed to solution containing half this concentration showed considerable inhibition of root development. A system of 'stilt roots' was produced as a result of the failure of newly formed laterals to penetrate the medium containing Al (Read and Kerley, 1995). Plants colonized by H. ericae produce normal root systems at the same concentration of Al. Similar effects are seen when ericaceous plants are exposed to Zn and Cu (Bradley et al, 1981, 1982). In these cases improvement of growth of C. vulgaris, V. macrocarpon and Rhododendron ponticum, associated with colonization by H. ericae, appears to arise by a process of avoidance, involving sequestration of the metals in the fungal mycelia and consequent reduction of transfer to the shoot. The apparent indifference of the ericoid fungi to high concentrations of metals such as Fe, Al, Cu and Zn, and their ability to accumulate these elements without adverse consequences, raises questions as to their localization on, or in, the mycelium. In the case of the extraradical hyphae, Bradley et al. (1982) suggested that the metal ions might be bound to carboxyl groups of the hyphal walls. By excluding the metal from the intracellular environment, this feature would avoid toxicity to the fungus as well as the plant. More recently, Denny and Ridge (1995) have provided indirect evidence for binding of Zn ions to the mucilaginous sheath surrounding the hyphae. The high concentrations of metals found in colonized roots indicate that some transfer to the plant or at least to the fungal component of the mycorrhiza also occurs. Ultrastructural observations reveal an abundance of pectin-like substances in the interfacial matrix separating plant and fungal plasmamembrane in the colonized epidermal cells (Duddridge and Read, 1982b). These molecules would also provide potential binding sites. None of these suggestions exclude the possibility of some accumulation of metals in fungal vacuoles and there is clearly a need for the application of accurate localization techniques to answer the questions precisely. Conclusions Emphasis in research on ericoid mycorrhizas has turned from speculation about the nature of colonization processes to experimental analysis of the function of the

346

Mycorrhizas in the Ericales

symbiosis in heathland environments. Some debate continues as to the taxonomic position of the hingi which normally form this type of mycorrhiza, although there is common consent that they are ascomycetes. One of these, H. ericae, has been identified and used extensively in experiments on function of the mycorrhizas. Members of the genus Oidiodendron are also regularly isolated from ericoid mycorrhizas. The application of molecular techniques promises to increase our knowledge of the taxonomy and interrelationships of ericoid endophytes. It is already clear that there is considerable genetic diversity amongst isolates, even those taken from the same root, but the taxonomic and functional significance of this diversity remains to be elucidated. Detailed analysis by light, transmission and scanning electron microscopy has gone some way to elucidating the major events associated with attachment, penetration and internal proliferation of the fungus in the epidermal cells of the root. Cytohistochemical and molecular tools are now being deployed to enable localization of some of the processes involved in nutrient transfer. These methods promise exciting increases in our understanding of these fundamentally important events. Experimental examination of the nutrition of ericoid endophytes growing in monoxenic culture have revealed that they possess a wide range of saprotrophic capabilities. A number of simple and complex organic forms of N and P are readily assimilated by the fungi. When grown in symbiosis with ericaceous plants, the fimgus facilitates transfer of the nutrient originally contained in the polymers, to the plant. Progress has been made both towards characterization of the fungal exoenzymes involved in nutrient mobilization and understanding of the environmental conditions leading to their induction and repression. In addition to the direct nutritional benefits arising from colonization, there are other advantages that are likely to be of ecological significance. The ability of the fungal partner to sequester metal ions that are toxic to the plant appears to be important, since this excludes the toxins from the shoots where they would interfere with photosynthesis. This combination of nutritional and non-nutritional attributes of mycorrhizal associations in with members of the Ericales contributes significantly to the ability of these plants to grow on heathland soils which are characterized by their high organic content and C:N ratio and low pH.

13 Orchid mycorrhizas

Introduction The fact that members of the Orchidaceae are mycorrhizal has been known for well over 100 years. Both Wahrlich (1886) and Janse (1897) surveyed a large number of temperate and tropical species and noted the regular occurrence of fungal colonization of the roots. It was Bernard who carried out the first major experimental work on orchid mycorrhizas which encompassed isolation and identification of the fungi involved, studies of symbiotic and asymbiotic germination, consideration of the unstable nature of the symbiosis and the plant defence responses, as well as the significance of such studies to horticultural production (e.g. Bernard, 1899, 1901, 1903,1904a,b,c, 1905,1907,1908,1909a,b, 1911; for a more complete list see Rayner, 1927). He isolated and identified Rhizoctonia orchid fungi (often referred to as endophytes) which he found could grow indefinitely on nutrient media and would form typical mycorrhizas in germination studies. Much of what Bernard observed has been confirmed in later research (see Burgeff, 1936; Hadley, 1982; Arditti, 1992). The orchids have considerable fascination for scientists and amateurs alike because of their great diversity, the extraordinary form and beauty of their flowers, and their varied and sometimes bizarre methods of pollination. Consequently, they are horticulturally very important. In addition. Vanilla is grown commercially for the flavouring agent vanilla produced from its pods and has an annual value of perhaps $1 billion (Prince and Gunson, 1994). Many orchid products are believed to have medicinal properties and are used as aphrodisiacs, treatments for sores, emetics and vermifuges (Arditti, 1966; Lawler, 1984). The diversity within the Orchidaceae is perhaps not surprising, as the family is one of the largest (up to 17500 spp.; Mabberley, 1987). The family is cosmopolitan and believed to be undergoing rapid diversification and speciation, with members found in a vast range of habitats from tropical forests to deserts. The species include a varied range of life forms, including many terrestrial and epiphytic species and a few lianas, like Vanilla. The smallest orchids, Rhizanthella spp., are completely subterranean, including their flowerbuds (George, 1980; Dixon et ah, 1990). Whereas most adult orchids are green and photosynthetic, the underground orchids, as well as perhaps 200 other species in 45 genera, are achlorophyllous and are clearly myco-heterotrophic (Leake, 1994). Indeed, all orchid species pass through a prolonged seedling stage

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during which they are unable to photosynthesize and depend on an exogenous supply of carbohydrate, provided in nature by the mycorrhizal fungi. The fungi are probably also involved in uptake of mineral nutrients but this has not been extensively investigated and the physiological interactions between the fungi and adult photosynthetic species are not well understood. For the myco-heterotrophs it is assumed that the fungus continues to supply carbohydrate and minerals throughout the life of the plant. So far there is no evidence of the plant supplying anything to the fungus in the mycorrhizal interaction, so that the symbiosis cannot be regarded as mutualistic. There is a vast literature on the germination of orchid seeds, which are very small and undifferentiated (0.3-14 |ig) and have few reserves. Colonization by an appropriate fungus is a requirement for germination in nature, although many species, particularly epiphytes, can develop fully if supplied with an exogenous source of sugar (asymbiotic culture). Symbiotic germination and development are only successful if the fungus has access to a soluble or insoluble source of carbohydrate, which it absorbs and translocates to the orchid. Much of the impetus for the work on germination has come from the horticultural industries which, after some initial failures of symbiotic propagation (e.g. see Bernard, 1904b, 1907), have mainly adopted asymbiotic methods. The early part of the twentieth century saw controversy about whether or not symbiosis was a prerequisite to germination and complete development of the plants (Knudson, 1922, 1927, 1930; Arditti, 1967, 1992; Soutamire, 1974; Withner, 1974). There is no doubt that some orchid species and hybrids can be grown to maturity and flowering in a non-mycorrhizal condition (Laelia-Cattleya: Knudson, 1930; Miltonia sp.: Bultel, 1926) but in nature this does not occur. Difficulties in transferring asymbiotic plantlets to soil can be encountered (Clements et al., 1986) and numerous species will not germinate in the absence of an appropriate mycorrhizal fungus. These are not widely grown or used in hybridization, but some are rare or threatened species (including many myco-heterotrophs), for which knowledge of the conditions favourable to symbiotic germination may be a prerequisite for conservation. Land clearance and altered land management practices are having an impact on wild orchid populations, so that in many places conservation is an important issue, and one for which knowledge of the role played by mycorrhizal fungi in germination may contribute (Clements, 1982a; Rasmussen and Rasmussen, 1991; Zettler and Mclnnes, 1992; Rasmussen and Wigham, 1994).

The Fungi of Orchid Mycorrhizas Isolation and Identity Fungi are readily isolated from roots of many adult orchids using unspecialized media and can be grown easily in pure culture. Bernard (1904a) was the first to record regular isolations of the Rhizoctonia-like fungi which are the main causal agents of orchid mycorrhizas (Fig. 13.1). Although the surveys of orchid fungi in different geographical areas have been limited, the species obtained from Australia, Italy, Japan and Canada appear similar (Warcup, 1981; Marchisio et al., 1985;

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351

Figure 13.1 Drawing of a typical orchid mycorrhizal fungus, Rhizoctonia repens {Tulasnella calospora)y isolated In pure culture. From Bernard (1909).

Terashita 1985; Currah, 1987; Currah et al, 1987,1990; Ramsay et ah, 1987; Terashita and Chuman, 1987; Clements, 1988; Currah and Zelmer, 1992; Richardson et al, 1993; Masuhara and Katsuya, 1994). Techniques employed for isolation include plating fragments of surface-sterilized root on nutrient agar (with attendant problems of contamination and difficulties of relating the fungi to actual mycorrhizaforming species) and careful separation and plating of individual fungal pelotons (intracellular hyphal coils; see below). Growth from the latter can be followed microscopically and in consequence there is considerable confidence that the isolates originated from mycorrhizas. In many cases the fungi have been subjected to germination tests with orchid seeds providing more information on their mycorrhizal status (e.g. Harvais and Hadley, 1967; Smreciu and Currah, 1989). A very large number of the fungi have been referred to the form genus Rhizoctonia (e.g. Burgeff, 1936; Warcup and Talbot, 1967, 1970, 1980; Marchisio et al, 1985; Currah, 1987; Ramsay et ah, 1987). These include Rhizoctonia repens (perfect stage Tulasnella calospora) which was much used by Bernard, R. goodyerae-repentis {Ceratobasidium cornigerum) isolated from Goodyera repens and R. solani {Thanatephorus cucumeris), a very active parasite of herbaceous plants isolated from Dactylorchis purpurella (Downie, 1957,1959b; Masuhara et ah, 1993). Some rhizoctonias have now been transferred to other genera, such as Ceratorhiza for those with presumed perfect stages in Ceratobasidium (see Currah and Zelmer, 1992). Perfect stages of many Rhizoctonia isolates have been obtained, especially by Warcup and Talbot (19621970; Warcup, 1981, 1988; and see Currah, 1987). The basidiomycete genera to which they belong include Thanatephorus (Corticium), Ceratobasidium, Ypsilonidium, Sebacina and Tulasnella and a preliminary key for these is provided by Currah and Zelmer (1992). Whereas Marchisio et al. (1985) failed to obtain perfect stages of the fungi they isolated, they were able to classify them into three groups and considered that a number of binucleate and multinucleate isolates could be tentatively assigned to the Rhizoctonia solani complex {Thanatephorus and Ceratobasidium). The underground orchid, Rhizanthella gardneri, is symbiotic with Rhizoctonia species and at least one of these appears to form ectomycorrhizas with the

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myrtaceous shrub Melaleuca, with which R. gardneri is consistently associated (Warcup, 1985). Whether ectomycorrhizas are commonly formed by rhizoctonias is not currently known but the application of molecular methods of identification has the potential to determine this relatively rapidly. These methods may also solve problems of identification of many orchid endophytes arising from the difficulties of inducing perfect stages in culture. Conidia-producing hyphomycetes have also been isolated from orchid roots, although their symbiotic abilities have not been confirmed in all cases (Currah et al, 1987, 1990; Richardson et al, 1993). Obvious basidiomycetous mycelia bearing clamp connections have been obtained from many achlorophyllous or leafless orchids. Some are agarics and bracket fungi, for example, Marasmius coniatus, Xerotus javanicus, Hymenochaete sp., Armillaria mellea (agg.), Fomes sp. and Favolaschia dybowskyana (Kusano, 1911a,b; Burgeff, 1932, 1936; Hamada, 1940; Hamada and Nakamura, 1963; Jonsson and Nylund, 1979) and most have active woodrotting or parasitic life styles. Rhizomorphs of A. mellea have been found growing from mycorrhizal roots of some Gastrodia spp. (e.g. Kusano, 1911a,b; Campbell, 1962; Masuhara, unpublished; see also Masuhara and Katsuya, 1991). Warcup (1981) tested 65 wood-rotting fungi with seeds of Galeola foliata, an achlorophyllous liane. Seven white-rot species, including Coriolus versicolor and two species of Fomes, successfully encouraged germination, as did three species isolated from G. sesamoides. As with the ectomycorrhizal Rhizoctonia, there is a distinct possibility of the fimgi forming two types of mycorrhizas, because Zelmer and Currah (1995) have demonstrated that Corallorhiza and Pinus are associated with a common, clamp-forming fungus. Recently, molecular identification has indicated that the fungal associates of the achlorophyllous orchid Cephalanthera austinae belong to the genera Thelephora and Tomentella, and were present in ectomycorrhizal root tips in the cores below the orchid plants (Taylor and Bruns, personal commimication). The implications are twofold: the orchid may be epiparasitic (myco-heterotrophic) on the ectomycorrhizal plant; and, if so, the structure of the mycorrhiza is clearly modified by the plant, with typical pelotons in the orchid, and sheath and Hartig net in the ectomycorrhiza. Similar myco-heterotrophic associations occur in members of the Arbutoideae (see Chapter 11). In the main, the isolations have been made from root material, with protocorms being used only rarely, for the obvious reason that they are very small and are difficult to find in the field (but see Masuhara and Katsuya, 1989, 1991, 1994; Rasmussen et al., 1990a,b; Rasmussen and Wigham, 1994). The assumption has been that fungi from roots are the same as those colonizing protocorms, so that tests of symbiotic ability with the latter were believed to be valid for both and useful to determine specificity. The results have been somewhat confusing, and there is clearly a large number of root-colonizing species which do not form mycorrhizal associations with the protocorms of the orchid from which they were isolated (at least under the laboratory conditions of the germination tests), although they may do so with other orchid species (Harvais and Hadley, 1967; Milligan and Williams, 1988; Smreciu and Currah, 1989; Masuhara and Katsuya, 1994). For some associations the explanation for this discrepancy may lie in the need for some orchids to associate with different fungi at different stages of development. For example, early stages of protocorm development of Gastrodia elata (Xu and Mu, 1988; Xu et al., 1989, reported in Leake, 1994) require typical

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Rhizoctonia-like fungi, but full development of the plant only follows if secondary colonization by Armillaria takes place. Seedlings of G. septentrionalis are also colonized by Rhizoctonia species some of which form coils, suggesting a mycorrhizal interaction (Masuhara and Katsuya, 1991). Whether fungal succession is common during the life of other orchid species is not known at present, but it has the potential to add another level of complexity to the determination of the specificity of orchid-fungus interactions. Nutritional Ciiaracteristics All orchid mycorrhizal fungi are able to obtain carbohydrate from outside the orchid, although there is some diversity of source. Most are relatively fast-growing saprophytes which, in culture, can use complex polymers such as starch, pectin and cellulose and occasionally lignin, as well as soluble sugars (Burgeff, 1936; Harley, 1969; Perombelon and Hadley, 1965; Smith, 1966; Hadley and Ong, 1978). Others, such as Rhizoctonia solani and Armillaria mellea, grow saprophytically in culture but are better known as parasites. There is also mounting evidence of mycelial links between orchids and ectomycorrhizal plant species. Warcup (1985, 1991) has demonstrated that a Rhizoctonia which forms orchid mycorrhizas with Rhizanthella is also ectomycorrhizal with Melaleuca uncinata, while Zelmer and Currah (1995) have shown that Corallorhiza trifida is linked to ectomycorrhizal Pinus contorta by a slow-growing, bright yellow, clamp-forming fungus. In these examples, as well as the case of Cephalanthera austinae mentioned above, the implication is that both fungus and orchid are using organic C from the photosynthetic mycorrhizal species. Orchid fungi have unspecialized requirements for nutrients other than C sources. Most can use a wide range of N compounds, at least in pure culture (Hollander, in Burgeff, 1936; see also Arditti, 1979,1992) and it seems likely that organic sources of N, and possibly also P, are important for these fungi growing in soil, although direct investigations have not been carried out. The significance in nature of the effects of B vitamins, yeast extract and root exudates which have been observed in culture, has not been followed up, although it may have relevance to the horticultural production of orchids by symbiotic methods (Vermeulen, 1946; de Silva and Wood, 1964; Perombelon and Hadley, 1965; Hadley and Ong, 1978).

Seed Germination and Protocorm Development Asymbiotic Germination All orchids, whether they are chlorophyllous or achlorophyllous as adults, pass through a phase where they are non-photosynthetic and dependent on an external supply of nutrients including organic C. The minute seeds (Fig. 13.2) contain very small amounts of high-energy protein and lipid, and very little sugar (Harrison, 1977; Arditti, 1979; Manning and van Standen, 1987; Richardson et al, 1992). Some species also contain small numbers of starch grains (Hadley and Williamson, 1971; Purves and Hadley, 1975). If the seeds are spread on a moist substratum the

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354

.V. Hernanl. ./,•/.

Phaldenopsis. Figure 13.2 Stages in the development of PhalaenopsiSy showing the minute seeds (I), undifferentiated embryos (2 and 3) and mycorrhizal protocorm (5). Rhizolds produced by germinating protocorms are shown (10 and 13). From Bernard (1909).

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Orchid mycorrhizas

Seed

Symbiotic infection with Tulosnelic colospofQ

Uninfected

Figure 13.3 Development of Spathigottis plicata after 5 weeks' growth on two C sources, in relation to colonization by Tulasnella calospora. From Hadley (1969), with permission.

undifferentiated embryos (Figs 13.2, 13.3 and 13.4a) absorb water, swell slightly and may burst the testa, and sometimes produce epidermal hairs. The embryo does not develop further unless it receives an exogenous supply of carbohydrate or is infected by a compatible mycorrhizal fungus. In some species, vitamins or growth factors are also required for the embryos to develop asymbiotically. Lipid reserves are not mobilized, probably because of the absence of glyoxysomes and the very low concentrations of endogenous sugars (Harrison, 1977; Manning and van Standen, 1987). Starch is not hydrolysed in those species containing it, and like the lipid and protein, it does not support ongoing protocorm development. If sugars (such as glucose, fructose, sucrose, maltose and trehalose; see Ernst, 1967; Ernst et al, 1971; Smith, 1973; Nakamura, 1982) are supplied, together with vitamins for those that require them, then many species will develop further, and although growth is slow, mature plants can be obtained. The seeds of Disa, Disperis, Cymbidium and Huttonia, when supplied with sucrose, synthesized small amounts of starch, developed glyoxysomes and began to use endogenous lipid (Manning and van Standen, 1987). In nature, continuing supplies of soluble sugars, vitamins, amino acids and growth factors are not available from the soil and the conclusion reached by Bernard (1904c) that the fungus is required for successful germination, remains valid. Fungal colonization is required both to stimulate gluconeogenesis and mobilization of reserves and to provide ongoing nutritional support, before photosynthesis commences. Mycorrhizal Colonization of Protocorms Most of the experimental work on fungal colonization of orchid tissue has been carried out using protocorms in axenic culture. The interaction between embryo and fungus can have three basic outcomes: (a) a mycorrhizal interaction, with the

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Figure 13.4 Light microscopy of orchid mycorrhizas. (a) Protocorm of Dactylorchis sambucina inoculated with Ceratobasidium cereale. Bar, I mm. From Smreciu and Currah (1989), with permission, (b) Transverse section through a root of Tipularia discolor, showing extensive infection in the cortex, but fungus free zones adjacent to the stele. Bar = 0.44mm. From Rasmussen, 1995.

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formation of pelotons (Fig. 13.5a) which are later lysed; (b) a parasitic interaction, in which the orchid cells are invaded by relatively disorganized hyphal growth and death of the protocorm results; and (c) a resistant, rejection response in which the fungus is excluded from the orchid tissues (Hadley, 1970; Beyrle ei ah, 1995). These interactions may occur simultaneously in a population of protocorms, emphasizing the dynamic and relatively unstable nature of the plant-fungus association. The proportion in any category is influenced by nutritional and environmental factors, at least under laboratory culture conditions. The imbibed and swollen embryos are rapidly colonized by hyphae of suitable fungi. The most usual route appears to be via the suspensor, but in some species the fungus enters via epidermal hairs (Williamson and Hadley, 1970; Hadley, 1982; Clements, 1988; Peterson and Currah, 1990). There is no good evidence for specific mechanisms of attraction between the symbionts or directional growth of hyphae prior to penetration (Clements, 1988). As it penetrates the cell of the embryo, the infecting hypha invaginates the plasma membrane of the orchid cell and becomes surrounded by a thin layer of cytoplasm (Fig. 13.5b) which remains healthy and continues to show protoplasmic streaming (Williamson and Hadley, 1970). In electron micrographs, the plant cells appear physiologically active at all stages of colonization and contain numerous mitochondria, well developed endoplasmic reticulum, Golgi bodies and vacuoles of various sizes (Hadley et ah, 1971; StruUu and Gourret, 1974; Dexheimer and Serrigny, 1983). The nuclei of both colonized and non-colonized cells of a mycorrhizal protocorm are obviously hypertrophied (Burgeff, 1932), and are reported to have higher DNA contents than those of uninfected ones (Alvarez, 1968; Williamson and Hadley, 1969; Williamson, 1970). Another important physiological change is the disappearance of starch grains from the colonized cells and often from other cells in the mycorrhizal protocorm. This is clearly associated with the presence of the fungus, because it does not happen in axenically grown protocorms (Burgeff, 1959; Peterson and Currah, 1990; Beyrle et al, 1995). Within the protocorm the fungus spreads from cell to cell so that the basal region becomes extensively colonized. Hyphae penetrating the plant cell wall narrow and do not distort the wall, nor induce thickening (Peterson and Currah, 1990; Beyrle ei ah, 1995; and see Fig. 13.5d), suggesting that localized hydrolysis rather than pressure is important in penetration. Growth and anastomosis of the intracellular hyphae result in the formation of complex pelotons which very much increase the interfacial area between the symbionts (Fig. 13.5a). In recently colonized cells few vacuoles are present in the hyphae, but in mature coils the cytoplasm of the fungus becomes vacuolated and contains nuclei, mitochondria, ribosomes, lipid globules and glycogen rosettes, but little endoplasmic reticulum (Hadley et al, 1971; Strullu and Gourret, 1974; Hadley, 1975; Peterson and Currah, 1990). The walls of the fungus do not appear to undergo changes during primary colonization and development of the pelotons. They are described as being formed of two layers: an electron-dense inner layer and an outer less dense, more 'flocculent' layer which is always present and of variable thickness (Hadley et ah, 1971; Strullu and Gourret, 1974). It was at first thought that the outer layer was of fungal origin, but Hadley (1975) concluded that it was comparable to the interfacial matrix in other plantfungus interactions. The composition of the walls has not been analysed as thoroughly as in vesicular-arbuscular (VA) mycorrhizas. However, Peterson and Currah

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Figure 13.5 (Caption opposite)

Orchid mycorrhizas

Figure 13.5 Ultrastructure of orchid mycorrhiza. (a) Scanning electron micrograph of pelotons (p) of Rhizoctonia in a protocorm of Orchis morio. Bar, 100 |im. From Beyrle et al. (1995), with permission, (b) Hyphae of Rhizoctonia in the host cytoplasm of a cell of Dactylorhiza purpurella. Both the living hyphae (h) and dead collapsed hyphae (dh) are surrounded by an encasement layer (e), which is probably of host origin. Paramural bodies (pb) occur in the interfacial matrix between the host plasma membrane and the encasement layer. Host cytoplasm contains endoplasmic reticulum (er), mitochondria (m) and a crystal (X). Host vacuole (vac) is also indicated, (c) Inset: Detail of the endoplasmic reticulum and encasement layer. From Hadley (1975), with permission, (d) Penetration of a host cell wall (w) by a fungal hypha (h) in a normal mycorrhizal interaction between Goodyera repens and Ceratobasidium cereale. Note the dissolution of the host wall (arrowed). Bar, l.0|im. From Peterson and Currah (1990), with permission, (e) Resistant response of Orchis morio to penetration by Rhizoctor)ia under culture conditions in which the fungus is excluded and fails to form a mycorrhiza. Note the thickening of the plant wall (arrowed) beneath the penetration peg (p). Fungal hypha (h), host wall (w). Bar, 5.0 |xm. From Beyrle et al. (1995), with permission. (1990) suggest that changes in the reaction of walls to cellufluor (see below) indicate that chitin staining is normally blocked, but that during lysis of the pelotons the blocking material is removed, resulting in a very strong reaction. They pointed out that many polysaccharides, including chitin, react strongly with cellufluor and did not agree with the conclusions of Barroso and Pais (1985) that the matrix material was cellulose. Hadley and his colleagues (Hadley et ah, 1971; Hadley, 1975) observed that the fungal plasma membrane was invaginated in places to form vesicles and tubules.

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Orchid mycorrhizas

while the fungal wall bore protuberances adjacent to the plant (Dactylorhiza purpurella) plasma membrane. Both these features increase the surface area across which transfer of nutrients might take place. However, Peterson and Currah (1990) did not find similar structures in protocorms of Goodyera repens colonized by Ceratobasidium cereale. Other modifications in the interfacial region include the presence of ATPases on both plant and fungal membranes, with the former remaining active in the plant plasma membrane surrounding the collapsing pelotons. Neutral phosphatase activity in the interface and on the plant membrane surrounding active pelotons may be involved with the synthesis of interfacial matrix by the plant (Serrigny and Dexheimer, 1985). The intracellular pelotons have a limited life even in the mycorrhizal interaction. Some of the earliest workers (Wahrlich, 1886; Janse, 1897) noted the characteristic clumping of the hyphal coils, which was thought by Bernard (1905b, 1909b) to be the result of defensive phagocytosis. Electron microscopy has revealed that during lysis (sometimes called digestion) the hyphal contents become disorganized and the walls take on flattened or angular profiles. In the final stages the wall material clumps together, forming an irregular mass (Figs 13.2, 13.4b). The plasma membrane of the plant remains intact and is separated from the clumped fungal material by an electron-lucent layer which has now been shown to contain callose, pectins and a small amount of cellulose (Peterson and Currah, 1990). During this process the plant cells remain alive and active, and may be recolonized by hyphae either apparently surviving the lytic process or invading from an adjacent cell (Burgeff, 1936; Burges, 1939; StruUu and Gourret, 1974). The timing of colonization, development of pelotons and subsequent lysis have been followed in a number of orchid-fungus combinations, usually with potentially photosynthetic orchid species. The myco-heterotrophic orchids have proved difficult to germinate and much less is known about them. In culture, fungal penetration occurs within a few days of the symbionts coming into contact. For Goodyera repens, MoUison (1943) recorded that suspensors were invaded by Rhizoctonia goodyerae-repentis {Ceratobasidium cornigerum) within 5 days, pelotons were formed within 7 days and had lysed by 11 days. The symbiotic stimulus to protocorm growth occurred before lysis of pelotons. She also noted that if the protocorms were already imbibed, the process occurred more quickly. Hadley and Williamson (1971) followed colonization in Dactylorhiza purpurella continuously for several days, using an inverted slide technique (Fig. 13.6). There was considerable variation within the population but, on average, colonization via the epidermal hairs occurred within 14.5 hours of the first contact and pelotons were formed by 29 hours. The number of pelotons and the number of lysed pelotons increased linearly with time. Lysis of pelotons was observed by 3 0 ^ 0 hours and continuous observation of a single, peloton-containing cell showed that lysis took less than 24 hours. The growth of protocorms (measured as volume) was followed at the same time as colonization and although the number of pelotons was not linearly related to the rate of growth there was evidence that the mycorrhizal growth stimulus preceded any lysis of protocorms (compare Fig 13.6e and f), thus confirming MoUison's (1943) observations. The causes of the hyphal collapse and lysis are not really known. Many workers have believed it to be the result of activity of the cells of the orchid and a manifestation of a defence reaction against fungal invasion (e.g. Burges, 1939), or

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a means by which the orchid cell causes a release of nutrients from the fungus. Evidence still does not clearly distinguish between these two hypotheses but on balance the former seems the most likely, especially in view of the results cited above showing that a growth stimulus occurs before any lysis is apparent (Fig. 13.6). Williamson (1973) showed that acid phosphatase activity, often thought to be a marker for lysosome activity, increases in cells where hyphal collapse is taking place. Subsequently, it was shown that this activity was never found in the interface of the plant with young active hyphae but, even in the same cell, was markedly increased around old, highly vacuolated hyphae. These old hyphae themselves contained acid phosphatases, which disappeared when the hyphae finally collapsed (Dexheimer and Serrigny, 1983; Serrigny and Dexheimer, 1986). There are reports of chitinase and p(l,3)glucanase activity in corms of Gastrodia elata which, by comparison with plant-pathogen interactions, might be an indication of deployment of defence responses (Zengming and Zhong, 1990). Increased activities of oxidase systems (e.g. polyphenol oxidase, catalase and ascorbic acid oxidase) have been observed during lysis but their roles in the process are not clear (Blakeman et al, 1976; Pais and Barroso, 1983). Orchids also produce fungitoxic phytoalexins such as orchinol, lorroglossol and hircinol (Stoessl and Arditti, 1984), mainly found in tubers where they are believed to be involved in controlling or excluding fungal colonization. Recently, the presence of orchinol in protocorms of Orchis morio has been confirmed, so that a role in the more dynamic stages of plant-fungus interaction is possible (Beyrle et ah, 1995). Once colonization is established in a mycorrhizal (rather than parasitic) pattern, growth of protocorms proceeds rapidly, with different phases of colonization restricted to well defined regions. The protocorms differentiate into distinct tissues, including uncolonized storage cortex, vascular tissue, shoot meristem and (in the potentially photosynthetic orchids used) young leaves, as well as basal colonized regions contain both living and lysed pelotons (see Figs 13.2 and 13.7). Fungal hyphae connect the internal protocorm to the substrate, with the only entry/exit points being the suspensor or the epidermal hairs (Clements, 1988). The growth of mycorrhizal protocorms is generally much faster than asymbiotic protocorms raised under the same conditions. For example, plantlets of Goodyera repens, colonized by Ceratobasidium cornigerum, reached a height of 5-10 cm in 12 months on cellulose agar, whereas asymbiotic protocorms were only 2-3 cm tall (Alexander and Hadley, 1984; Fig. 13.3). The sequence and timing of different stages of development have rarely been followed under natural conditions. However, preliminary observations using seeds of several terrestrial species buried in mesh bags, suggest that colonization probably occurs in spring-summer and may be much slower than in laboratory culture (Rasmussen and Wigham, 1994).

Transfer of Nutrients from Fungus to Orchid Carbon The increase in growth following mycorrhizal colonization of protocorms is based on the fungi supplying organic C to the orchid. The ability of many saprophytic

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•

Olh

20

40 No. of pttlotons

60

80

Figure 13.6 Development of the interaction between Dactylorhiza purpurella and Rhizoctonia sp. in inverted slide cultures, (a) Grovsrth of protocorms when grown on water agar in the absence of a fungal symbiont. (b) Growth on water agar following inoculation, (c) Growth on Pfeffer's medium plus 0.1% glucose, following inoculation, (d) Growth on Pfeffer's medium plus cellulose, following inoculation, (e) Relationship between time after contact and development of pelotons. P, Mean penetration time, 14.5 h; D, first digested pelotons observed, 30-40 h. (f) Relationship between time after contact and volume of protocorms. From Hadley and Williamson (1971), with permission.

orchid mycorrhizal fungi to hydrolyse insoluble carbohydrates means that the external mycelium has access to abundant supplies in the soil that can be transported to the associated orchids. The orchid mycorrhizal fungi, symbiotic (parasitic or mutualistic) with other plants, can obtain a continuing supply of recent photosynthate that again can be delivered to orchids linked into the same mycelium. Much of the work on symbiotic growth of orchid protocorms has employed soluble sugars in the medium that can be used by both fungus and protocorm. However, growth responses to mycorrhiza establishment are regularly obtained on oatmeal-, starch- or cellulose-containing media, where hydrolysis by the fungus would be a prerequisite for use by either symbiont and on which no growth of asymbiotic protocorms occurs. Indeed, establishment of a mycorrhizal interaction and continued protocorm growth is more certain if the fungus is provided with cellulose than if presented with a medium rich in soluble carbohydrate (Figs 13.3, 13.6). In the latter case, a parasitic interaction is more likely to occur (Smith, 1966; Harvais and Hadley, 1967; Hadley, 1969). As with other mycorrhizal systems, translocation of nutrients is a prerequisite for effective delivery to the symbiotic partner. The first evidence of translocating ability in orchid endophytes was provided by Beau (1920) using a split-plate system which

364

Orchid mycorrhizas EMBRYO

DEVELOPING PROTOCORM

SEEDLING

Rhizoid Suspensor 1

-^ ^""^

I Meristem

Epidermis Outer cortex Inner digestion cortex Inner storage cortex Vascular tissue

Direction of fungal growth Intact hyphae Fully or partially digested hyphae

F i g u r e 13.7 A schematic diagram showing the cell layers found in the protocorms and regions of fungal colonization. From Clements (1988), with permission.

Inoculum disc

Orchid protocorms a nono

^ Cellulose agar

nonno Mineral nutrient agar

F i g u r e 13.8 Modified Petri dish, used to study translocation to orchid seedlings. Seeds were sown on mineral nutrient agar in the outer dish. The fungus was inoculated onto the cellulose-containing medium in the inner dish; it grew over the barrier into the outer compartment and formed mycorrhizas with the seedlings.

separated organic substrates for the fungus from the colonized protocorms. Growth only occurred if an intact mycelium was present. If the hyphae connecting the protocorms to the nutrient supply were cut, growth ceased. Later, Smith (1966) used the experimental system illustrated in Figure 13.8 to show that R. solani (Thanatephorus cucumeris), was capable not only of hydrolysing cellulose and absorbing its products, but also of translocating them to orchid seedlings in sufficient quantities for growth to occur. The results are given in Table 13.1. Harley and Smith (1983) used data for the growth of protocorms of Dactylorhiza purpurella and assumptions about the number of hyphal connections with a cellulose substrate to calculate an approximate (hexose) flux of 2.5 X 10"^ mol m"^ s"^. This flux is considerably higher than the fluxes of inorganic nutrients in VA mycorrhizal hyphae (see Chapter 14).

365

Orchid mycorrhizas T a b l e 13.1 G r o w t h of seedlings of Dactylorhiza

purpurella

in 14 weeks on

substrates w i t h o r w i t h o u t cellulose in the presence o r absence of Thanatephorus cucumeris at 22.5 °C, in the dark Not inoculated

Number of seedlings* Length (|im)^ Width (|im)+

14 248 ± 9 206 ± 10

Inoculated + Cellulose

— Cellulose

226 1170 ± 119 692 ± 44

324 800 ± 72 519 ± 39

* Seedlings remaining healt w out of approximately 800. ^ ± Standard error. Data from Smith (1966).

(a) ^

1600

E Q> Q.

1200

Q>

C

800

*E a. c O

O

400

L^ 2

4

!_ 6

8

J \ \ L 10 12 14 16 18 Days after addition of I4rC-glucose

F i g u r e 13.9 Translocation of '"^C-labelled compounds by nnycorrhizal fungi into orchid p r o t o c o r m s after the fungi had been supplied w i t h '"^C-glucose on split-plates, (a) Goodyera repens colonized by Rhizoctonia goodyerae-repentis. Redrawn f r o m Purves and Hadley, 1975. (b) Dactylorhiza purpurella colonized by R solar)!. • , Alcohol-soluble fraction; • , alcohol-insoluble fraction. Redrawn f r o m Smith (1967).

The translocating ability of orchid mycorrhizal fungi and delivery of sugars to symbiotic protocorms of Dactylorhiza purpurella and Goodyera repens has been confirmed using ^^C-glucose (Smith, 1967; Purves and Hadley, 1975; Alexander and Hadley, 1985; Fig. 13.9). Both alcohol-soluble and insoluble fractions of the seedlings became labelled in D. purpurella and chromatographic analysis of the soluble fractions showed that uncolonized orchid tissues contained sucrose, glucose and fructose, whereas the fungi contained trehalose accompanied by glucose and occasionally by mannitol, but no sucrose. Seedlings fed with [^^C]glucose via the fungus in split-plates became labelled in the fungal sugars and also in the orchid sugar, sucrose. Changes in the pattern of labelling with time in D. purpurella are shown in Figure 13.10 and indicate that the fungal sugar, trehalose, is the most

366

Orchid mycorrhizas

lOOr

Days after addition of

14,

C-glucose

Figure 13.10 Distribution of ''^C in the components of the neutral ethanol-soluble fraction of mycorrhizal protocorms of Dactylorhiza purpurella after the mycorrhizal fungus, Rhlzoctonia solani, had been supplied with '^C-glucose on split-plates. A , Trehalose; • , sucrose; • , glucose; A , mannitol; O, fructose. Redrawn from Smith (1967).

heavily labelled in the early samples but, as time elapses, sucrose becomes proportionally more heavily labelled as trehalose labelling declines (Smith, 1967). There is thus reasonably good evidence that carbohydrate is translocated in the fungus, and that during or following transfer to the orchid cells it is converted to sucrose. Since trehalose can support asymbiotic growth of the seedlings of several orchids it is possible that it is transferred directly from the fungus, but hydrolysis to glucose before absorption, by the action of a fungal or orchid trehalase, cannot be ruled out and might occur in the symbiotic interface within the protocorms. Trehalose is a suitable source of carbohydrate for germination of D. purpurella (Smith, 1973), G. repens (Purves and Hadley, 1975), Bletilla hyacintha, Dendrobium sp. and Phalaenopsis sp. (Ernst, 1967; Ernst et a/., 1971; Smith, 1973). It can also be absorbed and metabolized by the leaves of B. hyacintha. The labelling patterns following absorption of ^^C-trehalose were identical to those following [^^C]-

Orchid mycorrhizas

367

glucose absorption (Smith and Smith, 1973). Ernst et al (1971) observed low concentrations of glucose in the medium following incubation of trehalose with asymbiotic Phalaenopsis seedlings, possibly indicating that hydrolysis precedes absorption by the protocorms. Mannitol is less likely to be important as a carbohydrate for translocation and transfer. It occurs in only a proportion of the fungi examined and is suitable for the asymbiotic germination of only a few species of orchid. The leaves of Bletilla hyacintha absorb and accumulate mannitol but do not metabolize it; nor is it suitable for the germination of the seeds of this species (Smith, 1973; Smith and Smith, 1973). It would be most interesting to know whether orchids such as Phalaenopsis spp. and Dendrobium spp., which germinate on mannitol, can use it in adult tissues (Ernst, 1967). Metabolism of mannitol might possibly provide a basis of crude specificity between the fungi producing it and the orchids capable of using it. Vitamins and growth factors synthesized by the fungi may also be important or essential for the growth of the seedlings of some orchids and there is reasonable circumstantial evidence that the requirements of seedlings in asymbiotic culture are provided by the fungus in symbiotic systems. However, direct evidence for their transfer from fungus to plant is lacking. Mineral Nutrients The experiments of Hollander and Burgeff (see Burgeff, 1936) indicate major mycorrhizal effects on N nutrition. The increase in weight of Cymbidium seedlings on polypodium fibre was about 10-fold in three months, whereas the increase in N content was 25-fold. More direct evidence for uptake and translocation of N is not available, although split-plate techniques using ^^N-sources (both organic and inorganic) would be relatively simple to carry out. ^^P is translocated by the fungi and accumulates in protocorms of Dactylorhiza purpurella in split-plates (Smith, 1967). Furthermore, P inflow and accumulation in plantlets of Goodyera repens depends on the mycorrhizal association and is discussed below (Alexander and Hadley, 1984; Alexander et al, 1984). Mechanisms of Transfer Although lysis of the fungus in orchid mycorrhizas is generally regarded as a manifestation of defence of the plant against invasion, it has also been assumed that it is important in the transfer of nutrients. There is no clear evidence against this hypothesis, except that the growth response to colonization appears to start before any lysis of pelotons is observed (Fig. 13.6). At present, it seems more likely that nutrients are transferred across the intact membranes of fungus and orchid, with potential for separate control of movement of different nutrients, which is clearly required, as shown by the work of Alexander and Hadley (1984,1985) on G. repens at different stages of development (see Chapter 14). However, Serrigny and Dexheimer (1985) have shown that diethyl stilbestrol(DES)-sensitive ATPases are active on the plasma membranes of both fungus and plant in the peloton interface and on the plant plasma membranes surrounding the lysed clumps of fungus, but not on the uninvaginated plasma membrane. This distribution might be taken as

368

Orchid mycorrhizas

evidence that the membrane is energized, as would be required for nutrient absorption by the plant from both intact and digested pelotons. Further work is clearly required to clarify this.

Transfer of Carbohydrate from Orchid to Fungus Authoritative statements have been made on fairly slender grounds that C transfer from fungus to orchid seedlings is reversed when the orchid becomes photosynthetic (Burgeff, 1959; Scott, 1969). These assertions were unsupported by experimental evidence and C movement from photosynthetic orchid (fed with ^^COz) to the fungus has subsequently been shown to be negligible. Early experiments by Smith and by Lewis and Hadley (unpublished) failed to detect any radioactivity in the fungal sugars within the protocorms or in the hyphae growing out on to carbohydrate-free medium after green mycorrhizal seedlings of D. purpurella had assimilated ^^C02 in the light. In later work with the same orchid, Lewis et al. (see Purves and Hadley, 1975) showed that fungal metabolites sometimes became labelled in similar experiments, but since the control (asymbiotic plants) released ^^C into the medium, carbohydrates released in a similar way by mycorrhizal protocorms might have been the source of ^^C in the mycelium around them. Moreover, dark fixation of ^^C02 by the fungus might also have occurred. Experiments with Goodyera rq)ens showed that no ^^C-labelled metabolites leaked into the medium and in this case when ^^C02 was applied to the green top alone, radioactivity passed to the rhizome but none passed into the growing hyphae in the medium (Hadley and Purves, 1974). When the rhizomes alone were exposed to ^^C02, small quantities of radioactivity appeared in the emerging mycelium. The conclusion that C movement from photosynthetic seedlings to the fungus does not occur was later confirmed by Alexander and Hadley (1985) using both plantlets and plants of G. repens, neither of which received any ^ C-labelled compounds from the fungus.

Plant-fungus Interactions: Mycorrhizal or Not? The outcome of the interaction between potentially mycorrhizal fungi and orchid protocorms is quite variable. In any population raised under apparently controlled conditions with a single endophyte, there may be actively growing mycorrhizal protocorms and also protocorms completely invaded and parasitized by the fungus or from which the fungus has been excluded (rejection). In the last two situations little or no growth of protocorms occurs. Figure 13.11 shows a scheme of possible pathways of fungal interaction with orchid protocorms drawn up by Hadley (1970) after experiments with 32 strains of 10 species of fungus (isolated from five north temperate and three tropical species of orchid) with 10 species of protocorm in culture. Similar variations in response have been found for Australian orchids by Warcup (e.g. 1975) and Clements (1981). Manipulation of culture conditions, especially N and C composition and concentration in the medium, can alter the proportions of protocorms in the different categories (Hadley, 1970; Beyrle et ah, 1991, 1995) and can be used to analyse the interactions, although the very artificial conditions are unlikely to relate to the situation in soil. Rejection of the fungus is

369

Orchid mycorrhlzas Germinated seed + fungus

O No infection (no growth)

Parasitism of protocorms (death)

Compatible infection

SP Breakaway parasitism (death)

Orchid reaction eliminates fungus (growth stimulation limited)

SS or SSS Stable symbiosis (good growth)

SSP Parasitism (growth ceases: death)

Normal plant development

Figure 13.11 Scheme of symbiotic development in orchids. O, Non-colonized, no growth; S, SS, SSS, greater numbers of protocorms develop; S*, initial colonization followed by hypersensitive reaction or complete fungal disintegration; P, fungus becomes an aggressive parasite, killing cells; —, no infection, but protocorms die for other reasons. Modified from Hadley (1970).

accompanied by thickening of cell walls (Fig. 13.5e) and deposition of phenolics, while uncontrolled parasitism is characterized by soft rot, with the hyphae ramifying through the tissues and no evidence of mobilization of structural defence responses (Masuhara and Katsuya, 1991; Beyrle et ah, 1995). To add to the complexity, mycorrhizal protocorms may develop normally for some weeks but then succumb to what Hadley (1970) referred to as 'breakaway parasitism', in which the fungus changes its behaviour and ramifies through the tissues. The proportion of protocorms in each class depends upon the strain of fungus and the species of orchid, as well as upon the environmental conditions. Little is known of what controls the balance between the two organisms but the symbiosis is clearly much less stable than other types of mycorrhizal interaction and can be

370

Orchid mycorrhizas

likened to a situation where attack by a potential pathogen is controlled by defence responses in the plant. A few of the fungi, such as Rhizoctonia solani and Armillaria mellea, are already known as destructive necrotrophic parasites and all those that have been investigated produce cellulases and pectinases which may be deployed in colonization of plant tissues. The amounts of pectinases produced in culture by different species of Rhizoctonia are not related to their pathogenicity towards seedlings of Dactylorhiza purpurella (Perombelon and Hadley, 1965). A considerable range of cellulolytic ability has also been found (Smith, 1966; Hadley, 1969; and see Burgeff, 1936), so that control of activity in mycorrhizal interactions must occur, perhaps via end-product repression in tissues with a relatively high hexose content compared with non-mycorrhizal orchid tissue (Purves and Hadley, 1975). During parasitism the cell walls are degraded (Beyrle et al., 1995), suggesting that control of hydrolytic activity no longer operates. In the mycorrhizal interaction, limited hydrolytic activity in the tissues might be significant in permitting penetration of cell walls (as in Fig. 13.5d) and in releasing small quantities of oligosaccharides which would elicit defence responses in the plant and lead to control of invasion. Evidence for the deployment of defence responses by orchids is quite extensive and dates from the initial observations of Bernard (1909b), who suggested that the absence of fungal infection from the tubers of some orchids and the resistance of some seeds to fungal attack was due to the presence within them of an antifungal principle. He showed that tubers of Loroglossum contained a substance which was toxic to many orchid endophytes, including Rhizoctonia repens (Tulasnella calospora) but not to a strain of R. solani. Subsequently, it has been confirmed that orchids produce dihydroxyphenanthrene phytoalexins such as hircinol, loroglossol and orchinol, which inhibit the growth of mycorrhizal fungi and many other fungi and bacteria (Arditti, 1979; Stoessl and Arditti, 1984). It is not completely clear whether these phytoalexins are present before contact with the fungi, but Gaumann and his colleagues (see Nuesch, 1963) have shown that orchinol is formed in tubers of Orchis militaris when incubated with Rhizoctonia repens, not only by the cells immediately in contact with the fungus, but also by those up to 12 mm away. It seems certain that these substances play a part in controlling or restricting fungal invasion, particularly in tubers where they are produced in high concentrations. However, their role in the fine-timing of the interactions in protocorms and roots is not at all clear and until recently neither orchinol nor related phytoalexins had been detected in these structures. Beyrle et al. (1995) showed that low concentrations of orchinol were present in asymbiotic protocorms of Orchis morio, and that levels increased on contact with a mycorrhizal Rhizoctonia. Neither the amoimt of orchinol synthesized nor the activity of phenylalanine ammonia lyase (PAL, a key enzyme in the synthetic pathway for phytoalexins) was related to the type of symbiotic response. In contrast, Reinecke and Kindle (1994), working with Phalaenopsis, detected no 9,10-dihydrophenanthrenes in young sterile plantlets, but contact with Botrytis cinerea and a Rhizoctonia caused synthesis of phytoalexins, including hircinol, and concomitant rises in PAL and bibenzyl synthase activity. Unfortunately, the type of symbiotic interaction with the Rhizoctonia was not recorded, so the picture remains incomplete. Production of hydrolytic enzymes by the plant may be important in lysis of the fungus. The possible involvement of acid phosphatases and of P(l,3)glucanases and chitinases has already been mentioned, but their activities in the different symbiotic

Orchid mycorrhizas

371

responses have not been closely analysed. As the last two are recognized as pathogenesis-related (PR) proteins, produced in many plant-pathogen interactions and under conditions of stress, they may have considerable significance in orchid symbioses. It is quite clear that the dynamic and variable nature of the interactions between protocorms of orchids and their endophytic fungi sets them apart from the reactions in other mycorrhizal associations. Compared with VA mycorrhizas, for example, in which plant defence responses are hardly apparent (see Chapter 3), the situation in orchids looks like a battlefield in which attack and defence mechanisms are mobilized by both partners. There is clearly scope for further work which would provide a clearer picture of gene expression and protein accumulation in asymbiotic protocorms and in the different symbiotic interactions. DNA probes are available for many of the genes coding for PR proteins and for enzymes in the shikimic acid pathway, which leads to the synthesis of orchid phytoalexins as well as lignin and other phenolics. In addition, antibodies for the protein products of many of these genes are available, so there is considerable scope for the analysis of the interactions both at the levels of gene expression and protein synthesis and with respect to accumulation of antimicrobial products. Polymerase chain reaction (PCR)-based methods, in situ hybridization and immunolabelling should help to overcome the problems arising from the small quantities of symbiotic tissue available. One advantage of the orchid mycorrhizal system is that both symbionts can be cultured axenically.

Mycorrhizas in Adult Orchids Adult orchids, whether or not they contain chlorophyll, usually have mycorrhizal roots or tubers although the extent of colonization is variable. Temperate and tropical species of terrestrial orchids are usually heavily colonized (Hadley, 1982; Goh et ah, 1992; and see Fig. 13.4b). Roots are frequently produced anew each season and rapidly become mycorrhizal, with the fungus entering from the soil. In Ophrys this coincides with the production of new tubers in autumn (Bernard, 1901). Roots of Goodyera repens are also colonized as soon as they are produced (4 mm long) and no difference in the extent of colonization at different seasons has been observed (MoUison, 1943; Alexander and Alexander, 1984). In Bletilla striata root growth begins in early summer (May) and colonization by living, undigested fungal pelotons was observed to be highest in young roots. As the rate of root growth increased and the season progressed, both active and total percentage colonization declined. Seasonal isolations showed that Rhizoctonia repens was the most common endophyte of B. striata at the site surveyed (Masuhara et ah, 1988). A few investigations have compared colonization in different orchid tissues. In the evergreen Goodyera repens colonization of rhizomes (up to 90%) occurred independently of that of roots and was at a maximum in winter (November to April) when the rate of elongation was lowest (Alexander and Alexander, 1984). Spiranthes sinensis var. amoena produces true roots in autumn which, as in the species described above, become rapidly colonized up to a maximum the following summer. During flowering the amount of living fungus declined and the roots decomposed. In contrast, tuberous roots were produced in spring and, although anatomically similar to the true roots, were only locally colonized. The fungus did

372

Orchid mycorrhizas

not spread and the extent of colonization was always less than for the true roots (Masuhara and Katsuya, 1992). Five patterns of colonization have been found in Western Australian species from arid environments (Ramsay et al, 1986). In species with stem and root tubers the fungus was found in these structures throughout the year, although the extent of colonization was lowest during the summer period of aestivation. In other species, seasonally produced stem collars and roots were colonized anew each season (as in temperate species), with a peak coinciding with the period of maximum vegetative development. In epiphytic orchids colonization appears, with a few exceptions, to be relatively sparse and sporadic compared with terrestrial species. Not all plants examined contain typical mycorrhizal structures and colonization, where present, was often limited to the regions of the roots adjacent to the support (Ruinen, 1953; Hadley and Williamson, 1972; Bermudes and Benzing, 1989; Lesica and Antibus, 1990; Richardson et al, 1993). At the cellular level, fimgal colonization of tissues of adult orchids is generally similar to protocorms. A fungal hypha penetrates the epidermis and enters the cells of the cortical parenchyma, forming characteristic and complex hyphal coils. Penetration of the suberized hypodermis, which is often well developed in orchids, is via the short passage cells (Janse, 1897; Mejstrik, 1970; Esnault et ah, 1994). Colonization spreads either as a result of repeated colonization from the soil or by hyphae penetrating from cell to cell in the root cortex. More than one species of fungus can form pelotons in a root or even within the same cell (Warcup, 1971). The ultrastructure of mycorrhizal roots of orchids does not differ markedly from the picture obtained with protocorms (Dorr and Kollmann, 1969; Mejstrik, 1970; StruUu and Gourret, 1974; Barmicheva, 1989) and within the cortex the cells may be uncolonized, or contain active pelotons or clumps of degenerating hyphae in different proportions (Fig. 13.4b). The function of mycorrhizas in adult orchids is not well understood. For a long time it was assumed that the fungus was involved in supplying organic C, and its potential role in absorption of nutrients such as P or N was ignored, despite the fact that the root systems are frequently poorly developed. There is certainly some indirect evidence that a few terrestrial species must continue to gain C via the fungi. These include achlorophyllous myco-heterotrophs (e.g. Galeola, Gastrodia, Corallorhiza, Rhizanthella and many others; see Leake, 1994) and possibly some green species which, like Spiranthes spiralis, Cephalanthera rubra and Goodyera repens, may spend several years undergroimd before producing flowering scapes (Summerhayes, 1951; Wells, 1967). Experiments with epiphytic orchids by Ruinen (1953) led to the same conclusion. She found that many were connected by their mycorrhizal hyphae to the living tissues of their supports, which showed improved growth when the epiphytes were removed. Epiphytes resemble achlorophyllous orchids like Gastrodia elata (Kusano, 1911a,b), G. minor (Campbell, 1963) and Rhizanthella (Warcup, 1985) in that they are attached to living plants or to dead organic matter by their fungal associates. Burgeff (1932,1936) gives many examples which can be rationally interpreted only in the sense that many adult orchids with green foliage are to a significant extent reliant on their fungi for C nutrition. Clearly, this is a subject which needs much more research and the indirect evidence certainly needs confirmation, particularly in the light of the tracer experiments

Orchid mycorrhizas

373

which showed that young plantlets of Goodyera repens did not receive any ^^C from the fungus (Alexander and Hadley, 1985). More recently, the possible role of mycorrhizal fungi in mineral nutrition of adult plants has been examined experimentally. In G. repens, mycorrhizal colonization is associated with increased growth and concentrations of N and P in the tissues, as well as with increased P inflow. These effects were reduced if the plants (collected in the field) were treated with thiabenzadole to reduce fungal colonization (Alexander and Hadley, 1983). The role of the external mycelium in transporting ^^P to the plants from distances of up to 9 cm was also confirmed (Alexander et ah, 1984). This is an area that deserves much greater attention, particularly with respect to organic sources of P, N and other nutrients. Compartmented or mesh systems used to investigate similar problems in VA and ectomycorrhizal associations (see Chapters 5 and 8) could be applied to orchids, although there might be difficulties in obtaining sufficient experimental material. Specificity and Ecology of Orchid Fungi Considerable controversy has been generated over the specificity of the association between orchids and their mycorrhizal fungi. The analysis of specificity is complicated for a number of reasons. Isolation methods and experiments to verify the mycorrhizal potential of the fungi have been based on the assumption that those associated with adult orchids and with protocorms are the same. The frequency of isolation of different species or strains from roots of plants growing in the field provides one picture of specificity. A different picture is obtained from the results of germination tests. Fungi obtained from the roots of a particular orchid species may not form mycorrhizal associations (with pelotons) with the protocorms of the same species, although it may do so with others. Furthermore, it has not always been recognized that the outcome of germination tests is markedly influenced by the composition of the media and the environmental conditions. A few examples will illustrate the complexities of the situation. Several typical orchid fungi, for example Rhizoctonia repens {Tulasnella calospora) and R. goodyeraerepentis {Ceratobasidium cornigerum) are of worldwide distribution. They have been isolated from and stimulate the germination of many orchid species which have much more localized distributions than the fungi themselves. These fungi and their orchid associates appear to have rather unspecific relationships. Downie (1943) used seed germination tests to establish the presence or absence of R. goodyeraerepentis in untreated pine wood soils and found that the distribution of the fungus was more widespread than the orchid. In aseptic tests there was no evidence for a specific relationship between these two organisms or between a number of fungi and many European terrestrial orchids, although there was considerable variation in response (Downie, 1941). Curtis (1939) was also of the opinion that most orchid endophytes are widespread soil saprophytes and that their presence in roots of particular species reflected the distribution in soil rather than any specific relationship with the plants. However, again using G. repens, Rasmussen and Wigham (1994) observed germination of buried seed only at sites where the adult plants (and presumably appropriate fungi) occurred, so that even for this species the picture is still confused.

374

Orchid mycorrhizas

Some orchids do seem to have a more specific interaction with mycorrhizal fimgi. Bernard (1905) isolated a number of distinct fungi from roots of different orchids and, in germination tests on a variety of synthetic media, showed that while some orchid species were stimulated to grow by several different endophytes, others seemed to require a specific one. Warcup (1971,1973,1975) also showed that some fungi appear to be fairly specific to a given genus or species of orchid. Isolation of fungi fi-om hyphal coils in the roots showed that each species of the genera Pterostylis, Caladenia and Thelymitra growing within a few centimetres of each other in dry sclerophyll forests of Australia had its own endophyte. Rarely were there 'cross-overs' and then only in roots infected by two fungi. In germination tests, two species of Pterostylis were stimulated only by Ceratobasidium cornigerum, and two species of Diuris only by Tulasnella calospora. Species of Caladenia, Glossodia, Elythranthera and Eriochilus (which are related to each other) all harboured Sebacina vermifera in their roots, but this fungus was not conspicuously successful in stimulating the growth of seedlings. On the other hand, seedlings of Thelymitra were stimulated by several species of Tulasnella and not by C. cornigerum (Warcup, 1973). In Japan, Rhizoctonia repens (T. calospora) was the main fungus isolated from the roots of adult plants of Spiranthes sinensis var. amoena and Bletilla striata. Soil was baited with seeds of S. sinensis buried in gauze and the same fungus was isolated from the resulting protocorms 8 weeks later. However, baiting with buckwheat stems revealed a number of other Rhizoctonia species, which were not foimd in the orchid protocorms, but which induced germination of the orchid in tests in vitro (Masuhara and Katsuya, 1994). Rasmussen and Wigham (1994) have used a similar technique to follow germination of seeds of several orchid species in soil in orchid habitats. Some species did not germinate at all, while others (Goodyera repens and Corallorhiza odontorhiza) only germinated at sites where adults of the same species occurred, and not at sites where other orchid species grew. Assuming that the differences were dependent on the occurrence of potentially mycorrhizal fungi, the results of these studies indicate a strong degree of ecological selectivity in the colonization of both developmental stages of the orchid and highlight the problems of deducing relationships in the field from the results of germination tests in the laboratorj^ In any event, there appear to be grounds for investigations of specificity which take into account the presence of an alternative (photosynthetic) symbiont for the fungi, as with the tripartate systems involving mycorrhizal fungi and Corallorhiza-Pinus and Rhizanthella-Melaleuca. Determination of specificity requires precise identification of the fungi, and the recognition that those associated with adult and seedling orchids may be different and that the responses observed in germination tests are highly variable and influenced by nutritional and environmental conditions. The techniques developed to study germination in the field (Masuhara and Katsuya, 1994; Rasmussen and Wigham, 1994), if more widely applied, will give valuable and ecologically relevant data, not only on specificity but also on the whole process of germination and development of orchids. If these are coupled with the precise identification of the fungi, using molecular methods to overcome the difficulties of obtaining perfect stages in culture, considerable advances may be anticipated (White et al., 1990; Gardes and Bruns, 1993; CuUings et al., 1996; and see Chapter 1). The work of Taylor and Bruns (personal communication), showing apparently high specificity of the ectomycorrhizal fungi Thelephora and Tomentella with Cephalanthera austinae,

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demonstrates the power of these molecular methods of fungal identification in unravelling problems of epiparasitism, mycoheterotrophy and specificity. Conclusions Although the widespread occurrence of mycorrhizas in adult orchids has been recognized for well over a century, most of the work on orchid mycorrhizas has been directed towards understanding the interactions that take place during the early stages of seedling germination and growth under laboratory culture conditions. All orchids have a relatively prolonged heterotrophic stage during germination and early growth, and some species are heterotrophic throughout their life cycles. During this phase all are dependent on carbohydrate translocated to them via the mycelium of a mycorrhizal fungus. The source of the carbohydrate is either dead organic matter or recent photosynthate, depending on the nutritional interactions of the fungus with other plants. Although the seeds of many species can be induced to germinate and grow in the absence of a mycorrhizal fungus, as long as sugars and various growth factors are supplied, this does not occur in nature. In the soil, colonization by a fungus is required, but only a few studies have followed the early stages of orchid development imder field conditions. Carbohydrate transfer to the orchid continues throughout the life of the plant in myco-heterotrophic orchids but the extent to which this occurs in photosynthetic species is unknown. The fungi probably also supply N and P and other nutrients to both seedlings and adult plants, but this aspect of the physiology of the association should be investigated with a wider range of plants and, again, under ecologically relevant conditions. Orchid mycorrhizal fungi are capable of translocating both ^^C- and '^^P-labelled compounds in their hyphae and delivering them to protocorms. The mechanisms by which nutrients are transferred from fungus to plant are not well understood but it seems likely that controlled transport occurs across the interface between pelotons and orchid cells. There is no good evidence that transfer in the opposite direction, from orchid to fungus, takes place at any stage. Whether or not the fungus gains anything from the mycorrhizal relationship remains one of the mysteries of the association. However, the unstable nature of the symbiosis means that there may be situations in which the fungus successfully obtains nutrients by parasitizing the orchid, mainly in the seedling stages. Orchid mycorrhizas are different from other mycorrhizal associations in many respects. They are certainly not mutualistic symbioses and attempts to classify them on nutritional bases have not been very successful. It has been suggested that the orchid is a necrotrophic parasite of the fungus (Lewis, 1973) and this might indeed be the case in the strictly mycorrhizal phase of the associations during which pelotons are lysed. However, in other phases the fungus can clearly be a necrotrophic parasite on the plant, and its hyphae can grow in an uncontrolled way throughout a protocorm, inducing soft rot. In recent years mycorrhizal associations in orchids have been neglected relative to the enormous amount of research on VA and ectomycorrhizas. We hope this chapter has shown how a combination of conventional and molecular methods has the potential to unravel some of the complexities of plant-fungus interactions at both physiological and taxonomic levels and provide data relevant to ecological situations.

14 Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

Introduction The function of all mycorrhizal systems depends on the ability of the fungal symbionts to absorb nutrients available in inorganic and/or organic form in soil and to translocate them (or their metabolites) to the symbiotic roots through the extensive vegetative mycelium. In all systems these processes are followed by transfer of nutrients from the fungus to the plant across one or more symbiotic interfaces where the two symbionts are in close contact. In most mycorrhizal types, organic C derived from photosynthesis is also transferred from the plant to the fungus, followed by translocation to the growing margins of the extraradical mycelium and to developing spores and fruit bodies. These essential features of fimctional mycorrhizas have long been recognized (see Harley and Smith, 1983) and bidirectional nutrient movement - C from plant to fungus and soil-derived nutrients from fungus to plant - is believed to be the basis for mutualism as well as being one of the main factors favouring the low specificity of the plant-fungus relationships observed in most mycorrhizal types. Exceptions are to be found in orchids and other myco-heterotrophs, in which transfer of both organic C and soilderived nutrients is polarized in the direction of the non-photosynthetic plant (see Chapters 11 and 13). Although the processes of uptake, translocation and transfer are understood in broad terms and some mechanisms are inferred by comparison with other organisms and symbioses, there are still considerable gaps in our knowledge of even the simplest systems involving one fungus and one autotrophic host. Mycorrhizal systems have been used successfully to determine rates of translocation but the underlying mechanisms, in rhizomorphs or individual hyphae are still not understood. Moreover, it is presumed that there may be modifications at both tissue and cellular levels which facilitate nutrient transfer across the plant-fungus interface(s), but the details are only now being elucidated. On top of these imcertainties, research in the last ten years has altered the way we think about the function of

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mycorrhizas in natural ecosystems, so that much more emphasis is now placed on the development and operation of the external mycelium and its role in linking plants together in mycorrhizal guilds. The possibilities of interplant transfer of nutrients (see Chapters 4, 5 and 15) need to be considered in terms of the ways in which the membranes in the symbiotic interfaces function, because it is at this level that the interactions between whole plants will ultimately be controlled and the efficiency of the symbioses determined. The sites of nutrient transfer between the symbionts are also being critically discussed. Whereas bidirectional nutrient transfer almost certainly occurs across the same plant-fungus interface in ectomycorrhizas (the Hartig net) and ericoid mycorrhizas (the intracellular hyphal complexes), the importance of the arbuscule as the sole site for bidirectional transfer of different solutes in vesicular-arbuscular (VA) mycorrhizas has been questioned. In these mycorrhizas there are usually at least two types of interface: the intracellular, arbuscular interface, which may be rather transitory, and more robust hyphal interfaces, either intercellular (in Arumtype mycorrhizas) or intracellular (in Paris-types; see Chapter 2). This differentiation offers the possibility of specialization of function and spatial separation of different transport processes. Dependence of achlorophyllous mycorrhizal species (myco-heterotrophs) on 'normal' mycorrhizal plants, mediated via a common mycorrhizal mycelium, provides a further challenge to elucidate the mechanisms of nutrient transfer and the controls that may determine the direction and amounts of material transferred through the external mycelium and across the symbiotic interfaces. This chapter outlines what we know of the processes leading to nutrient transfer between mycorrhizal symbionts, highlights new developments and considers avenues of research that may be productive. The detailed information presented in Harley and Smith (1983) is not repeated but is augmented and corrected where appropriate.

Nutrient Uptake by Mycorrhizal Fungi and Mycorrhizas The external mycelium of all mycorrhizal types plays a key role in uptake of nutrients by plants, proliferating in nutrient-rich sites and presumably competing effectively with other soil microorganisms. All the fungi absorb inorganic nutrients, and it has also been confirmed that ericoid and ectomycorrhizal fungi have the ability to mobilize organic sources of N and P, thus making a significant contribution to N nutrition and N cycling in ecosystems (see Chapters 8, 9, 11 and 15). The exploratory and exploitative phases of mycelial development in soil are adapted to this role and contrast with the regions in close association with the plants where differentiation into specialized interfacial structures is important in nutrient transfer and the acquisition of organic C. Intact mycorrhizal systems are difficult to manipulate experimentally, so most of the detailed work on the mechanisms and rates of uptake of nutrients by mycorrhizas has been done with excised ectomycorrhizal rootlets, detached from any mycelium, or with mycelial cultures of ericoid and ectomycorrhizal fungi. Pioneering studies, demonstrating the capacity of ectomycorrhizal roots to accumulate and store nutrients and subsequently to transfer them to the plant, were carried out by

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Harley, McCready and co-workers in the 1950s and 1960s. Harley and Smith (1983) summarized this extensive work particularly with respect to P, N and K (see Chapters 8 and 9). The conclusions are that uptake by mycorrhizal rootlets essentially does not differ from uptake by other absorbing systems: it is active, dependent on metabolic energy and reduced in the presence of inhibitors. P is rapidly accumulated in the ectomycorrhizal sheath, whether or not the mycorrhizal rootlets are excised or remain attached to the plant, and only later and more slowly passes to the root cells. It seems probable that P is absorbed as H2PO4, as in most other cell types. Dual uptake systems (high and low affinity) have been shown to operate in VA (Thomson et ah, 1990a) and ericoid mycorrhizal fungi (Straker and Mitchell, 1987), as they do in cells of higher plants and saprophytic fungi. A phosphate transporter (GvPT) has been cloned from Glomus versiforme (Harrison and van Buuren 1996; see below). In the cultured mycelium of the ectomycorrhizal fungus Pisolithus tinctorius, uptake is partially controlled by the P status of the mycelium, being rapid into mycelium of low intracellular P concentration, but slow when the P concentration is high (Cairney and Smith, 1992), again showing similarities to non-mycorrhizal basidiomycetes and Neurospora crassa (Beever and Burns, 1980; Clipson et ah, 1987). Absorption must be active in all cases, because the electrochemical potential difference between soil and hypha is strongly in favour of efflux, there being a large (inside negative) electric potential difference (PD) and high internal inorganic phosphorus (Pi) concentration relative to the soil solution. By comparison with other cell types, including other fungi, uptake is likely to be via proton co-transport, with the necessary proton motive force (PMF) generated by a membrane-bound H"'-ATPase (Beever and Burns, 1980; Sanders, 1988; Smith and Smith, 1990; Garrill, 1995; Michelet and Boutry, 1995; and see Fig. 14.1). A cDNA representing a POJ transporter (GvPT) from Glomus versiforme has been obtained from a library prepared from roots of Medicago iruncatula, mycorrhizal with that fungus (Harrison and van Buuren, 1995). The gene is expressed in external hyphae of mycorrhizal roots and when transformed into mutant yeast (p/zo84), which lacks the high-affinity P transporter, wild-type transport properties are restored. Other data are also consistent with the identification of GvPT as a high-affinity P transporter. The work on POJ uptake by germ tubes of Gigaspora margarita is somewhat contradictory. Although Thomson et al. (1990a) were able to demonstrate the existence of two PO^ uptake systems in 14-day-old germlings. Lei et ah (1991) found no evidence of '^^P accumulation in germinating spores using autoradiography, unless they had been stimulated by the presence of root volatiles and CO2 (see Chapter 2). The unstimulated germ tubes showed no H'^-ATPase activity on their plasma membranes (essential for generation of PMF), while stimulated germ tubes did. Growth of the stimulated germ tubes was inhibited by application of the H"^ATPase inhibitor diethyl stilbestrol (DBS; Lei et ah, 1991). These findings may indicate that an inability to accumulate nutrients from the medium might be one factor accounting for the failure of VA mycorrhizal fungi to grow extensively in the absence of a host plant. Further work is required to confirm this suggestion, particularly as the two research groups used material of different ages. There have been very few measurements of the rate of uptake of P (mol m~^ hyphae s~^) by the mycelium of any mycorrhizal fungus in association with the

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Figure 14.1 Schematic representation of transport processes operating in the fungal plasma membrane and tonoplast. Evidence for the existence of the different processes and appropriate references are given in the original article. | , channels; 0 , pumps. From Garrill (1995), The Growing FunguSy Figure 8.4, Chapman and Hall, with permission.

plant. In VA mycorrhizas uptake per unit length of hyphae has been calculated from amounts of P absorbed to be between 2 and 4 X 10~^^ mol m~^ s~^ (see Table 5.2). Sukarno (1994) estimated the surface area of hyphae of a Glomus species as 12 X 10~^ m^ m~^ hyphal length, a value which can be used to derive uptake rates by the hyphae of 20-40 X 10"^ mol m"^ s"^. These values are quite high, but of the same order of magnitude as rates of uptake by other cell types. Sukarno et al. (1996) calculated the relative rates of uptake by external hyphae and of transfer from fungus to plant and found that uptake per unit area of absorbing hyphae was about 10 times slower than transfer per imit area of symbiotic interface. The difference emphasizes that a VA mycelium must absorb nutrients over a large surface area in soil to supply P through the entry points to the interfacial regions and from there to the plant. No comparative data are available for other mycorrhizal systems. Following uptake, mycorrhizal fungi both in culture and in symbiosis synthesize polyphosphate. This is probably of relatively short chain length and is stored in the vacuoles, thus maintaining relatively low cytoplasmic Pi concentration. The importance of polyphosphate in storage and translocation is discussed below. Uptake of other solutes has received less attention. In general, ammonium (NH4) is absorbed more rapidly than nitrate (NO^) by ectomycorrhizas, as is also the case

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383

for non-mycorrhizal roots (Botton and Chalot, 1995). NH4 absorption might be passive, via a specific carrier protein, with rapid assimilation into amino acids and amides (see Chapter 8) playing an important role in maintaiiung the necessary electrochemical potential difference for continuing absorption; or absorption might be active via proton co-transport (Fig. 14.1). It must be emphasized that the best data have been obtained for ectomycorrhizas and that there is considerable variation between different plant-fungus combinations (see Fig. 8.5; and Chapter 8). In Fagus-Laccaria mycorrhizas incorporation of NH4 is via glutamine synthetaseglutamate synthase (GS-GOGAT), whereas in Abies-Hebeloma mycorrhizas the NADP-dependent glutamate dehydrogenase (NADP-GDH) and GS are important. In any event, the major products are glutamate and glutamine, from which the N can be redistributed to other organic N compounds (see Botton and Chalot, 1995, for a comprehensive review of the enzymology). Mechanisms of uptake of amino acids and amides, following protein hydrolysis by ericoid or ectomycorrhizal fungi, are likely to be via proton co-transport, with the thermodynamic driving force generated by an H'^-ATPase, as for POJ (Reinhold and Kaplan, 1984; Bush, 1993; Botton and Chalot, 1995; and see Fig. 14.1). So far there have been few investigations of the intracellular redistribution of N from absorbed amino acids to others in the cytoplasmic pool. However, mechanisms of absorption of amino acids and amides would be relevant to the form in which N is acquired by the fungus and transferred to the plant (see below) and might also indicate whether the C skeletons contribute to fungal or plant nutrition (or both). Efflux of Nutrients from the Mycelium The factors influencing efflux are important because this process must play a key role in transfer of nutrients from the fungus to the plant at the symbiotic interface and the rate of movement of P across the plant-fungus interface in VA mycorrhizas at least, far exceeds that which might be expected if the process were dependent on 'normal' efflux (Smith et al, 1994a,b). Efflux of solutes from free-living mycelium is usually low, reflecting the low permeability of the membranes even when there is an electrochemical potential difference that favours efflux. Rates of loss of P from cultured mycelium of ericoid and ectomycorrhizal fungi have been shown to be similar to those from other fungi; 8-11% of the rates of uptake (Beever and Bums, 1980; Caimey and Smith, 1993b; Smith et a/., 1995). In P. tinctorius ^^P efflux was higher (and uptake lower, see above) when the intracellular P concentration was high, so that net efflux of P from the mycelium might be expected under these conditions. The '^^P efflux was also increased in the presence of 10 mM (or more) KCl in the bathing medium, with a smaller but similar effect of NaCl (Caimey and Smith, 1993b). Further research focused on mechanisms that promote efflux, such as enhanced opening of ion channels, would be valuable because the rates may well control the overall rates of transfer from fungus to plant in the interfacial apoplasts (Tester et al, 1992).

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Translocation in Mycorrhizal Fungi The Pathway and Direction of Translocation Use of radioactive tracers has demonstrated the importance of the external mycorrhizal mycelium in transferring nutrients between sources and sinks below ground. Following the work of Melin and Nilsson and a number of others, who used a variety of compartmented systems, Harley and Smith (1983) were able to compile a list of mycorrhizal fungi shown to have long-distance translocating ability. This included VA, ericoid, orchid and ectomycorrhizal fungi, with the nutrients involved being derived either from the plant (via fixation of ^^۩2), or from the soil (e.g. '^^P, ^^N, ^^S, ^^Ca, ^^Zn). Mycelia have frequently been shown to be capable of bidirectional translocation of individual nutrients, from young to old regions of the mycelium and vice versa. Furthermore, ^^C derived from photosynthesis reaches the hyphal tips, whereas labelled mineral nutrients such as ^^P are transported from hyphal tips to the plants (e.g. Pearson and Read, 1973b; Finlay and Read, 1986a,b; Francis et al, 1986; Duddridge et ah, 1988). The functional links in the mycelial network as it grows into the soil and connects neighbouring plants have been elegantly demonstrated by autoradiography (see Figs 4.2, 9.1). The complexity of the external mycelium and its distribution in soil varies with the mycorrhizal type and the species of fimgus involved, and this again may have consequences for the mechanisms of translocation that can be envisaged. In endomycorrhizas (VA, orchid and ericoid) the mycelium is a network of more or less separate hyphae which may branch and anastomose or form loose bundles (see Fig 2.2), but which do not form closely aggregated rhizomorphs. The details of the distribution of this simple mycelium in soil are hard to determine because of the fragility of the hyphae, but it certainly extends many centimetres from the roots and may proliferate in nutrient-rich microsites, such as soil organic matter. There is differentiation into hyphal types, at least in VA mycorrhizal fungi (see Chapter 2; and Fig. 2.3b), and of these the large-diameter, runner hyphae have been suggested (with little experimental evidence) to be the main routes of nutrient translocation in the external mycelium, whereas the fine side branches are envisaged as playing a key role in nutrient absorption. In the runner hyphae bidirectional cytoplasmic streaming is easy to observe (Rhodes and Gerdemann, 1980), with rates of between 2.7 X 10~^ and 3.5 X 10"^ m s"^ (Cox et al, 1980; Ka et al, 1994), although the importance of this process in translocation has yet to be clarified. In contrast, many ectomycorrhizal fungi form complex rhizomorphs, in which hyphal growth is coordinated and in which differentiation can result in the formation of large central 'vessel' hyphae (6-20 |Lim diameter) which lack cytoplasm, surrounded by densely cytoplasmic hyphae with relatively thick cell walls (e.g. Duddridge et al., 1980, 1988; and see Chapter 6). These are similar to the rhizomorphs of saprophytic fungi such as Serpula lacrymans, which have been used for many experiments on fungal translocation (Jennings, 1987). The different hyphal types in the rhizomorphs are probably involved in long distance translocation, with mass flow of solution occurring in the vessel hyphae and symplastic translocation in the hyphae filled with cytoplasm. Water transport in mycelium of Suillus bovinus to Pinus sylvestris seedlings has been demonstrated with ^H-labelling (Duddridge et

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al, 1980), but there is no convincing evidence that mass flow of solution in vessel hyphae (driven by the water potential gradient) is important in translocating nutrients in mycorrhizal mycelium. In autoradiographic studies of ^^C movement in the same plant-fungus combination, the tracer was confined to the hyphae containing cytoplasm and did not appear in the vessel hyphae (Duddridge et aL, 1988), indicating symplastic, rather than apoplastic translocation of organic solutes, even in these differentiated rhizomorphs. Furthermore, mass flow (in this case towards the transpiring plant) does not allow for the bidirectional translocation of nutrients which certainly occurs in the mycelium as a whole. At the level of individual hyphae, the occurrence of septa and septal pores could be important in influencing cytoplasmic continuity and the potential for long distance, symplastic translocation. Mycorrhizal fungi come from many different taxonomic groups with different septation, yet all can translocate. In zygomycetes, including VA mycorrhizal fungi, the healthy vegetative hyphae are aseptate and cytoplasm is continuous throughout the multinucleate mycelium. Old parts of the mycelium may be isolated by complete septa which are assumed to prevent not only cytoplasmic but also nutrient movements. In ascomycetes (most ericoid and some ectomycorrhizal fungi), the frequent septa are perforated by simple pores with or without Woronin bodies, which allow the passage of cytoplasm and organelles including nuclei (Burnett, 1976; Marchant, 1976; Rees et ah, 1994). The cytoplasm is continuous through these pores with cytoplasmic streaming between the compartments. The dolipore septum characteristic of basidiomycetes and therefore of most ectomycorrhizal and orchid mycorrhizal fungi is much more complex. Its development in the ectomycorrhizal fungus P. tinctorius has recently been described in detail by Orlovich and Ashford (1994), following freeze substitution which causes less cellular distortion than the chemical fixation used in most earlier investigations. They have confirmed that there is symplastic continuity through the septal pores and associated parenthosomes (septal caps), and that the pores in the parenthosomes are of the appropriate dimensions to exclude organelles from the septal pores, preventing blockage. However, tubular cystemae do pass through both the parenthosomal and septal pores and are connected with the hyphal vacuole system (Fig. 14.2). The tubules transfer the fluorochrome 6-carboxyfluorescein between adjacent cells at the tips of the hyphae (Shepherd et ah, 1993a,b). The tubules (likened to endosomes in cultured animal cells; Ashford and Orlovich, 1994) have received the most attention in the apical and subapical cells of hyphae of P. tinctorius. They move in both directions across mature septa independently of cytoplasmic streaming, with peristaltic movements appearing to transfer material between the hyphal compartments (Shepherd et ah, 1993a,b). The behaviour of the tubules close to dolipore septa in the living system suggests that their orientation and direction of movement are controlled (Shepherd et ah, 1993b), and electron microscopy (Orlovich and Ashford, 1994) indicates that this may be brought about by the pores in the parenthosomes which guide the tubular cysternae to the septal pore 'like the spokes of an umbrella'. According to Rees et al. (1994), the basidiomycetes, including some other mycorrhizal fungi such as Paxillus involutus and Suillus granulatus, and also ascomycetes, including Hymenoscyphus ericae, all had vacuole and tubule systems similar to P. tinctorius. The saprophytic zygomycetes surveyed also had tubule systems associated with vacuoles, but these

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Figure 14.2 Time-lapse photomicrographs in the same focal plane showing movements of the tubular reticulum in Pisolithus tinctoriuSy following loading with carboxyfluorescein. Note changes in the location of tubules (t) and vacuoles (v). Bar, 20 |xm. From Shepherd et o/. (1993b), Journo/ of Cell SurfacCy with permission of the Company of Biologists.

appeared smaller and more fragile and did not form an extensive reticulum as in the basidiomycetes and ascomycetes. Germ tubes of Gigaspora margarita contain tubules similar to other zygomycetes (S. Dickson and A.E. Ashford, unpublished observations). Although tubules are most active in the apical cells of the hyphae.

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they are also present in older regions, leading Ashford and Orlovich (1994) to suggest that they may have a role in long distance translocation of solutes in the mycelium. Discovering the roles of tubules and cytoplasmic streaming in translocation should be a fascinating area of research in the next few years. The Rates of Translocation Measurements of the rates of translocation of nutrients in hyphae, expressed as specific mass transfer, mol m~^ s~^ are essential before problems of the relative efficiency of translocation in different directions by different fungi in various environmental conditions can be resolved satisfactorily. Such rates are also important when considering the role of mycorrhizal fungi in plant nutrition. Expression of the rate of translocation as m h~^ (Jennings et ah, 1974; Burnett, 1976; and see Harley and Smith, 1983, for discussion) is useful if mass flow of solution is the mechanism involved, but can only provide information on relative efficiencies of translocation if the concentrations of the solutes are also known (Jennings, 1987). Moreover, estimates of the rate of movement by determining the time of first appearance of a dye or tracer at a particular point in a hypha are generally unhelpful. This is strongly influenced by the sensitivity of detection and only useful for making comparisons if the translocation profile is sharp, which is not the case for any mycorrhizal fungus investigated so far (Canny, 1960; Harley and Smith, 1983). Rates of translocation of nutrients in hyphae of Glomus mosseae to Trifolium repens growing in split-plates are given in Table 5.3. For P, specific mass transfer was between 2 and 20 X 10~^ mol m~^ s~^ (Pearson and Tinker, 1975; Cooper and Tinker, 1978, 1981). Reduction in transpiration of the plants reduced the rate of translocation and, if we can assume that uptake of ^^P by the hyphae was not affected by changed water relations, then the results indicate that mass flow of solution in the fungal hyphae can contribute to translocation. Similarly, the distance over which Hymenoscyphus ericae could translocate ^^F was increased from 6 to 25 mm when the fungus was associated with transpiring seedlings of Vaccinium macrocarpum (Pearson and Read, 1973b). However, mass flow was discounted as a major mechanism of translocation in ectomycorrhizal mycelium by Finlay and Read (1986b), who showed bidirectional translocation of ^^P in the mycelium of Suillus bovinus associated with Pinus sylvestris. In the rhizomorphs of this fungus the specific mass transfer of P was 2-11 X 10~^ mol m~^s~^ - close to the values for VA mycorrhizal fungi. Sanders and Tinker (1973) used total uptake of P by plants of Allium cepa (rather than tracers) to calculate the specific mass transfer of P through the wide entry-point hyphae of G. mosseae. The rates were one to two orders of magnitude higher (see Table 5.3) than the estimates for the mycelium as a whole, probably reflecting the channelling of P through the entry points where the flux would be expected to be maximal. If there is a significant effect of transpiration it might also contribute to the high fluxes, because the plants used by Sanders and Tinker (1973) were grown in soil, rather than in agar plates. An additional feature of experimental results obtained using tracers, which needs to be explained in any mechanism of translocation, is the lag phase that frequently occurs after application of ^^P to split-plates and before a steady rate of appearance in the host (Fig. 14.3). Suggestions that this lag is due to a delay between arbuscule

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formation and degeneration (Cox and Tinker, 1976) can, as Cooper and Tinker (1978) point out, be discounted, for the lag occurs not only in appearance of ^^P in the shoots but also in whole plants. It seems likely that the lag phase comprises two factors: a simple delay in time of appearance of ^^P owing to the distance between the source and the site of detection; and a delay caused by the equilibration of tracer (^^P) with ^^P already present in the translocation pathway. In all mycorrhizal types the large amounts of polyphosphate present in the mycelium and in mycorrhizal roots would constitute a considerable reserve of "^^P. Thus, although translocation of total P [^^P + ^^P] occurs at a constant rate, this will not be apparent from measurements of radioactivity until the specific activity of P is uniform throughout the translocation pathway (see Harley et ah, 1954, for discussion of this problem with respect to movement of ^^P through the sheath of ectomycorrhizas). Orchid mycorrhizas offer an opportunity of studying both mineral nutrient and C translocation into a host plant. Translocation of both ^^P and ^^C has been demonstrated (see Chapter 13), but unfortunately the published studies do not include calculation of fluxes. However, as shown in Chapter 13, a rough indication of C translocation in hyphae of Rhizoctonia species can be obtained from the growth of protocorms by making assumptions about the number of mycelial connections with the medium. The transfer of trehalose was estimated very approximately as between 0.12 and 1.2 X 10~^ mol m~^ s~^ and is similar to values obtained for Armillaria mellea and Serpula lacrymans, of between 0.32 and 1.0 X 10~^ mol m~^ s~^ (Jennings, 1987). Data for the rates of translocation of other solutes are few, but what there is

I

2

3

Days after addition of '^P Figure 14.3 Transport of ^^P across a diffusion barrier by Glomus mosseae in split-plate. The host plant was Allium cepa. # , Total P transported (detected in roots, shoots, soil and external mycelium); O, P transported to the shoots. Drawn from data presented as a table in Cooper and Tinker (1978).

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indicates relatively rapid rates for ^^N (0.74 X 10"^ mol m"^ s"^) and lower rates for ^^Zn and ^^S (0.02 and 0.17 X 10"^ mol m~^ s " \ respectively), in agreement with the relative amounts absorbed by the VA mycorrhizal plants with which the fungi were associated.

Which Solutes are Translocated? The identity of the solutes (organic or inorganic) translocated by mycorrhizal fungi is not known with any certainty. There are some indications that trehalose is the form in which sugars move, at least in orchid mycorrhizal fungi. Trehalose was the first fungal sugar to appear in protocorms of Dactylorhiza purpurella, after feeding the associated mycelium of Rhizoctonia with ^^C-glucose (Smith, 1967; and see Fig. 13.10). Trehalose, mannitol and arabitol were the predominant forms of organic C in the ectomycorrhizal fungi investigated by Soderstrom et al. (1988), leading them to suggest that while trehalose was probably the main storage carbohydrate, the others were probably involved in translocation. This evidence is circumstantial at best, and experiments designed to determine which solutes arrive first at discrete sinks are required. Glutamate together with its amide, glutamine, are probably important in N translocation, as they are the forms into which inorganic N is assimilated in the external mycelium and in excised ectomycorrhizal roots. Glutamine (plus alanine in some systems) is probably the main N compound transferred from fungus to plant across the interface. There is no information about the form of N translocated in other mycorrhizal types.

Polyphosphate in Mycorrhizal Systems Uptake of Pi by mycorrhizal fungi is followed (as in many microorganisms) by synthesis of a large amount of polyphosphate which is stored in the fungal vacuoles. Much early work suggested that the polyphosphate occurred as granules, stabilized by Ca^"^ or arginine (Ashford et al., 1975; Ling-Lee et al., 1975; Callow et al, 1978; Cox et al, 1980; Capaccio and Callow, 1982). Hypotheses were formulated involving these polyphosphate granules in both storage and translocation in fungal hyphae and in the sheath tissues of ectomycorrhizas. It is very important to stress that the granules are probably artefacts of specimen preparation (Beever and Burns, 1980; Orlovich and Ashford, 1993; and see Fig. 14.4) and hypotheses depending on their behaviour must be re-evaluated. There were early indications from nuclear magnetic resonance (NMR) spectroscopy (Harley and Smith, 1983; B.C Loughman, personal communication) that relatively short-chain, soluble polyphosphate occurs in the fungal sheath of mycorrhizas of Fagus. The data of Martin et al. (1985), again using ^^P NMR, also indicated a large, soluble polyphosphate fraction in the mycelium of Cenococcum geophilum and Hebeloma crustuliniforme. Polyphosphate present as insoluble precipitated material (granules) would not be detected by NMR spectroscopy. Importantly, the behaviour of polyphosphate in the fungal mycelium was similar to that revealed by counting the (artefactual) granules in excised mycorrhizal roots (Chilvers and Harley, 1980; StruUu et al, 1981a,b, 1982). Polyphosphate accumulation

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General themes

Figure 14.4 Effect of fixation methods on the preservation and distribution of polyphosphate in Pisolithus tinaorius. (a) Transmission electron micrographs of a glutaraldehyde-fixed hypha embedded in Spurr's resin and triple stained. Note the spherical opaque granules (*) in vacuoles (V). (b) Transmission electron micrograph of a stained ultrathin section of part of a freeze-substituted hypha near a clamp connection. The vacuoles (V) contain electronopaque material which is evenly dispersed. There are no discrete granules. Bars, I ^m. From Orlovich and Ashford (1993), Protoplasma, with permission.

varied with different stages of growth, being low when growth was rapid in young myceUa and linear in the early and late stages of the stationary phase when P in the medium was relatively abundant compared with N. When the mycelium was transferred to low-P medivim, the NMR spectra indicated mobilization of polyphosphate - rapid in H. crustuliniforme and more slowly in C. geophilum. Martin et al (1985) discussed the apparently conflicting evidence on the form of polyphosphate, concluding that the spin-lattice relaxation times of the ^^P nuclei in both fungi were consistent with a single pool of relatively fluid polyphosphate, possibly in the form of 'macromolecular aggregates'. They certainly found no evidence for granules and considered the investigation of purified granules by NMR spectroscopy to be 'particularly urgent'! Recently, Orlovich and Ashford (1993) have obtained data from anhydrous freeze-substituted material, which indicate that in P. tinctorius polyphosphate is uniformly distributed in the fungal vacuoles and is stabilized by K^ (Fig. 14,5). Polyphosphate of about 15 Pi units was extracted from the mycelium and identified by chromatography, gel electrophoresis and ^^P NMR. This investigation also illustrated the formation of granules stabilized by Ca^^ during chemical fixation, explaining the widespread observation of these 'structures'. No reinvestigations of the form of polyphosphate in VA or ericoid mycor-

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

1

p

391

320 cnt 1 K

Ir iiiii iiiiiiwillll 320[ cnt

h

^..jkiAitfaui

Figure 14.5 Distribution of P and K in a hypha of Pisolithus tinctorius. (a) and (b) Energy-dispersive X-ray spectra from the analysis of an unstained, freeze-substituted hypha cut dry at I \im. The full vertical scale, measured in Xray counts, is at the top right of each spectrum, (a) Vacuole showing large peaks for P and K. (b) Cytoplasm, showing relatively small peaks for P and K. (c) Transmission electron micrograph of a hypha near a clamp connection. Note the large vacuole (V). Bar, 2 |im. (d) and (e) X-ray maps of the hypha in (c), showing the similar distribution of: (d) P and (e) K in the vacuole. From Orlovich and Ashford (1993), Protoplasma, with permission.

392

General themes

rhizas have yet been carried out. Polyphosphate is located in vacuoles and in the tubular cystemae of mycorrhizal fungi (Cox et al., 1980; Orlovich and Ashford, 1993; Ashford et al, 1994). This compartmentation is important in discussions of the possible mechanisms of translocation.

The Mechanism of Translocation Harley and Smith (1983) discussed the evidence for the hypothesis that translocation in fungi might involve the movement of material in discrete compartments or 'packets' of material, powered by cytoplasmic streaming. The ideas were developed from the suggestion of Cox et al. (1980), that the polyphosphate 'granules' in vacuoles of VA mycorrhizal hyphae contained enough P to explain the measured rates of P translocation if they vy^ere moved directionally by cytoplasmic streaming. This cannot now be regarded as at all credible. However, vacuoles containing polyphosphate (and of course other solutes) could be involved in net directional movement (translocation) as long as certain conditions are met. Translocation must occur down a concentration gradient, between a source and a sink. Streaming (either unidirectional or bidirectional) could result in movement of polyphosphate-containing vacuoles, but well organized 'loading' and 'unloading' systems must be envisaged at the source and sink, the rates of which will be important in determining the rate of translocation. The system of motile tubules, described above, provides another possible translocation pathway which would be independent of cytoplasmic streaming. These tubules appear to provide a means whereby their contents (including polyphosphate) can be moved between vacuole clusters, potentially for long distances in the mycelium. The fact that tubules can operate peristaltically in both directions in a hypha also provides a possible mechanism for bidirectional translocation of whatever is contained in them (Ashford and Orlovich, 1994), The tubules, as we have said, certainly do accumulate, retain and transfer carboxyfluorescein over short distances (Fig. 14.2). Unfortunately for any hypothesis involving movement of polyphosphate in the vacuoles, there is little evidence to support the idea of localization in a form which does not equilibrate reasonably rapidly with the cytoplasm. In most organisms in which polyphosphate metabolism has been studied, the synthesis of polyphosphate is regarded as a method of 'mopping u p ' large amounts of Pi from the cytoplasm (see Harold, 1966; Beever and Bums, 1980), and the data of Chilvers and Harley (1980) and Martin et al (1985) are consistent with this idea. However, there is some slender evidence for spatial separation of different aspects of polyphosphate metabolism in the intraradical and extraradical mycelium of VA mycorrhizas. The breakdown of polyphosphate can be envisaged as occurring by two reactions: by hydrolysis mediated by polyphosphatase, or by a reversal of polyphosphate kinase with the formation of ATP in the presence of ADP. Enzymes of polyphosphate metabolism have been detected in VA mycorrhizas by Capaccio and Callow (1982). They found low activities of exopolyphosphatase in non-mycorrhizal roots of onion. Mycorrhizal roots contained higher activities of this enzyme and also of endopolyphosphatase, but no polyphosphatase activity was found in external hyphae. Polyphosphate kinase was detectable in mycorrhizal roots and external hyphae, but not in non-mycorrhizal roots, while polyphosphate hexokinase was

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

393

found only in the external hyphae. The absence of polyphosphatases in external hyphae is interesting, as it suggests that polyphosphate turnover might be low and the vacuoles therefore 'tight', but much more work on the control of polyphosphate metabolism is required before this could be regarded as certain or indeed likely. At this stage there are two possibilities: (a) the vacuoles and interconnecting tubules are sufficiently impermeable to effect long distance translocation; or (b) the vacuoles equilibrate relatively rapidly with the cytoplasm. Even in the second case they could still be involved in translocation by increasing the rate of mixing of solutes along a concentration gradient in the hypha, thus facilitating a basically diffusive mechanism. If concentration gradients for different solutes are differently polarized in the mycelium, bidirectional translocation would occur as long as the mixing mechanism is also bidirectional. No doubt the discovery of the motile system of vacuolar tubules will act as a spur to revitalize investigations of the mechanisms of translocation in fungi in general and mycorrhizal systems in particular. It will be important to obtain more data on the identity and amounts of the solutes translocated and contained within the vacuolar systems and to ascertain whether they operate consistently throughout the mycelial networks.

Transfer of Nutrients Between the Symbionts Development of Mycorrhizal Interfaces In the majority of mycorrhizal types, organic C (probably sugar; see below and Chapters 4 and 7) moves from the autotroph to the heterotroph, while nutrients derived from the soil pass in the opposite direction. The molecules transferred may be organic or inorganic, but the result must be net movement of P, N, Zn, Cu and so on, into the plant (see Chapters 5, 8 and 9) and of C to the fungus. It has generally been assumed that this transfer takes place at the same time, across the same interface. In other words, it is envisaged that each functional interface is individually specialized for bidirectional nutrient transfer. However, in some mycorrhizal types there are several different and distinct interfaces and no mycorrhizal organ of any type has a static form or structure. They all have an initial stage of colonization, and then their constituent tissues, both of host and of fungus, undergo a sequence of development to a mature form before they senesce, either both components simultaneously or in sequence (see Chapters 2, 6, 11, 12 and 13). The most evanescent structures are the intracellular arbuscules of Arum-type VA mycorrhizas, which may grow, mature and degenerate all within about 15 days, and at the same time as new ones are formed in other cells (see Chapter 2). In these mycorrhizas, the intercellular hyphae a n d / o r the relatively robust intracellular coils probably form a persistent living 'skeleton' for the fungus. Ericoid mycorrhizas are similar in that the intracellular hyphal complexes are short-lived (see Chapter 12) but in this case the whole cortex of the hair root is also a much more transitory structure than that of most roots, and the protoplasts of the cell and of the fungus seem to degenerate almost simultaneously, although the fungal hyphae may outlast the host. The fungi, including the arbuscules and coils, of Paris-type VA mycor-

General themes

394

Table 14.1 Types of interface present in different mycorrhizal types, showing the occurrence of intracellular and intercellular Interfaces, together with the occurrence of ATPases on the interfacial membranes (see text) Mycorrhlza

Structure

VA

Intercellular

Intracellular

Structure

ATPase

Plant

Plant

Fungus Hyphae

Arbuscules

ATPase Fungus +a.b (+)

Orchid

Coils

ND +

(+) ND +

None

NA

NA

Ericoid

Coils

ND

ND

None

NA

NA

Monotropoid

Pegs and

ND

ND

Hartig net

ND

ND

Coils

menfibranous sac Ecto

None

NA

NA

Hartig net

+*>

+''

Ectendo

Hyphal

ND

ND

Hartig net

ND

ND

ND

ND

Hartig net

ND

ND

complexes Arbutoid

Hyphal complexes

NA, not applicable; ND, no data available; -^, ATPase activity, demonstrated cytochemically, always present; (+), ATPase activity not always present *, Vanadate-sensitiv€5; **, DES-sensitive.

rhizas are reported to be relatively long-lived but more work on these associations is needed. In the mycorrhizas of orchids the fungus forms physiologically active coils in the cells which later degenerate. The cell of the host outlives the fungus and may be recolonized (Chapter 13). Ecto and monotropoid-mycorrhizal roots appear to have greater longevity than other mycorrhizal types (Chapters 2 and 6). However, not only are there distinct stages during development which exhibit different cellular interactions in the interface, but many root systems are also differentiated into long and short roots which have differing life spans and, in the most extreme examples, the short roots persist for months only This diversity of structure and development (see Table 14.1) offers the potential for differentiation of function, both temporally and spatially in a root system. Any of the stages of development or degeneration may be involved in transfer of nutrients and might contribute to this specialization. The exact location of the symbiotic interfaces within the tissues of the root may also be relevant to the mechanisms and controls of nutrient transfer. Localization of the Fungus in an Apoplastic Exchange Compartment Despite the variations in structure and development, the interfaces at the cellular level have the same basic structure (Fig. 14.6). In all instances the fungus actually colonizes the root apoplast and remains strictly outside the plant protoplast. The fungus may be located in the intercellular spaces of the root, or closely associated with the walls of the epidermis and cortical cells, or it may penetrate intracellularly and create a new apoplastic compartment between the fungal cell wall and the

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses INTERFACIAL '^-jmrri-z^ APOPLAST

FUNGUS

efflux

395

PLANT

influx

mineral nutrients P, N, Zn, S etc

—-•

influx

efflux 'carbohydrate

FPM

FW

PPM

Figure 14.6 A highly simplified diagrann of a symbiotic interface, indicating the key components including the interfacial apoplast bounded by the fungal plasma membrane (FPM) and plant plasma membrane (PPM). The arrows indicate membrane transport processes involved in transfer of carbohydrate and soil-derived nutrients. Dashed lines, efflux; solid lines, influx. Wall of the fungus is indicated by shading, but plant wall and walllike components not shown.

plant plasma membrane (Smith and Smith, 1990). In the mature state, cells of both symbionts are alive and the interface is therefore delimited by two plasma membranes, one from the plant and one from the fungus, separated by an interfacial zone or apoplast. There are, of course, differences in detail between different types of interface, particularly in the wall components and other materials found in the interfacial apoplast, as well as variations in membrane activity deduced from structure and ATPase activity. However, in all cases the fungus actually colonizes the apoplast and the environmental conditions there will be very important for all aspects of fungal development and also for nutrient transfer. Indeed, the need for an apoplastic step is recognized as a means of exercising control at the membrane transport level in phloem unloading and in the transfer of sugars to fruits and seeds (e.g. Humphreys, 1988; and see Patrick, 1989). Transfer of sugars from the plant to the fungus is obviously similar to these processes in many ways and the transfer of mineral nutrients needs similar control steps operating in the opposite direction. The interfacial zone normally includes the wall of the fungal hypha which may or may not show some structural modification and/or reduction, and also material derived from the activity of the plasma membrane of the host. In ectomycorrhizas and in the intercellular interfaces of other kinds of mycorrhizas, the interfacial apoplast may consist of a relatively unmodified host wall and a more or less modified middle lamella; or, it may be extremely modified to form a specialized contact layer referred to as the 'involving layer' (see Chapter 6). In the interfaces formed within cells there is a matrix between the plasma membrane of the host and the fungal wall which under the electron microscope is unlike the plant cell wall. At the base, where the trunk of the arbuscule or

396

General themes

penetrating haustorium or hypha passes through the wall of the host, it is usual to find that a layer of host wall, the collar, encloses the hyphae. This wall layer becomes progressively thinner away from the peripheral cell wall and is very thin or absent over the surface of most of the intracellular fungus. Instead, an interfacial kyer, often containing vesicles and fibres derived from the host plasma membrane, may be present. It appears as though the plant maintains some ability to secrete carbohydrate and polymerize it into fibres, but is unable to organize them into recognizable wall. This ability may be regained as the fungus senesces and becomes encapsulated in a thick fibrous layer. How far the wall and wall-like materials are involved in or influence transfer of nutrients across the interface is not at all clear. In the interfaces where transfer is assumed to occur there is no real evidence as to the permeability of wall-like materials, although the assumption must be that, like primary cellulose walls of plants and chitinous walls of fungi, they are permeable to the solutes that pass from one organism to the other. Impermeable structures, like the 'neckbands' associated with the haustoria of some biotrophic fungal pathogens, have rarely been observed (Smith and Smith, 1990). However, an electron-dense ring occurs in monotropoid mycorrhizas where the membranous sac bursts through the 'peg' (see Fig. 11.24) and at the point of entry of ericoid hyphae into cells of the root. These neckbandlike structures may be important in isolating part of the plant-fungus interface, permitting tighter control of conditions in the interfacial apoplast, but their effectiveness in sealing the fungus into an intracellular exchange compartment has not been tested. It could be significant that both these examples involve situations where the fimgus colonizes the epidermis of the plant and is thus topographically outside the hypodermis (see below). The apoplastic phase of the fungal sheath of some ectomycorrhizas is impermeable, and hence must be a barrier to nutrient movement between soil and root (Ashford et al, 1988,1989). Plate 6 shows the way in which entry of cellufluor to the roots of Eucalyptus pilularis is prevented by the fungal mantle of Pisolithus tinctorius (Plate 6a) unless the rootlets were deliberately damaged to remove the outer, unwettable region of the mantle (Plate 6b). Sections to which cellufluor was applied directly (Plate 6c) showed the characteristic turquoise fluorescence of this dye. The presence of the impermeable layer means that all solutes reach the root cells via the fungal symplast of the sheath, first by translocation in the external mycelium and subsequently by efflux to the interfacial apoplast in the Hartig net region. Conversely, solutes from the root cells effluxing to the apoplast must pass to the fungal symplast and cannot 'leak' to the soil via the apoplast of the fungal sheath. The impermeable layers thus offer an opportimity for control of conditions and solute concentrations in the cortical apoplast and Hartig net region, where transport between the ectomycorrhizal symbionts must occur. As Ashford et al. (1989) point out, maximum efficiency requires that material must not be allowed to escape from the 'exchange compartment' (Fig. 14.7). The initial work on apoplastic impermeability was carried out with Pisonia grandis, in the mycorrhizas of which there is no Hartig net. A similar, but slightly differently organized exchange compartment could exist where the Hartig net penetrates as far as the outer layer cortical cells; in this case the inner boundary would be the endodermis, which again provides a block to apoplastic transfer. In this context it is relevant that a fungal hydrophobin gene is up-regulated during ectomycorrhizal development (Martin

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

wall

outer apoplastic barrier sheath or hypodermis

397

inner apoplastic barrier hypodemriis or endodermis exchange compartment

cytoplasi

fungus in soil

fungus in root (intracellular or intercellular)

plant cell associated with intracellular fungus or Hartig

net

Figure 14.7 Diagramnnatic representation of the proposed apoplastic exchange compartments in mycorrhizal roots (see text), v, Vacuolar compartments; light-shaded areas, cytoplasm (symplast); dark-shaded areas, apoplast; black areas, impermeable apoplastic barriers. Transport of phosphate (P) and sugar (C) through the apoplast (dashed lines and arrows) and symplast (solid lines and arrows).

and Tagu, 1995; and see Chapter 6). The products of this gene are likely to play a significant role in cementing together the fungal hyphae and forming an impermeable and unwettable layer. The fungal sheath has not been reported to be impermeable in all investigations and the discrepancies may relate to different plant-fungus combinations as well as to different experimental methods. The external mycelium of some species of fungi is also covered with non-wettable material (Unestam, 1991). It is not yet clear whether this is important in long distance translocation within the rhizomorphs or whether it might play a role in preventing desiccation as has been suggested for the aerial hyphae of some saprophytic fungi, or indeed whether both these attributes are important. In mycorrhizas without a fungal mantle, any control of apoplastic conditions in the root cortex must lie with the root cells themselves. Here, the suberized cell walls in a well developed hypodermal layer beneath the epidermis may play the same role as an impermeable sheath in ectomycorrhizas. This hypodermis has been known for at least a century (e.g. Janse, 1897) and its widespread occurrence and significance has been emphasized in recent reviews (Peterson, 1988). The tangential walls of the hypodermis become increasingly impermeable as the root matures, essentially insulating the cortex from the soil. Entry (or exit) of solutes must occur in very young regions, or through the symplast of non-suberized 'passage cells', or via the hyphae of mycorrhizal fungi, which themselves enter via the passage cells (Peterson, 1987; Smith et al, 1989). Thus the fungus does not breach the suberized layers and may very well be exploiting the mechanism by which the non-mycorrhizal roots control the solute concentrations and conditions in the cortical apoplast. Again, the apoplast of the root cortex may function as a shared 'exchange compartment'.

398

General themes

Membrane Transport in the Interfaces The first step in transfer across a symbiotic interface must be efflux from the 'donor' organism into the interfacial apoplast. It is important here to correct the impression given by Harley and Smith (1983) that increased permeability, or 'leakiness', of a non-specific sort could be important in such mycorrhizal transfer systems. It is much more likely that increased rates of loss of individual solutes occurs to support overall rates of transfer between the symbionts, and that this is achieved by separate control of specific carriers or channels (Tester et al., 1992; and see above). No channels have yet been found, but techniques that will make the search easier are now becoming available. Conditions in the.apoplast that would promote efflux must be sought. It seems likely that mechanisms that are involved in phloem unloading in other tissues, could operate in the mycorrhizal exchange compartments, for loss of sucrose from the plant. This would require only a change in the tissues involved in expression of the relevant transport proteins. Mechanisms that might be involved in high efflux of P, Zn or amino acids from the fungus are unknown. Efflux must be followed by uptake from the interfacial apoplast by the 'receiver' organism. Membranes involved in uptake by proton co-transport must be energized by an H^-ATPase to generate the necessary PMF (Michelet and Boutry, 1995). The distribution of H^-ATPases on the membranes in the interfaces may therefore provide information on which membranes are important in uptake from the apoplastic compartments (Table 14.1). The distribution of H'^-ATPases in VA mycorrhizas has been determined cytochemically by Pb deposition (Fig. 14.8). The results of Marx et al. (1982) suggested that active ATPases were present on both plant and fungal membranes in the arbuscular interface and thus provided support for bidirectional transfer of nutrients (Fig. 14.9a). Later work has thrown some doubt on this interpretation. Some of the ATP-hydrolysing enzymes have been identified as H^-ATPases but others are probably non-specific phosphatases (Gianinazzi-Pearson et al., 1991a; see Fig. 14.8). Important points to note are that when non-specific phosphatases are inhibited with Mn^"*^, the periarbuscular membrane of the plant and the fungal plasma membrane of the intercellular hyphae retain consistent high ATPase activity. In both cases the opposed membranes have weak or absent activity (Table 14.1), which suggests that spatial separation of transfer functions may occur (Fig. 14.9b,c). The suggestion is that sugar is transferred from plant to fungus in the intercellular interface. Here, sucrose would efflux from the cortical cells of the root, be hydrolysed to hexose, which would then be actively absorbed by the fungus. In the arbuscular interface it is the plant that has an active H'^-ATPase. Here, efflux of P (or Zn, or organic N, etc.) to the apoplast would be followed by active uptake by the plant. The absence of H"^-ATPase activity on one of the membranes in each interface suggests a mechanism whereby reabsorption of solutes by the 'donor' cell (plant or fungus, depending on which interface) might be reduced, thus polarizing transfer across the interface as a whole. Similar methods have been applied to orchid and ectomycorrhizas, although work with the latter system did not involve inhibitors (Serrigny and Dexheimer, 1985; Lei and Dexheimer, 1988; and see Table 14.1). In these symbioses the interfaces were

399

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

B

:• \ s i * -

V ^^.^^

- . • /• / ' •

. — ^

y

..

^^,u

•

,

r .

Figure 14.8 Transmission electron micrographs of details of the mycorrhizal interaction between Allium cepa and Glomus intraradices. Sections of roots fixed and stained to demonstrate ATPase activity by the deposition of Pb phosphate which appears as fine electron-dense precipitates. (A), (B) and (C) No inhibitors; (D) in the presence of molybdate, which inhibits non-specific phosphatases but not H'*'-ATPases. (A) Extraradical hypha (h) at the root surface; epidermal cell (ec). (B) Intercellular hypha, in the intercellular space (is) between host cortical cells (he). (C) Arbuscular trunk (at) hypha in a cortical cell (he). (D) Fine arbuscular branches (ah) within a cortical cell (he) surrounded by periarbuscular membrane (RAM). Arrows in all parts of the figure indicate the presence of ATPase activity on fungal (A)-(C) and plant (D) membranes. Bars, O.Sjiim. The plate is reproduced from Smith and Smith (1996b), with permission. Origins of figures: (A)-(C), V. GianlnazziPearson, unpublished; (D), from Gianinazzi-Pearson et al, (1991a).

400

General themes

characterized by ATPase activity in opposed membranes of single interfaces. Data are not available for other mycorrhizal types, as indicated by the gaps in Table 14.1. What must emerge from a discussion of nutrient transfer between the symbionts is a picture of considerable complexity (see also Smith and Smith, 1990). For each solute molecule or ion transferred there must be a separate membrane protein, whether it be a carrier or pump or channel (Bush, 1993). This point is made forcibly in Figure 14.1 which shows a schematic representation of transport processes operating in fungi. Similar processes operate in plants, and in both organisms modifications to promote transfer between the symbionts are probable. Even if the complexities are initially intimidating. Fig. 14.1 at least emphasizes that the view of mycorrhizal transport systems as simple phosphate-sugar exchange must be unrealistic. Melin and Nilsson (1950-1958) found that P compounds, cations including Ca^"^ and N compounds derived from NH4 or glutamate are transferred from the substrate to the host via the fungus, and that ^^C-labelled photosynthates pass from the host to the fungus. Other observers have shown that ^^C-labelled compounds may also pass back across the interface into the host, a process which could have considerable significance in interplant transfer of organic C, if the transfer is shown to occur in nutritionally significant quantities. These general findings have been confirmed using a variety of plant-fungus combinations. In the early studies there were no real clues on the identity of the solutes moving across the interface, but there is now more information, especially for ectomycorrhizas. Organic C probably effluxes from the plant to the interfacial apoplast as sucrose, and data supporting this have been obtained for ectomycorrhizas. It is envisaged that sucrose is then hydrolysed by an acid invertase of plant origin and the hexoses resulting are absorbed by the fimgus. The way in which the transfer might be controlled by the pH of the apoplast and the concentrations of fructose has been discussed (see Chapter 7; and Fig. 7.3) and at this stage the hypotheses must be regarded as speculative, pending the availability of data on apoplastic conditions. NMR spectroscopy has shown that ^^C-glucose fed to VA mycorrhizal roots does pass to the fungus (Shachar-Hill et al., 1995; and see Chapter 4). In intact systems sucrose probably effluxes from the plant and is hydrolysed in the apoplast, so that Figure 14.9 Diagrammatic representation of the possible spatial distribution of H"*"ATPases and associated transport processes in VA mycorrhizal interfaces, based on data for molybdate-sensitive ATPase activity. (A) Bidirectional transfer of sugar and PO^ across the same arbuscular interface. Both plant and fungal membranes are shown with H'^-ATPase activity. (B) Transport of PO^ from fungus to plant across an arbuscular interface. Absence of H"*'-ATPase on the fungal plasma membrane (Fig. I4.8d) could be associated with passive loss from hyphae to interfacial apoplast, and presence with active uptake on the plant plasma membrane. (C) Transport of sugar from plant to fungus across the interface between cortical parenchyma cells and intercellular hypha. Little or no activity of H"^ATPase on the plant plasma membranes could be associated with passive loss of sugar to the interfacial (intercellular) apoplast and presence on the fungal membranes v^ith active uptake into the hyphae (Fig. 14.8c). fpm. Fungal plasma membrane; pam, periarbuscular membrane; rpm, plasma membrane of root cortical cell. From Gianinazzi-Pearson et 0/. (1991a), with permission.

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

401

the uptake of glucose by the fungus represents only part of the process and data on the fates of fructose and sucrose are required. A mechanism involving sucrose efflux and hydrolysis seems likely because plants possess the genetic information that results in specialized sucrose efflux in some tissues. Phloem unloading, for example, involves high rates of sucrose loss from cells to apoplastic compartments where, as in ectomycorrhizal systems, the operation of invertase helps to maintain a concentration gradient favouring further efflux (e.g. Humphreys, 1988). What is needed in a mycorrhizal system is expression of the relevant genes for sucrose

(A)

Soil

Arbuscular interface fpm pann

Fungus

fpm

- Sucrose or hexose

Hexose "

Fungal sugars H*

lATPasel

t

i

•P:

(B) Soil

t

i

lATPasel

lATPasel

t

i

^P:

- ^ PTranslocation

Arbuscular interface fpm pam

Fungus

fpm

Root

lATPasel

lATPasel

i

4^1

PT-

(C)

Root

•PT

Translocation'

Soil

Intercellular Interface

Fungus fpm

fpm

Hexose -4 i H^

Fungal sugars

i H^ lATPasel

F i g u r e 14.9 (Caption opposite)

"^^^

rpm Sucrose or hexose

402

General themes

transporters in cells associated, either inter- or intra-cellularly, with the fungal symbiont (Patrick, 1989). Uptake of hexoses by the fungal symbionts is presumably active, involving proton co-transport. The presence of H'^-ATPase activity on the fungal hyphae of the Hartig net region of Pinus sylvestris-Laccaria laccata ectomycorrhizas (Lei and Dexheimer, 1988) and in the intercellular hyphal interfaces of VA mycorrhizas formed between Allium and Glomus species, would be consistent with these being the sites of hexose absorption. However, ATPase activity on the fungal membranes in the arbuscules is patchy at best (Gianinazzi-Pearson et al., 1991a; and see Fig. 14.8), so that it is unlikely that they play a continuing role in uptake of sugars (Smith and Smith, 1995). More evidence on the localization of different ATPases and sugar transporters in the interfaces, and of their activity, is urgently required. In symbioses like ectomycorrhizas in which a single interface is presumably involved in bidirectional transfer, mechanisms which essentially maintain polarized transfer of different solutes need to be sought. For sugar transfer there is evidence for the operation of a ^biochemical valve' (Lewis and Harley, 1965c; Smith et al., 1969). The first step is the hydrolysis of sucrose by a plant invertase in the apoplast, which maintains sucrose efflux (see above and Chapter 7). This is followed by absorption of hexoses by the fungus and conversion to forn\s such as mannitol and glycogen, which are unavailable to the plant. Thus not only does the synthesis of fungal metabolites maintain a concentration gradient in favour of continued uptake, but any reversal of transfer is also prevented. If any solute did move back to the apoplast it would not be absorbed by the plant, but would accumulate and rapidly dissipate any concentration gradient and prevent further passive efflux. With respect to movement of soil-derived nutrients from fungus to plant, there are data for P and for N. Harley and Loughman (1963), through short-term labelling experiments with excised roots of Fagus, showed that orthophosphate passed from fungus to host and it is generally assumed (with little or no experimental data to support the contention) that the same applies to all mycorrhizal systems in which FO4 transfer takes place. Again, the electrochemical potential difference between the hyphae (Hartig net, hyphal complexes, arbuscules and so on) and apoplast will favour passive efflux from the fungus. Despite this, efflux of P from cells, including fungi (see above) is usually much lower than the measured rates of POJ transfer from fungus to plant in VA mycorrhizas. Mechanisms which promote efflux, and at the same time reduce retrieval or reabsorption, seem likely and the low expression of the fungal PO^ transporter (GvPT) within roots, compared with external mycelium (Harrison and van Buuren, 1996; and see above), is consistent with this suggestion. Data for rates of transfer across the symbiotic interfaces of other types of mycorrhizas are urgently required. There is little doubt that in ectomycorrhizas assimilation of N (either inorganic or organic) by the fungus is followed by transfer of organic N to the plant. In Fagus mycorrhizas, the important role of glutamine in this process was indicated the incorporation into it of ^^C, following dark fixation of ^^۩2 in the presence of NH4 and rapid transfer to the plant. The enzymic pathways show that there is a glutamine-glutamate shuttle which results in the net transfer across the interface of one N per glutamine, which is then transferred to a-ketoglutarate in the plant with the formation of glutamate (see Chapter 8 and Fig. 8.5a). In other plant-fungus

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

403

combinations, different distributions of enzymes involved in N assimilation give somewhat different pictures (see Fig. 8.5b,c). In Abies mycorrhizas there is also a glutamine-glutamate shuttle, with the N of glutamine being used in the formation of asparagine from aspartate in the root cells. Alanine is also transferred from fungus to plant, with the C skeletons required for a-ketoglutarate synthesis in the fungus being derived from sugars transferred from the plant. In mycorrhizas involving P. tinctorius a simpler picture emerges, with glutamine again the major organic N molecule transferred. However, as all the enzymes for organic N conversions are present in the fungus, there is in this case no cycling of N across the interface and the C skeletons are provided by sugars from the plant. The transfer of glutamine is important because its use does not perturb intracellular pH (Raven and Smith, 1976; Smith, 1980; Smith and Smith, 1990). In the mycorrhizal context this means that all export of H"^ that would be required to maintain intracellular pH during NH4 assimilation would be from the fungus, which has a large surface area in contact with the soil solution. If large amounts of H"^ were generated in the plant, export to the soil would have to be via the symplast of the fungal sheath. Figure 14.10 illustrates movements of K^ and H"^ that would be expected to be transferred across the interface concurrently with organic N in order to maintain charge balance across each membrane. Of course, charge balance also needs to be maintained during transfer of Pi, Zn or Cu. Given that we now believe that polyphosphate is stabilized by K^, it seems logical to propose that both Pi and K might be transferred to the plant, thus maintaining charge balance in both symbionts. The Rates of Transfer There is surprisingly little information on the flux of solutes across the interface. Calculations based on the uptake of P via the fungus and the area of interface available for transfer have only been made for VA mycorrhizas. Values for this flux are in the range 3-30 nmol m~^ s~^ (see Chapter 5), which suggests that abnormally high efflux from the fungus may be required (Smith et ah, 1994a,b; and see above). Data are required for other mycorrhizal types and other nutrients. Bidirectional Nutrient Transfer: Does it Occur at the Same Interface? Simultaneous bidirectional transfer of molecules or ions between the symbionts can only be envisaged when the interface separates two living cells. In mycorrhizas where only one type of interface exists (see Table 14.1) it is likely that the interface and component membranes are modified to promote this transfer. However, where there are two or more different interfaces they could individually be involved in unidirectional transfer, with the combined outcome of bidirectional transfer at the level of the whole mycorrhizal root (Smith and Smith, 1990, 1995, 1996a,b,c; Gianinazzi-Pearson et ah, 1991a; and see above). This idea of spatial separation of function has some attractions for the membrane transport processes that may operate to achieve polarized transfer. What must be emphasized is that whether transfer is bidirectional or unidirectional it is controlled by the two interfacial membranes. Moreover, transfer of any solute will involve efflux from one symbiont

404

General themes

{a)

(b) Soil

fp

Fungus

fp rp

Root

Soil

fp

Fungus

fp

rp

Root Sugar

Sugar

*
2H*4—t

i-^

2H*

2H*4—•

2H*

i

l-j

i-^

aKG^Gin

t—4

•-

^

.^—\

NH4

Glu" i

N

f _y1

1—4

aKG' 2Gln-

A

2NH/ •

Glu'

K*

->

K*

-¥

Soil

fp

Fungus

2H* 4 -

fp

2H* i

rp

Root

\

^

Soil

fp

1—t

2Glu'

•

J

Sugar

Gin

2K*

NH4.

•> 2K*

1

•-

rp

Root

•

^

-

4—\—•Gin

^

Glu" aKG^

NH4* — t

• - ^

2H* i—\

4-Nt

aKG' Glu"

1

fp

IAI

^

NH4^

Fungus

f

Gin

NH/

Gin

(d)

IA

2K*

2Glu" <

K*

{c)

>Gln-J

t—IN

2Glu-

•-^

Sugar4-

f

Sugar

crKG^ 2H* 4 -

r^

Sugar 4

f

- Sugar

F i g u r e 14.10 Possible mechanisms f o r compartmentation of NH4-assimilating enzymes in mycorrhizas (compare w i t h Fig. 8.6). A , interfacial apoplast; G l u ~ , glutamate; G i n , glutamine; a K G 2 ~ , «-ketoglutarate. (a) Glutamine synthetase (GS) in the fungus and glutamate synthase ( G O G A T ) in the roots, w i t h net gain of glutamate in the roots, (b) GS in the fungus and G O G A T in the r o o t , w i t h net gain of glutamine in the r o o t (c) glutamate dehydrogenase ( G D H ) and GS in the fungus and G O G A T in the r o o t , w i t h net gain of glutamine in the r o o t , (d) G D H and GS in the fungus w i t h net gain of glutamine in the r o o t . N e t fluxes of N H 4 , H"^ and K^ are shown and give charge balance across each membrane. In (c) fluxes of N H 4 and H"*^ associated w i t h different parts of the pathway are ( f o r clarity) s h o w n separately at the plasma membrane of the fungus, but it is n o t implied that m o r e than one transporter f o r each ion is required there. Active and passive fluxes are not distinguished. From Smith and Smith (1990), w i t h permission.

into the interfacial apoplast and uptake by the other symbiont (Fig. 14.6). We need to consider whether these processes are modified in symbiosis and if so, how. The concentrations of the molecules transferred will be important, as will conditions such as membrane PD, p H and so on, which could markedly affect membrane function. Furthermore, mechanisms that reduce retrieval of solutes from the apoplast by the donor symbiont are likely to play an important role in polarizing

Uptake, translocation and transfer of nutrients in mycorrhizal symbioses

405

transfer. One of these is the 'biochemical valve' discussed above; others are the altered distribution of H^-ATPases and the low expression of GvPT, the fungal high affinity P transporter, in fungal structures within the root, compared with extraradical mycelium (Harrison and van Buuren, 1996). In situations when one or other symbiont is senescent or dead, only unidirectional transfer can be envisaged. For example, in the pre-Hartig net zone of ectomycorrhizas the cells of the root cap and epidermis are crushed and destroyed so that the fungus might derive material from the dying cells, but transport in the reverse direction to the host would be very unlikely. Similarly, the endophytes of Calluna colonize the mucigel layer of the root surface before penetrating the tissues and may derive C from this source. Conversely, nutrients may be transferred from fungus to root cells following hyphal or arbuscular collapse in orchid, ericoid and VA mycorrhizas, but the amounts transferred would be restricted to the contents of the collapsed hj^hae and, as already mentioned (Chapter 5) are probably insufficient to account for the measured rates of transport. The spatial separation of transfer functions does provide opportunities for differences in efficiency of the symbioses either at different stages of development or in different plant-fungus combinations, depending on the relative abundance and activity of the intercellular and arbuscular interfaces (Smith and Smith, 1995, 1996a,c). The fact that there are differences in efficiency of P transferred per unit C (see Chapters 5 and 8), indicates that the conceptually attractive model outlined by Woolhouse (1975), in which P and organic C were exchanged on a single transporter at the interface, as they are at the chloroplast envelope, is unlikely to provide the correct picture. This was certainly realized by both Clarkson (1985) and Smith and Smith (1986), who appreciated the sigiuficance of the interfacial apoplast and showed separate membrane transport systems operating for FO4 and sugars. More recently, Clarkson (personal communication) has suggested that if, as outlined above, the root apoplast can be viewed as an exchange zone in which conditions can be controlled and solute concentrations are relatively high, then it may be that FO4 uptake by the plant is via the low-affinity uptake system. If, as now seems possible, the high-affinity system is switched off in the fungus, this would provide a way of polarizing transfer in the direction of the plant. Again, probes for these transporters would provide useful data, especially if they could be used at a high level of resolution to show localization on the different membranes. It must be reiterated that the model of spatial separation of hexose and mineral nutrient transfer on different interfaces is still hypothetical. It is based on the distribution of H^-ATPase activity revealed by cytochemistry and on the observations that VA mycorrhizal fungi grow intercellularly in mutant plants of Pisum, in which arbuscules are reduced or absent, implying that the arbuscular interface is not required for C transfer. The idea should be useful in stimulating investigations of transfer processes but it must not be taken as established fact. Indeed, there are a number of problems with it, not least relating to the diversity of structures formed by glomalean fungi in roots. Intercellular interfaces are not present in all associations (Smith, 1995; Smith and Smith, 1996a; and see chapter 2), so that alternative ways of establishing spatial separation of function may need to be sought, perhaps involving intracellular hyphal coils.

406

General themes

Interplant Transfer The widely held belief that nutrients are transferred between plants linked by mycorrhizal mycelium is based on much uncritical discussion and does not recognize the essential difference between net transfer, which might be nutritionally important to the plants, and apparent transfer, brought about by equilibration of tracers with pools of unlabelled solutes (Newman, 1988). It is clear from the discussion above that a mechanism does exist at the membrane transport level which explains tracer movement of ^^C in both directions across a symbiotic interface. Sugars or organic N move from plant to fungus and organic N moves in the reverse direction. In a system involving a single plant and a fungal mycelium, net C movement must occur in the direction of the fungus, because C skeletons are required for synthesis of the organic N. Of course, if the fungus obtains these C skeletons from another source then there can be net C transfer as amino compoimds from the fungus into the plant (Fig. 14.11). Potential alternative sources of C are either another photosynthetic plant linked into the mycelium, or organic N from the soil. It is very difficult to differentiate experimentally between the situation outlined above (which actually involves net transfer across the symbiotic interfaces) and one in which the two photosynthetic plants contribute different amounts of sugar to support the mycelium (see Chapter 4). However, there is now one report in^vhich double labelling with ^^C and ^^C has been used to investigate transfer between Betula and Abies; the results are consistent with an important role for Donor plant

(active interface)

Receiver plant

Soil

NH4"

(active interface)

Al

Pi" -

Pi"

(senescent interface)

Gin

- • Gin

-• Pi"

- • Pi"

-•

Sugar-

1 ^ ^''

U

r

Fungus

A (senescent root)

F i g u r e 14.11 Possible nnechanisnns f o r transfer of organic C and P O ^ between mycorrhizal plants. Possible shuttles across the Veceiver' Interface are not shown. From Smith and Smith (1990), w i t h permission.

Uptake, translocation and transfer of nutrients in mycorrhizal synnbioses

407

glutamine in net transfer both N and C between plants (Simard, 1995; and see Chapter 15). As discussed in Chapter 5, it is less easy to envisage mechanisms at the membrane transport level which would promote reversal of transfer of PO^, so that it occurred from plant to fungus. We have suggested (see above) that the 'normal' symbiotic situation involves considerable modification of fungal membrane transport, so that PO^ efflux is markedly increased and uptake switched off in order to maintain polarized net transfer to the plant. Until we know how this change is achieved and whether it is readily reversible, it seems simplest to agree with Newman (1988) that the apparent transfer could be the result of effective exploitation by the fungus of P in senescent roots, which would indeed be likely to be generally permeable (leaky) to all solutes including P (Fig. 14.11).

Myco-heterotrophs Myco-heterotrophic plants obtain their supplies of organic C from a fungus, which in turn obtains it from a photosynthetic plant or, in the case of some orchids (see Chapter 13), from organic matter in soil. They also obtain mineral nutrients in this way, so that transfer of all solutes is polarized in their favour. Symbiotic modifications to membrane transport processes would certainly be different in the two 'host' plants, because in one (the autotroph) net C transfer would be in the direction of the fimgus and in the other (the heterotroph) it would be in the direction of the plant. Even in green orchids there is as yet no good evidence for organic C transfer to the fungus (see Chapter 13), and it is possible that in the adult photosynthetic stages the main role of the fungus is to supply mineral nutrients to plants which frequently have very poorly developed root systems. Transfer of mineral nutrients (N, P and so on) to the heterotroph could be by mechanisms similar to those operating in the autotroph. We know almost nothing about what actually happens in these symbioses and it will be a fertile but difficult area for experimentation. Measurement of rates of transfer and investigations of distribution and activity of H^ATPases and other transport proteins would be most interesting.

Conclusions Transport processes are central to the function of all mycorrhizal symbioses because they are nutritionally based. In the most common mycorrhizal types, explanations for mycorrhizal function must be sought which promote polarized transfer of different nutrients in opposite directions. Mechanisms that enhance efflux and maintain influx must coexist, at least in the same plant if not in the same cells, and consequently increased efflux cannot be based on general increases in permeability (leakiness) and loss of membrane integrity. Transport processes, whether at the whole-plant or at the cellular level, have been studied in only a few examples of the major mycorrhizal types. There is a wealth of information yet to be gained both in terms of the efficiency of the symbioses (nutrients gained for C expended) and the mechanisms involved. The increasing realization that within each major mycor-

408

General themes

rhizal type there is considerable diversity in structure and efficiency means that there is also diversity at the levels of uptake, translocation and transfer of nutrients. Myco-heterotrophic associations pose a particular challenge with respect to mechanisms of transport between the symbionts. The problems should become easier to address as the identity of the fungal symbionts and their links with autotrophic plants become clearer. This information will pave the way for realistic experiments on the 'unusual' transport processes that support the growth of nonphotosynthetic plants.

Plate 6. Penetration of cellufluor into the fungal sheath of Pisonia grandis. (a) Intact rootlet showing absence of turquoise fluorescence in the cortex, (b) Rootlet showing penetration of cellufluor where the fungal sheath has been damaged, (c) Section bathed in cellufluor, showing turquoise fluorescence of cellufluor throughout. Bars, 100 |im. From Ashford et al. {Australian Journal of Plant Physiology^ 1989), with permission.

15 The roles of mycorrhizas in ecosystems

Introduction In the century following the first experimental investigations of mycorrhizal function by Frank (1894) a wealth of information has accumulated, much of which is covered in this book. The basic structural and physiological attributes of mycorrhizal fungi, and of colonized roots, have been elucidated in detailed laboratory studies. The growth responses of individual species to colonization, usually in pots and as pure stands, have been subjected to innumerable investigations. There has been a small number of experiments investigating the impact of mycorrhizal fimgi upon interactions between different species of plant, grown in pots, but there remains a conspicuous gap in our understanding of the function of mycorrhizas at the community level. The mycorrhizal symbiosis, in all likelihood, co-evolved with the first land plants (Chapter 1) and invariably in nature the plants have occurred in communities of mixed species. The fungi themselves also coexist in similar communities, so that very diverse and interlinked populations are the norm. The questions which arise, and which will in all probability increasingly occupy the thoughts of those investigating the biology of mycorrhizas in the next century, concern the role of the symbiosis, if any, in the biology of plants growing in natural substrates and mixed communities in the field. Are there impacts of mycorrhizal colonization upon the fitness of individual plants? Do mycorrhizal fungi influence the outcomes of competitive interaction between species of plant? If so, under what circumstances and in what way are the influences mediated? Moreover, we may also ask how the populations of plants influence similar features of the fungal community. These are the sorts of questions which need to be addressed and for which, when we find positive or negative interactions, explanations will need to be sought at the molecular, biochemical and physiological levels. In other words, the detailed investigations directed at understanding mechanisms, which frequently must be done in simplified and ecologically unrealistic experimental systems, must be to an increasing extent focused on answering questions of ecological relevance.

410

General themes

Some researchers have already turned their thoughts in these directions. It is, for example, increasingly a prerequisite of any field-based ecological study to know what kind of mycorrhizal association predominates in a given community or ecosystem. This is frequently supplemented with information on seasonal and spatial distribution of mycorrhizal types and activity. Information of this kind is not difficult to obtain and we now have lists of 'occurrences' of broad mycorrhizal types in many different ecosystems, although details of structural variations are not always available. More difficult and time-consuming to obtain is information on function, so that investigations directed towards understanding the roles of mycorrhizas in community dynamics and ecological processes are in their infancies. What is emerging is a picture suggesting that the functions of the symbioses go far beyond the simple capture of mineral nutrients by individual plants of organic C by the associated fimgi. There is clearly considerable diversification in structure and in function, not only in the major types or even in mycorrhizas formed between different species of symbionts, but also imposed by genotypic variation of both fungus and plant. In any distinctive type of ecosystem, selection may have favoured specialized attributes of the symbiosis and symbionts that are appropriate to that particular set of environmental circumstances. What follows is a survey of the major types of biome, considered in a latitudinal gradient, with a view to determining the extent and distribution of mycorrhizas occurring within each, and to elucidate, as far as is known, the functions that might be important in the circumstances prevailing in each system. Inevitably, at this early stage of development, there are more questions than there are answers, but what is emerging is a picture in which the symbioses are multifunctional. It is to be hoped that identification of the questions can at least be of assistance to the next generation of researchers who will be seeking answers relevant to the real world.

Mycorrhizas in High Arctic and Alpine Biomes The small number of species that occur, usually as individual plants, in the nival zone of the Alps and in the very high Arctic are only intermittently uncovered by snow, grow in mineral soils and appear to be largely uncolonized by mycorrhizal fungi. Read and Haselwandter (1981) found that in this zone of the Austrian Alps, Ranunculus glacialis appeared to be free of all fungal colonization. Vare et ah (1992) reported that none of the six Ranunculus spp. examined in the Arctic at Spitsbergen were colonized by vesicular-arbuscular (VA) fungi. This contrasts with the situation for this genus below the nival zone (Mullen and Schmidt, 1993) and in temperate latitudes (e.g. Harley and Harley, 1987) where colonization by VA mycorrhizal fungi appears to be the norm. Similarly, in the maritime Antarctic, Christie and Nicolson (1983) could find no VA colonization on plants such as Deschampsia antarctica which were mycorrhizal at less extreme sub-Antarctic sites in the Falkland Islands. Haselwandter et al (1983) point out that during the short growing season of R. glacialis in the nival zone the plant is supplied with N and P contained in melt-water, so that climatic rather than nutritional factors are likely to determine success at the highest elevations. Below the nival zone of alpine areas and in those regions of the Arctic which have a consistent snow-free growing season, there is a continuous vegetation cover and

The roles of mycorrhizas in ecosystems

41 I

some accumulation of organic matter, at least at the surface. In their study of Ranunculus adoneus, which was carried out at 3500 metres in the alpine zone of the Colorado Front Range, Mullen and Schmidt (1993) showed that while the plant was lightly colonized by coarse and fine VA endophytes throughout the year, arbuscules were present only during the short growing season. Arbuscule formation was followed by increases of P concentration in both shoots and roots. It was proposed that P acquired in this period was stored for use during growth and flowering the following spring, both of which occur before soils thaw to release nutrients. More studies of this kind, in which the dynamics of colonization and nutrient acquisition are followed through the year in the natural environment of the plant, are much needed. They provide not only ecologically relevant information, but also valuable pointers for physiological investigations of arbuscule and hyphal function. Often, especially in those regions of the Arctic where the water-table lies permanently near the surface, plant communities contain a preponderance of species in families like the Cyperaceae, which are typically non-mycorrhizal. While this may help to explain the observation made both in alpine (Haselwandter and Read, 1980; Read and Haselwandter, 1981) and Arctic (Bledsoe et al, 1990; Vare et al, 1992) environments that VA mycorrhizal colonization is low, it does not provide a full explanation, since again many species which are 'hosts' to VA fungi at lower altitudes, are uncolonized or only lightly so in arctic-alpine situations. Even where VA mycorrhizas are observed, they are often formed by 'fine' rather than 'coarse' endophytes, there being a progressive increase in colonization by Glomus tenuis with altitude (Crush, 1973; Haselwandter and Read, 1980). One striking feature to emerge from studies of arctic-alpine plants is the extensive occurrence on their roots of fungi with dark septate (DS) hyphae (Haselwandter and Read, 1980; Christie and Nicolson, 1983; Kohn and Stasovski, 1990; Vare et al, 1992; Treu et al, 1996). In a study of 179 vascular plant species of Alberta, Currah and Van Dyk (1986) found that roots of 87% of alpine species were colonized by DS fungi, in contrast to only 9% in non-alpine environments. The preponderance of fungi of this general type on plants growing in alpine conditions is reflected in analyses of soil microflora, which suggest that in Antarctic (Heal et al, 1967), Arctic (Vare et al, 1992) and alpine soils (Haselwandter and Read, 1980), fungi with DS hyphae dominate the soil microbial community. Their quantitative importance in these habitats means that attention must be directed towards their possible taxonomic and functional status. Based upon the presence of sclerotium-like bodies or of aggregates of pigmented swollen hyphae with pores, Haselwandter and Read (1982) tentatively identified DS fungi of alpine plants as being of the genera Rhizoctonia and Phialophora, respectively. Fungi of the 'Rhizoctonia' type have been known, since early studies of Peyronel (1924), to be capable of colonizing roots, even those also occupied by VA mycorrhizal fungi, while Phialophora is recognized as a casual occupant or weak parasite of many roots, particularly of grasses, where they appear to be associated with programmed cortical senescence (Deacon, 1987). Stoyke and Currah (1991) observed that some cultures of DS fungi, originally isolated from roots of alpine plants, produced the large fan-shaped conidial appendages typical of Phialocephala fortinii (see Fig. 10.4), but only after prolonged storage at low temperature. They suggest that many of the DS isolates hitherto ascribed to Phialophora are congeneric

412

General themes

with Phialocephala and may even be conspecific with Rfortinii, If so, this contributes to an understanding of the status of these associations. Rfortinii can be, albeit under culture conditions of high C content, a pathogen of Pinus (Wilcox and Wang, 1987b) and is one of a number of dematiaceous fungi, previously included in Mycelium radicis atrovirens, which have been regarded as weakly pathogenic (see Chapter 10). It has also been considered to be a commensal saprotroph (O'Dell et ah, 1993) when growing on roots of Lupinus latifolius in temperate environments. Cluster analysis and ordination based on RFLPs of ribosomal DNA extracted from 117 dematiaceous and 10 sterile, hyaline fungi isolated from the roots of 26 species of subalpine plants indicated that two-thirds of the fimgi were closely related to or conspecific to P. fortinii (Stoyke et al, 1992). The prominence of DS fungi in general and of P. fortinii in particular, is clearly a feature of high elevations and latitudes, but the presence alone of these fungi in and aroimd roots is not sufficient to justify claims of mycorrhizal status. The fact is that we know virtually nothing of the biology of these associations in their natural environments and there is an urgent need for experimental analysis of their functional attributes. Haselwandter and Read (1982) isolated DS fungi from healthy, field-collected roots of the alpine sedges Carex firma and C. sempervirens, and obtained a positive growth response in C. firma when the plant was inoculated and grown in sand with one of the isolates. However, since neither the substrate nor the conditions employed in the experiment reflected those of the alpine habitat, they urged caution in the interpretation of these responses, referring to them as being evidence of an 'association' rather than of a typical mycorrhizal relationship. Of equally uncertain status are the ectomycorrhiza-like structures which are frequently, but not consistently, found on roots of herbaceous species such as Kobresia (Fontana, 1963; Haselwandter and Read, 1980; Kohn and Stasovski, 1990) and Polygonum viviparum (Hesselman, 1900; Read and Haselwandter, 1981; Lesica and Antibus, 1986). Where they occur, these associations too are normally formed by fungi with dark mycelia. Amongst these Cenococcum geophilum appears to be prominent, although the frequent presence of hyphae with clamp cormections indicates that basidiomycetous fungi may also be involved. There is a suggestion (Vare.ef al, 1992) that these species are more frequently colonized in this way when growing with typically ectomycorrhizal plants. Again, there is a need for experimental analysis of the status of these types of colonization. Trappe (1988) observed that when Pinus albicaulis occurred as a pioneer on recently exposed moraines in the Cascades Range (Oregon), it formed ectomycorrhizas predominantly with Cenococcum geophilum. The mechanisms of dispersal of fungal propagules at very high altitudes are not understood, but such occurrences, coupled with the observation that the surfaces of snow pack are frequently encrusted with soil particles, indicate that winds are sufficiently strong in these exposed environments to enable transfer of vegetative propagules of fungi. C. geophilum becomes progressively more important as a mycorrhizal colonist with altitude. In 'krunmiholz' formed by coniferous species above 2700 m in the Cascades, Trappe (1988) found that over 90% of ectomycorrhizas were formed by C. geophilum. This suggests that propagules of this fungus must be abundant in soils not far below mountain summits.

413

The roles of mycorrhizas in ecosystems

Mycorrhizas in Heathland Biomes Heathlands occur as major biomes under two environmental circumstances. In the first, the upland or montane heath is found in both continental and island locations (Fig. 15.1) at a distinct altitudinal position between the alpine zone and the tree line. The second, lowland heath, occupies areas of particularly impoverished acid soil at low elevation. These biomes are characterized by the presence of shrubby, sclerophyllous, evergreen plants of the families Ericaceae, Epacridaceae, Empetraceae, Diapensiaceae and Prionotaceae (Specht, 1979), many of which normally have hair roots colonized by ericoid mycorrhizal fungi (Read, 1983,1996; and see Chapter 12). Analysis of environmental gradients across which plants with ericoid mycorrhizas become increasingly prevalent, have shown that such communities arise primarily in response to nutrient impoverishment (Specht, 1981; Rundel, 1988; Read, 1989). Their occurrence in warm mediterranean climate zones as 'dry heath', or 'sand plain' formations as well as in subalpine environments, serves to emphasize the fact that nutritional rather than climatic factors play the primary role in determining the distribution of these communities. The response to low availability of N and P is to allocate increasing proportions of fixed C to the structural components lignin and cellulose rather than to molecules rich in protein or P, a process which leads directly to sclerophylly (Specht and Rundel, 1990) and to the release of residues of high C:N ratio and considerable recalcitrance. These accumulate at the soil surface to provide the matrix of complex residues in which ericoid mycorrhizal roots proliferate. In northern heaths, the hair roots of dominant plants such as Calluna vulgaris, Erica spp. and Vaccinium spp. are characteristically confined to the top 10 cm, or less, of the soil profile, where they are closely associated with the litter (Reiners, EUROPE 5000

TYROLEAN ALPS

ISLANDS FERNANDO PO

TRISTAN DA CUNHA

AFRICA O PICO

MT ELGON

MALAYA rSOOO

4000

3000

h 3000

2000

h 1000

sea level

sea level

Figure 15.1 Simplified global pattern of distribution of major biomes, highlighting the segregation of predominant mycorrhizal types in association with distinctive types of plant community. Mycorrhizal types: black shading, ericoid; grey shading, ecto; and no shading, VA. Note that in the dlpterocarp forests there will be important canopy and understory plants that are VA mycorrhizal (see text). From Read (1993), with permission.

414

General themes

1965; Gimingham, 1972; Persson, 1980). Interestingly, when herbaceous species such as Deschampsia flexuosa, Molinia caerulea, Eriophorum vaginatum and Carex spp. coexist with ericaceous shrubs, their roots are concentrated at greater depths in the soil profile (Gimingham, 1972), so the two groups of plants are not competing for the same resources. Similar mechanisms promoting coexistence in the sand plain communities of Western Australia have also been described. Indeed, the grasses may be colonized by VA mycorrhizal fungi, again emphasizing separate strategies of resource acquisition. Read (1993) presented a schematic view of the way in which distinctive mutualisms, together with modifications of root distribution and anatomy, might promote species diversity in northern heaths by enabling exploitation of different sources of the critical growth-limiting element N (Fig. 15.2). The coexistence of ericaceous, leguminous and carnivorous species, typically seen in heaths of moderate acidity, was facilitated by their abilities to use sources of N derived, respectively, from soil organic matter, the atmosphere and captured animals. Structural modifications, in particular production of aerenchyma, enables cyperaceous species like E. vaginatum to penetrate water-logged horizons where

(a) Ericoid mycorrhizal shrubs

(b) Insectiverous plants

(c) Leguminous plants

(d) Members of Cyperaceae, Restionaceae and Proteaceae, forming deep roots or cluster roots

F i g u r e 15.2 Schematic representation of compartmentation of resource acquisition in heathland ecosystems, based on the occurrence of distinctive mutualistic associations o r r o o t specializations, (a) Ericoid mycorrhizas occur in dwarf shrubs and play an important role in the mobilization of N in plant litter and microbial protein, (b) Insectivorous plants capture insects, f r o m which they release N . (c) Leguminous plants f o r m nodules which fix N . (d) Members of the Cyperaceae, Restionaceae and Proteaceae either produce deep roots that tap N in l o w soil horizons and / o r proteoid o r cluster roots that are important in capture of nutrients (particularly P, but also possibly N in the surface horizons). From Read (1993), w i t h permission.

415

The roles of mycorrhizas in ecosystems

they exploit N sources, including organic forms (Chapin et al, 1993), untapped by the other groups that are essentially surface rooting. Evidence in favour of a role for mycorrhizas in providing discrimination in nutrient use is increasing. Comparative analysis of 5^^N enrichment of leaf tissues of Picea, Vaccinium and Calamagrostis all growing in the tundra heathboreal forest transition zone of Alaska (Schulze et al, 1994) revealed significant difference of enrichment between these life forms, P. mariana having significantly lower 6^^N value (-6.496) than V. vitis idaea (-3.837) and C. canadensis (+0.585). Distinctive rooting depths may have contributed to these differences but isotope discrimination, facilitated by ecto-, ericoid and VA mycorrhizas respectively, is also a possibility (Schulze et al. 1994). Support for this suggestion has been obtained in a field-based study, in which the extent and type of mycorrhizal colonization was examined in subarctic, fellfield and heathland communities (Michelsen et ah, 1996). 6^^N enrichment of leaf tissue was determined in plants of known mycorrhizal status representative of ericoid, ecto-, VA and non-mycorrhizal categories. In the fellfield the mean 8^^N of the ericoid mycorrhizal species was -53, that of ectomycorrhizal species was -4.1 and of VA or non-mycorrhizal species, 0.0 (Fig. 15.3). In the heath the mean 5^^N values of the same groups were -7.6, -6.4 and -1.8, Leaf 5^^N (%o) -10

-8

-6

-4

-2

0 ERICOID MYCORRHIZAL DICOTS Cassiope tetragona Empetrum hermaphroditum Rhododendron lapponicum Vaccinium uliginosum ECTOMYCORRHIZAL DICOTS Arctostapiiylos alpinus Betula nana Dryas octopetala Salix myrsinites Salix reticulata NON-MYCORRHIZAL HEMIPARASITE Bartsia alpine NON-MYCORRHIZAL MONOCOTS Calamagrostis iapponica Carex vaginata Festuca ovina Tofieldia pusilla NON-MYCORRHIZAL NODULATED LEGUMES Astragalus alpinus Astragalus frigidus

Heath

LICHENS Cetraria nivalis Nephroma articum

ERICOID MYCORRHIZAL DICOTS Cassiope tetragona Empetrum hermaphroditum Vaccinium vitis-ideae ECTOMYCORRHIZAL DICOTS Betula nana Dryas octopetala Polygonum viviparum Salix herbacea\/ar.p '

Fellfield

NON- OR ARBUSCULAR MYCORRHIZAL SPR Calamagrostis Iapponica Carex bigelowii Festuca vivipara Luzula arcuata Lycopodium selago

F i g u r e 15.3 5 ' ^ N (0/00) of the most c o m m o n species of plant and t w o lichens in the treeline heath and fellfield in a subarctic ecosystem (Abisco, Sweden). Values are means of six replicate determinations ± standard e r r o r s . From Michelsen et al. (1996), w i t h permission.

416

General themes

respectively. In all cases values obtained from the ericoid and ectomycorrhizal plants were significantly different from those of the VA or non-mycorrhizal species. Although the differences between ericoid and ectomycorrhizal species were not significant (P = 0.051 in fellfield, and 0.270 in heath) the latter clearly appeared to occupy an intermediate position in the hierarchy. There was no evidence of segregation of roots by depth in these sites, almost all roots in the fellfield being restricted to the humus layer having a thickness of only 2-3 cm and to a similar zone in heathland of 10-15 cm. Ericoid and ectomycorrhizal colonization facilitates access to organic sources of N which are not only the predominant forms of N in the soil but frequently also have relatively low levels of 8 N enrichment. In addition to their abilities to use organic sources of N and P (Chapters 8, 9 and 12), it is increasingly evident that ericoid and some ectomycorrhizal fungi produce extracellular enzymes that break down complex polymers of C, thereby exposing new sources of organic N and P to attack (Table 15.1). A hitherto unrecognized association between the ericoid mycorrhizal fungus Hymenoscyphus ericae and the rhizoids of leafy liverworts in the families Adelanthaceae, Caphaloziellaceae, Cephaloziaceae, Calypogeiaceae and Lepidoziaceae has been described by Duckett and Read (1991, 1995). This relationship may be of considerable ecological importance because, whether in the moist acidic peats and mor-humus soils of high latitudes, or in the epiphytic cloud forest communities of tropical mountains, plants with ericoid mycorrhizas normally grow together with liverworts of these families. Microscopic analysis of the swollen tips of the rhizoids of the liverworts show that they are invariably occupied by dense hyphal complexes (Fig. 15.4). When these were exposed to the fluorescent dye 3,3'-dihexyloxacarbocyanine (DiOC6(3)), which selectively stains ascomycetes, they were seen to fluoresce brightly (Duckett and Read, 1991). Liverworts from these families were subsequently grown (Duckett and Read, 1995) in pure culture and inoculated either with H. ericae or with Oidiodendron spp., an orchid mycorrhizal fungus (Ceratohasidium cornigenim) and several ectomycorrhizal fimgi. H. ericae was capable of forming the typical hyphal complexes and swellings in the rhizoidal apices (Table 15.2) of all species seen to produce them in nature. The ericoid fungus failed to colonize those liverworts in families Jungermanniaceae, Amelliaceae and Aneuraceae which are normally associated with basidiomycetous fungi or those in the Pelliaceae, Fossombroniaceae, Lunulariaceae, Conocephalaceae and Marchantiaceae that have zygomycetous associates. None of the other fungi formed associations with any of the liverworts. The failure of Oidiodendron spp. to colonize the liverworts is of particular interest in view of their apparent ability to form ericoid mycorrhizas. Duckett and Read (1995) suggest that one of the important consequences of this pattern of colonization will be that the mats of leafy liverworts so extensively covering acidic organic surfaces will provide effective sources of inoculum for ericaceous seedlings germinating in them. The further possible ecological and physiological consequences of the susceptibility of these phylogenetically distinct groups of plants to colonization by the same fungal endophyte remain to be investigated. Heathlands in regions with a mediterranean-type climate experience a wet winter season, in which most of the root growth and mycorrhizal activity occurs (Ramsay et «/., 1986; Meney et aZ., 1993; Bell et al, 1994). These, and indeed most.

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Figure 15.4 Rhizoids of liverworts showing the association with fungi and alterations in morphology, (a) and (b) Cephalozia bicuspidata. The rhizoids retain normal morphology and are not colonized by either (a) Ceratobasidium, or (b) Oidiodendron griseum. Bar, 50 ^im. (c) and (d) Cephalozia loilesbergerL Young gametophytes showing colonization of rhizoids and mucilage papillae (arrowed) by Hymenoscyphus ericae. Bars, 50 ^im. (e)-(i). Nowellia curvifolia. (e) Non-colonized rhizoid, with packets of mucilage and acentrically located nucleus (arrowed). Bar, 25 jxm. (f) Early stages in colonization of rhizoids by Hymenoscyphus ericae, showing external hyphae (arrowed). Bar, 25 |im. (g) Numerous internal hyphae in a slightly distorted tip of a rhizoid. Bar, 25 jum. (h) Hyphae at the base of a rhizoid (arrowed). The adjacent rhizoid is not colonized. Bar, 20 ^im. (i) Rhizoid from fieldcollected plant, showing swollen tip packed with hyphae. Bar, 20 |im. From Duckett and Read (1995), with permission.

The roles of mycorrhizas in ecosystems

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lowland heaths of intermediate latitudes in the southern hemisphere experience long hot dry seasons during which evaporative conditions lead to surface accumulation of salts, maintaining soil pH over a range between 4.0 and 6.0, which is somewhat higher than that normally observed in wetter, northern heaths. This may also help to explain both their wider propensity to support nitrifying activity, and the greater floristic diversity often seen in them. The great species diversity seen in southern hemisphere heathlands containing epacrids (Pate and Hopper, 1993) is in striking contrast to the situation seen in northern heaths dominated by ericaceous species. It appears that with the diversity comes a commensurate increase in the range of root specializations (Lamont, 1982, 1984; Pate, 1994), suggesting that niche separation is even more marked here than it is in less stressed environments. Amongst the distinctive families found in these dry heathlands, the Proteaceae and Restionaceae are characterized by the production of cluster (proteoid) roots. These structures are formed in the same superficial horizons of the soil profile as are ericoid roots (Fig. 15.2), where they also exploit actively decomposing litter (Lamont, 1984). In this case, although not spatially segregated, the two types of roots probably have different functions. The main function of cluster roots is probably to enhance the capture of P the release of organic and phenolic acids acidifying the rhizosphere, thus making Pi (and possibly N), more available to the plants in the small volume of soil around the cluster (Grierson and Attiwill, 1989; Dinkelaker et al, 1995). There is no evidence that cluster roots are directly involved in the mobilization of either P or N from the organic forms in the litter. Consequently, while proteoid roots will scavenge for inorganic nutrients, the ericoid roots in the same substrate will exploit organic residues via the activities of the associated mycorrhizal fungi (see Chapter 12). Stewart et al. (1993) provide evidence of such functional segregation. In the fireprone habitats that they studied, NO^ was relatively abundant immediately after burning, although NH4 was also present. There was sufficient NO^ reductase (NR) activity in the shoots and roots of three proteaceous genera {Banksia, Petrophile and Stirlingia) to suggest that they would assimilate NO3 in nature when this form of inorganic N is available. In contrast, two epacrid species Astroloma macrocalyx and Conostephium pendulum, both of which have ericoid mycorrhizas, showed barely detectable NR activity, even after feeding with NO^ via the transpiration stream. These species would be dependent on assimilation of NH4, which was always present in soil and predominated at sites not burned for several years, or on utilization of organic N via ericoid mycorrhizal associates. Other plant families with widespread representation in epacridaceous heaths but which are absent or of little importance in the northern hemisphere, are the Rutaceae, Dilleniaceae and Compositae, most members of which would be expected to be colonized by VA mycorrhizal fungi. This type of mycorrhiza has been reported, for example, in Boronia (Rutaceae), Hibbertia (Dilleniaceae) and Helichrysum (Compositae; Lamont, 1984). These plants have a less fibrous root system than epacrids and, in addition, penetrate the sandy soils more deeply (Dodd et al., 1984; Pate, 1994), a feature providing spatial separation of root activity. Further, while VA mycorrhizal colonization will enhance their ability to scavenge for P, members of two of these genera, Helichrysum and Hibbertia, develop significant NR activity (Stewart et al., 1993), again suggesting the likelihood of nutritional as well as spatial niche differentiation between these plants and those

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with ericoid mycorrhizas. However, the recent demonstrations that VA mycorrhizal colonization may increase uptake of NH4 and, especially in dry soils, of NOs^ may be relevant here. It is in just such habitats as these that N acquisition is important (see Chapter 5). The presence of these patterns suggests that selection favouring a range of specializations has been an important factor in enabling the coexistence of taxonomically distinct species in Australian heathland systems, where the greatest diversity is foimd on the least fertile soils (Pate and Hopper, 1993). Tilman's (1982,1988) equilibrium model of plant competition predicts that in resource-poor environments, diversity will be low because few species can tolerate extremes of nutrient deprivation. In the sand plain heaths it appears that, on the contrary, selection of distinctive mutualisms, and also modifications of root morphology, have, over very long periods of evolution, facilitated coexistence of species in very diverse assemblages. Indeed, recent work, admittedly in artificial systems, suggests that mycorrhizal colonization may be of greatest advantage in competitive situations where resources are poor and plant density low. Whereas work on heathland plants of the northern hemisphere is relatively advanced, both with respect to mycorrhizal colonization and probable roles of the symbiosis in nature, the information on dry heathlands is scanty. However, enough is known to pose tantalizing questions relating to specialization and diversification of roots and symbioses and the survival of plants in extremely harsh environments.

Mycorrhizas in Boreal and Temperate Forest Biomes Communities of ectomycorrhizal trees are the natural dominants in most of the the boreal and temperate biomes of the world, particularly on acid soils. On base-rich soils, even within these regions, communities are dominated by species such as Acer, Fraxinus, Taxus, Juniperus and members of the Rosaceae, which are always or facultatively VA mycorrhizal and may have extensive VA mycorrhizal understories (Brundrett and Kendrick, 1988, 1990a,b). The diversity of plant species is relatively high, but the number of fungal species involved is probably lower than in ectomycorrhizal forests, reflecting the lower specificity of the VA mycorrhizal association. However, a note of caution needs to be soimded here, as our views on this specificity may have been coloured by the ease with which some species can be maintained in pot cultures. Other, less easily manipulated fungi may have been overlooked (Clapp et al., 1995). The ecological interactions between fungi and plants in these ecosystems have received little attention, although Girard and Forin (1984) were able to relate the incidence of different types of mycorrhiza to soil conditions in climax forests in southern Quebec. They showed that the soil in VAdominated maple-yellow birch and maple-hickory forests had a higher soil pH, lower C:N ratio and faster turnover times of soil organic matter than ectomycorrhizal forests dominated by black spruce. The data also showed much higher numbers of species in the maple forests, with 100 species recorded in maplehickory, as opposed to 76 in the maple-yellow birch and 16 in the black spruce forests. As in northern heathlands, the diversity of plant species in ectomycorrhizal forests

The roles of mycorrhizas in ecosystems

423

is characteristically low, but in contrast to the heathland situation, there is a very great diversity of fungal symbionts associated with the plants (see Table 6.2). The ectomycorrhizas develop in the upper organic layers of the soil or in the interface of organic and mineral layers (Meyer, 1973; Harley, 1978; Harvey et ah, 1978; Persson, 1978, 1980; Ehrenfeld et «/., 1992; Hahn, 1994; George and Marchner, 1996). Roots proliferate preferentially in the fermentation horizon (FH) immediately beneath the freshly fallen surface litter layer. There is even evidence of upward growth of lateral roots enabling them to exploit the organic horizons. In an extensive study of the occurrence of ectomycorrhizas on Eucalyptus in Australia, Chilvers and Pryor (1965) observed that litter accumulation was the single consistent requirement for the production of ectomycorrhizal roots. Despite recognition of the importance of ectomycorrhizas as components of surface organic horizons, relatively little attention has been given to their activities in these substrates. Thus while it is accepted that fungi are the most important agents of decomposition of acidic organic residues at infertile sites (Flanagan and van Cleve, 1977), the possible importance of the mycorrhizal component in these processes has not been widely acknowledged. Gadgil and Gadgil (1975), in an experiment in which plots under pine were trenched to exclude mycorrhizal roots, were the first both to recognize that ectomycorrhizal and decomposer fungi must coexist in forest litter and to investigate directly the nature of the interactions between these two groups of fungi. They observed that decomposition was more rapid where mycorrhizal roots were excluded than when they were present, a feature that they attributed to antagonism or competition for nutrients between the two fungal groups. Subsequent attempts to repeat these findings have not been entirely successful (Berg and Lindberg, 1980; Harmer and Alexander, 1985; Staaf, 1988) but considerable support for the existence of 'the Gadgil effect' has been provided by the observations of Bending and Read (1995a) and Entry et ah (1991) that intensive exploitation of litter by ectomycorrhizal mycelium leads to the mobilization and export of N and P from it. The implication of these observations is that ectomycorrhizal fungi will inhibit or reduce the activities of the decomposer population. Until recently, such interactions have been considered unlikely, not only by those who worked with decomposer organisms but also by students of mycorrhizas who, since the influential work of Lindeberg (1944), Norkrans (1950) and Lundeberg (1970) have largely held to the view that mycorrhizal fungi lacked the biochemical capabilities to enable them to mobilize nutrients from polymeric sources of C and so were themselves dependent upon decomposer fungi for such activities. An increasing body of evidence (Table 15.1) suggests that some ectomycorrhizal fungi release enzymes necessary to enable them to take part directly in decomposer activities. Studies of mycelial mats on the forest floor (Griffiths and Caldwell, 1992; Griffiths et ah, 1994) and of 'patches' formed in laboratory observation chambers (Bending and Read, 1995b), where ectomycorrhizal mycelium is the dominant component of the microbial biomass, have demonstrated that increases in amounts and activities of key nutrient mobilizing enzymes occur synchronously with intensive colonization of substrates by ectomycorrhizal fungi (see Chapters 8 and 9). Higher peroxidase and polyphenol oxidase activities suggest the potential to achieve ring cleavage of complex phenolic compounds, enhancing the release of peptides and amino acids within mycorrhizal mycelial aggregates. The ability of

424

General themes

many ectomycorrhizal fungi to assimilate these cleavage products was demonstrated earlier (Chapter 8). The FH layer, which is extensively occupied by fans of ectomycorrhizal mycelium (Ogawa, 1985), is also the most important site of N mobilization in the soil profile (Staaf and Berg, 1977). In pine litter a period of net immobilization of N during the first three years after deposition (Berg and Staaf, 1981), probably associated with colonization of the substrates by saprotrophic fungi (Berg and Soderstrom, 1979), is followed by a phase of release, which is normally considered to involve mineralization of N as the saprotrophs become starved of C (Berg and Staaf, 1981). Berg and McClaugherty (1989) concluded that in advanced stages of decomposition of forest litter this energy starvation will eliminate net immobilization of both N and P. It is at this point in the immobilization-mobilization process that ectomycorrhizal fungi, free of C limitation as a result of their association with the plant, are most likely to be able to compete effectively with 'decomposers' for N. The ability of ectomycorrhizal fungi to scavenge for organic N while being sustained by C from their plant associates could form the basis of the 'Gadgil effect'. While P is rarely the primary growth-limiting element in such communities, it is of interest that the processes involved in its immobilization and release in litter appear to be the same as those of N (Berg and McClaugherty, 1989). As in the case of N, therefore, the onset of energy limitation in the saprotroph population should enable ectomycorrhizal fungi, with their ability to produce a range of phosphomono- and diesterase (see Chapter 9), to compete effectively for P as well as N. Indeed, the retention of the mycorrhizal habit in mull-humus forests, in many of which active nitrification occurs (Ellenberg, 1988; Aber ei ah, 1989), may be attributable to the ability of such fungi to release P from the organic residues. In addition to the general observation that ectomycorrhizal mycelia proliferate most intensively in material from the FH horizon, there is evidence at a finer scale of hyphal growth patterns that are likely to provide intimate contact with resources of a particular quality. Ponge (1990), using the light microscope, observed selective exploitation of pine needles, Pteridium leaflets and animal corpses by hyphae of ectomycorrhizal fungi. In an attempt to simulate conditions prevailing in the ectomycorrhizal conifer-feather moss communities that cover large areas of the boreal forest zone, Carleton and Read (1991) grew mycorrhizal Pinus seedlings in association with the feather moss Pleurozium schreberi. Hyphae of the fungal symbiont Suillus bovinus selectively colonized and formed a sheath-like structure (Fig. 15.5) around senescing parts of the moss shoot, such colonization providing the potential for capture of resources from it. In nature this colonization is likely to provide a key link in the nutrient cycle since most of the elements arising at the forest floor are intercepted by the moss carpet. At an even finer scale, Agerer (1991b) has demonstrated structural modifications of the hyphal tips of the ectomycorrhizal fungus Sarcodon imbricatus at their point of contact with particular materials in soil. An appressorium-like structure attaches the tip to its substrate (Fig. 15.6) providing an enlarged surface for biochemical interaction. It seems that at their tips the hyphal walls are less hydrophobic than they are distal to the apex (Unestam and Sum, 1995) and there is increasing evidence both in basidiomycetous ectomycorrhizal fungi and in their counterparts among the wood-decomposing fungi (Rayner, 1991; Rayner et ah, 1994,1995) of phenotypic plasticity. This may provide differentiation of function: hyphal tips at the advancing

The roles of mycorrhizas in ecosystems

425

Figure 15.5 Observation chamber showing the colonization of senescent parts of the shoot of the feather moss Pleurozium schreberi by mycelium of Suillus bovinus (arrowed) growing from a colonized plant of Pinus contorta (not shown). From Carleton and Read (1991), with permission.

front or in intensively occupied patches having an absorptive role, while fully differentiated hyphal walls or rhizomorphs (probably impregnated with hydrophobin-like compounds) form the sealed conduits through which transport to the mycorrhizal sheath occurs (see also Chapters 6 and 14). There is a need, however, to verify these suggestions by more detailed analysis and experimentation. Having recognized the morphogenetic plasticity in ectomycorrhizal fungi, it is now necessary to examine further its role in attack upon colonized substrates. This in itself is a daunting task in view of the diversity of species, genotypes and hence physiological attributes likely to be encountered in the ectomycorrhizal fungi that occupy forest soil. There is no doubt of the great diversity of fungi capable of forming ectomycorrhizas with the roots of boreal and temperate forest trees (see Chapter 6) and significant progress has been made towards categorizing these fungi according to the level of specificity shown with regard to plant species (Molina et al., 1992). Fungi of narrow, intermediate and broad host range are all observed. Similarly, the 'receptivity' of plant species and genera can be analysed, and again a range is seen from genera such as Alnus which have few fungal associates (Molina, 1979, 1981),

426

General themes

Figure 15.6 (a) Thick-walled hyphae of Sarcodon imbricatus growing into a humus particle, (b) The hyphae in close contact with the soil debris are thick-walled and have somewhat swollen, appressorium-like tips adhering to the soil particles (arrowed). From Agerer (1991b), with permission.

to those such as Pinus which are receptive to many. It is also appreciated that environmental factors, most notably soil characteristics, can influence receptiveness, so producing ecological specificity. While recognition of diversity and the ability to classify types within it is valuable, and indeed constitutes one of the most important achievements of the last two decades, the challenge remains to understand its functional role in the ecosystem. Few have addressed this issue experimentally. In one study (Finlay, 1989), Larix and Pinus seedlings were grown together in an observation chamber, one of the Larix plants having first been grown in association with the fungus Boletinus clavipes which was thought to be specific to this tree genus. Mycelium of the fungus grew from this plant to form mycorrhizas on another Larix seedling, but colonized the pine only very sparsely. Autoradiography after ^^P had been fed to the mycelium of B. clavipes revealed that P was

The roles of mycorrhizas in ecosystems

427

translocated only to the mycorrhizal roots and to the shoots of Larix. Specificity in this case was clearly to the advantage of Larix. However, in nature Larix would probably also be colonized by fungi of low specificity, including those that form mycorrhizas with Pinus. Under these circumstances, unless only B. clavipes had access to the P source, it is likely that both plant species would have access to the nutrient. Some indirect evidence for the view that mixed populations of fungal symbionts facilitate sharing of resources was obtained in a study of competition between Pseudotsuga menziesii and Pinus ponderosa (Perry et ah, 1989b). The two species were grown together with a single fungus, Thelephora terrestris, or with a mixture of genus-specific Rhizopogon spp. and those of broad host range Laccaria laccata and Hebeloma crustuliniforme. Differences in growth between the plant species, which reflected their abilities to acquire nutrients, was much smaller in the presence of mixed inoculum. Many more experiments of the kind exemplified by Finlay (1989) and Perry et al, (1989b) are required if we are to understand the functional impacts of specificity phenomena. At the ecosystem level, some of the consequences of the low host specificity of many ectomycorrhizal fungi have been revealed in studies carried out in forests of Oregon, where some fungi, in addition to forming ectomycorrhizas on members of the Pinaceae and Fagaceae, also form arbutoid mycorrhizas on Arctostaphylos and Arbutus spp. (Zak, 1976a,b; Molina and Trappe, 1982a; and see Chapter 11). It was suggested by Molina and Trappe (1982a) that the arbutoid roots might be repositories of mycorrhizal inoculum, especially as these plants were relatively resistant to fire which extensively damages the coniferous components of the forest. The possible importance of this was further indicated by Amaranthus and Perry (1989), who planted seedlings of Pseudotsuga menziesii either close to Arctostaphylos viscida or in grassland at some distance froni the arbutoid species. Growth and survival was much greater in seedlings sown in proximity to the Arctostaphylos. Furthermore, conifer regeneration was considerably more extensive under Arbutus menziesii than beneath shrubs colonized by VA fungi or in open areas (Amaranthus et al., 1990). The seedlings regenerating in the rooting zone of A. menziesii had more mycorrhizal tips. A similar effect was obtained by Borchers and Perry (1990) who grew P. menziesii seedlings in pots of rhizosphere soil taken either from beneath the crowns of Quercus chrysolepis, Lithocarpus densiflora and Arbutus menziesii or from a site 4 m away from these trees. Seedlings grown in soil taken from beneath the crowns of these ecto- or arbutoid mycorrhizal plants produced 60% greater growth in height and double the weight of those grown in soil from the open areas. The greater productivity was associated with quantitative and qualitative differences in the mycorrhizal population on the conifer roots. In the rhizosphere soil taken from under tree canopies, root tips were colonized by Rhizopogon and Cenococcum, whereas with soil taken some distance from the canopies the mycorrhizas were of an undetermined brown morphotype. It appears that regeneration of forests after disturbances, whether caused by natural agents such as fire or by clear-cutting, will be favoured if some hosts to mycorrhizal fungi are enabled to survive on the site. These assemblages of plant species, which can have a number of mycorrhizal fungi in common, have been referred to as 'guilds' (Perry et al., 1989a; Read, 1990). Since significant benefits appear to arise from the broad range of compatibilities seen in nature, it is

428

General themes

important from both ecological and applied standpoints to determine the basis of the observed effects. It must be borne in mind that growth, used widely as a yardstick to judge mycorrhizal response, may in fact be a poor indicator of performance. Fitness, assessed in terms of increased survivorship or fecundity is of greater significance in ecological contexts. Reductions in growth are often observed in young plants as they are colonized by both ecto- and VA mycorrhizal fungi (Dosskey et al, 1990; Stenstrom et al, 1990; Newton, 1991; Colpaert et al, 1992; and see Chapter 4), and in these circumstances the challenge is to determine what, if any, benefits arise for the plant in terms of fitness. This question appears not to have been addressed to the same extent in ectomycorrhizal plants as for VA mycorrhizas (see Chapter 4). Increased survivorship of regenerating seedlings could be attributable simply to the provision of inoculum by surrogate plants, enabling them to function more effectively as independent individuals. An alternative possibility, and one that has caused some controversy, is that mycelial links between the individual plants are maintained after the initial colonization events and that these provide conduits through which resources may be transferred from one plant to another (see Chapters 4-7 and 14). In any case, the adult plants may, by maintaining the C supplies to the mycelium, effectively support the seedlings without any actual net transfer (see Chapter 4). In the context of establishment and maintenance in the population, any relief of C-drain on the seedlings would be an advantage. Increased growth would only be of benefit when adults were senescent, thereby opening gaps to be exploited, both above and below ground. The process of formation of mycelial interconnections between ectomycorrhizal plants has been observed in transparent observation chambers (Brownlee et al,, 1983; Read et al, 1985; Finlay and Read, 1986a,b; and see Chapter 6). Hyphae making up the advancing mycelial front or the established mycelial network rapidly form a mantle on uncolonized lateral roots of any compatible plants with which they make contact. These linkages are retained and the ongoing process of colonization of laterals, as they emerge from extending primary roots, inevitably leads to a situation where much of the root system of a plant becomes integrated into the mycelial network. This provides numerous potentially functional bridges between compatible plant species, so confirming that guilds of plants are not simply united by the possession of a common suite of fungal symbionts but that the individual plants are physically interconnected by these fungi. The ability of links of this kind to function as conduits for the transfer of C has been demonstrated by autoradiography after feeding ^^C02 to the shoots of a so-called 'donor'. Label was seen to be transported through the interconnecting mycelium and to accumulate in the colonized roots of 'receiver' plants (Finlay and Read, 1986a; and see Chapters 7 and 14). The fragility of these hyphal links is such that it is difficult to trace them in natural soil. However, indirect evidence for nutrient transfer in the field was provided by the observation that ^^C02 fed to an adult plant of Pinus contorta was subsequently detected in roots of neighbouring plants of the same or of different species, provided that they were also ectomycorrhizal, whereas only small amounts of activity were found in those species that were hosts to VA fungi (Read et ah, 1985). The greatest amounts of radioactivity were detected in a number of receiver plants that had been subjected to artificial shading during and after the

The roles of mycorrhizas in ecosystems

429

period of isotope feeding to the donor. This suggested that C transfer was influenced by sink strength. Experiments of the kinds described above have been justifiably criticized (Newman, 1988; Jakobsen, 1991; and see Chapters 4 and 14) on the grounds that they reveal only unidirectional transport and hence do not take into account the possibility of a flow of the same substance in the opposite direction. In other words, they cannot show whether net transfer of nutritionally relevant quantities of material occurs. Bidirectional transfer leading to exchange of resources is clearly a possibility. In order to establish unequivocally that net transfer is occurring, it is necessary to demonstrate that one of the interconnected plants gains more material than the other. In an enlightening study using double labelling methods, Simard (1995) has now demonstrated that significant net transfer of C can occur in interspecific combinations of plants colonized by shared ectomycorrhizal symbionts. The two ectomycorrhizal species used, birch (Betula papyrifera) and Douglas fir {Pseudotsuga menziesii), co-occur naturally in mixed wet forests of interior British Columbia, where they share seven mycorrhizal morphotypes, which occupy 90% of the root tips of both species. The likelihood of the occurrence of interconnections between the species is therefore great. For comparative purposes a further co-occurring species Thuja occidentalism which is colonized by VA fungi, was included in the experiments. Individual, one-year-old plants of each species were planted 50 cm apart in triangular groups. The groups were allowed to grow for one or two years before being subjected to reciprocal labelling with ^^C02 or ^^C02. Shading treatments (5% or 50% of full sunlight) were also applied in both years to selected individuals of P. menziesii, 3-6 weeks prior to labelling. After nine days of exposure to isotopes the test plants were harvested and their ^^C and ^'^C contents determined. Net transfer of C from B. papyrifera to P. menziesii was observed under several experimental circumstances (Fig. 15.7). One year after planting, net transfer to the conifer was found only when it was grown in full sun. The amount transferred represented 2% of the total isotope (^ C + ^^C) fixed by both species; 4% of the isotope assimilated by B. papyrifera and 7% of that assimilated by P. menziesii. In both shade treatments transfer to Pseudotsuga was balanced by transfer to B. papyrifera (i.e. there was no net transfer). In the second year after planting, net transfer to P. menziesii was around 6% of the total isotope fixed, averaged over all shading treatments. In the deepest shade (5% full sun) net transfer was double that in the 50% or full sun treatment. This amount of transfer represents a substantial C gain by Pseudotsuga and is similar to that which has been considered sufficient to improve growth and survival of connected ramets among clonal plants (Hutchings and Bradbury, 1986; Alpert et ah, 1991). No net transfer to Thuja occurred. Both spatial and temporal factors contributed to the increased net C transfer and to the effect of shading in the second year. In the spatial context, roots of potentially interacting species were likely to be closer together and the numbers of fungal interconnections between them larger two years after planting than one. Perhaps of greater importance was the fact that the application of isotope was carried out in August of the second year, fully one month after cessation of shoot elongation in P. menziesii, whereas the first feeding experiment took place in July when shoot activity was ongoing. The likely importance of seasonal effects upon C allocation below ground has been stressed earlier (Chapter 7).

General themes

430

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Figure 15.7 Net isotope transfer between Betula and Pseudotsuga, in full sun (100%) and in 50% and 5% of full sun. (a) 1993; (b) 1994. Means denoted by the same letter do not differ significantly (P = 0.01). See text for explanation. From Simard (1995), with permission.

Net transfer from Betula to Pseudotsuga coincided with net photosynthetic rates of the seedlings, which were 1.5 and 4.3 times greater for B. papyrifera than P. menziesii in full sun and full shade, respectively. Furthermore, foliar N concentrations were 1.2 and 6.7 times higher in B. papyrifera than in P. menziesii. It seems likely that net transfer was determined by the gradient of assimilate and nutrient concentrations between the two species. Since rates of photosynthesis of the Betula in full sun were so much greater than those of Pseudotsuga in shade it is likely that C supply to the colonized roots of Betula would be greater than to those of Pseudotsuga, This, combined with the fact that plants of Betula contained significantly more N than P. menziesii, may have contributed to the apparent sink effect. There is still much to be learned about the biochemical and biophysical pathways associated with transfer of C compounds between the symbionts, but where gradients of C and N cooccur the transfer of C in combination with N, perhaps as glutamine (see Chapters 8 and 14), is a possibility. This study should provide further impetus for investigation of transfer processes at the cellular level. Since gradients of nutrient availibility are the norm in nature, confirmation that mycorrhizal interconnections can facilitate the net transfer of C, which is one of the most patchily distributed of all resources, is important. The transfer demonstrated by Simard (1995) took place between plants of broadly equivalent age and size, but might be of even greater significance where the gradients of irradiance are from large, fully illuminated adult plants to small shaded individuals in the understory a situation typical of natural forest ecosystems. Not only might the gain to the receiver be greater but the relative costs to the donor will also be smaller. Again, there is a need to investigate these processes in communities of naturally regenerating plants.

The roles of mycorrhizas in ecosystems

431

The attributes of ectomycorrhizas revealed under simplified laboratory conditions may be constrained in nature where herbivory and fungivory are among numerous factors with the potential to affect mycorrhizal function. It has been shown (Gehring and Whitham, 1991, Del Vecchio et ah, 1993) that races of Pinus edulis that were genetically susceptible to chronic herbivory, either by a stem-boring moth, or a needle-feeding scale insect, suffered a 30% reduction in ectomycorrhizal colonization relative to non-susceptible races. This reduction was subsequently observed to lead to a 20% loss of shoot biomass (Gehring and Whitham, 1994). Such effects can persist for at least one year after removal of the herbivore and may be more severe in poor than in fertile soils (Gehring and Whitham, 1995). In view of the sensitivity of ectomycorrhizal fungi to reduction in assimilate supply, revealed in experiments involving artificial defoliation (Last et ah, 1979); shading (Lamhamedi et ah, 1994); and separation of mycelium from root (Soderstrom and Read, 1987), the negative impacts of shoot herbivory are predictable, but clearly they must be taken into account when evaluating likely responses to colonization in nature. Analysis of the effects of below-ground grazing upon ectomycorrhizas have concentrated upon fungivory. This is appropriate, because most of the youngest root tissue is enveloped in mycelium and therefore protected from direct attack by herbivores. These analyses may be flawed, however, if carried out under simplified conditions. CoUembolans, for example, show highly selective grazing habits in the laboratory, they are influenced by a variety of factors, even down to the culture medium used to support fungal growth (Leonard, 1984); analysis of field-collected animals, however, show a diverse range of food materials in their guts (Anderson and Healy, 1972). Obviously it is desirable to use intact mycorrhizal plants grown on natural substrates with realistic communities of animals when evaluating these effects. Ek et ah (1994) determined the effects of different densities of the coUembolan Onychiurus armatus upon ectomycorrhizas of P. contorta formed by Paxillus involutus. The impacts of fungivory upon nutrient uptake by the extramatrical mycelium was examined by placing cups, containing ^^NH4^ or phytin, to which the fungus alone had access, through the soil. Low densities induced greater development of the mycorrhizal mycelium and an enhancement of uptake and transfer of N to the plants by 76%. Mycelial growth was impeded only at a high density of O. armatus. Studies did not show a significant increase in the coUembolan population when mycorrhizal systems were compared to non-mycorrhizal systems. Setala (1995) exposed birch and pine plants, both colonized (as would be expected in nature) by a number of fungal symbionts, to a naturally complex or highly simplified microfaunal population. After 57 weeks, despite reduced colonization of both species in the presence of the complex animal assemblage, their shoot growth and N and P concentrations had significantly increased relative to those seen in the microcosms with simplified populations. This suggests that fungivory may not be harmful to the symbiosis provided that the composition of the microbial community is sufficiently complex to ensure efficient mobilization and turnover of soil resources. More experiments involving manipulation of target species in otherwise normal microfaunal populations are clearly needed.

432

General themes

VA Mycorrhizas in Temperate Biomes Plant communities which are dominated by trees or herbaceous plants that are colonized by VA mycorrhizal fungi progressively replace those with ectomycorrhizal roots on a global scale as, with decreasing latitude, mean annual temperatures and evapo-transpiration rates increase (Read, 1991a,b). The climatic changes lead to a reversal of the leaching tendencies prevalent in heath and boreal forest systems and to a consequent increase in base status and p H close to the soil surface. This, in turn, results in acceleration of the rates of turnover of organic matter and nitrification (EUenberg, 1988). As a consequence, the relatively mobile NO^ ion progressively replaces organic N or NH4 as the principal source of N for plants. As availability of the element increases, the proportion of nitrophilous species increases along the gradient and P, the mobility of which is low, can be predicted progressively to replace N as the major growth-limiting nutrient in the ecosystem (Read, 1991a,b). Whereas selection appears to have favoured the prevalence of VA rather than ectomycorrhizal colonization in many ecosystems which are primarily P limited, it has proved surprisingly difficult to demonstrate that plants growing in the field under natural conditions benefit from enhanced access to P (Fitter, 1985, 1990; and see Chapters 5 and 16). This situation contrasts strongly with that seen in pots under controlled conditions where reported values of P inflow to roots colonized by VA mycorrhizal fungi are an order of magnitude greater than those seen in the field (Fitter and Merryweather, 1992). A number of factors may combine to reduce the impacts of VA colonization in the field. Of primary importance is the fact that the rates of growth of many plants in nature are limited by environmental factors, for example water shortage, other than P deficiency. In stress-tolerant species, growth rates may be inherently so low that P requirements can be satisfied by diffusion, without involvement of mycorrhizal hyphae. In the case of VA mycorrhizal plants, as in that of plants with ectomycorrhizas, it is important to consider the extent to which the potential of the symbiosis, identified under controlled conditions, is influenced in nature by other biotic factors. One of the proposed reasons for the apparent 'ineffectiveness' of VA mycorrhiza in the field is grazing by coUembolans. Here, as in studies of their impact upon ectomycorrhizal fungi, much that has been written about the possible impacts of grazing coUembolans is based upon experiments of unrealistic design. It has been shown in pot experiments (Warnock et al., 1982; Finlay, 1985) that grazing by coUembolans can reduce or even eliminate the beneficial effects of mycorrhizal colonization, although at low densities of the animals the growth and P concentration of mycorrhizal plants can be increased (Harris and Boemer, 1990). Application of insecticides such as chlorfenvinphos with the aim of specifically reducing coUembolan populations has been shown to provide increases of growth and P inflow to Holcus lanatus in the field (McGonigle and Fitter, 1988). However, since this compound is known to be toxic to non-target animals such as earthworms (Rabatin and Stinner, 1991) and root feeding insects (Brown and Gange, 1990) the results of the experiment are confounded. Using a compound, chlorpyriphos, shown not to effect non-target organisms, Gange and Brown (1992) were, however, able to demonstrate that benefits of mycorrhizal colonization were significantly greater in the absence than in the presence of coUembolans.

The roles of mycorrhizas in ecosystems

433

While coUembolans may have important negative effects upon the mycorrhizal symbiosis under some circumstances, it is increasingly evident that a large number of animal species can influence the extent and outcome of colonization by VA fungi and that tripartite interactions between plant, fungal symbiont and microfauna are both complex and far-reaching. Only by assessing the impacts of all these interactions, many of which are occurring simultaneously in the ecosystem, can a balanced view of faunal effects on the mycorrhizal symbiosis be obtained. Application of the nematicide carbofuran to undisturbed prairie vegetation (Ingham et al, 1986) led to increases of VA mycorrhizal colonization in roots of the dominant grass Bouteloua gracilis, suggesting that nematodes had adverse effects upon the extraradical mycelium. There were no associated effects upon P concentration in shoots, but without data on plant growth and therefore total P uptake by the plants this cannot (as discussed in Chapter 5) provide unequivocal information on the contribution of mycorrhizas to P nutrition. A closer approach to the natural condition was obtained by Barker (1987) who grew Lolium perenne with or without its shoot (Acremonium loliae) and root {Glomus fasciculatum) endophytes, in order to determine their combined and individual contribution to defence against the Argentine stem weevil Listronatus bonariensis. The foliar endophyte, when present, deterred feeding and oviposition by the weevil but its effects were reduced by the presence of the mycorrhizal fungus. In the absence of the foliar endophyte the VA fungus had no effect upon herbivory. There is clearly the possibility that while the presence of shoot endophytes provides advantages to the plant in terms of direct anti-herbivore effects upon aboveground grazes, rhizophagous insects would be deterred by the presence of the mycorrhizal fungus. Obviously, experiments to investigate this more complete scenario would be worthwhile, especially in plants such as L. perenne, which have fibrous root systems and retain mycorrhizal colonization despite the likelihood that they are able to scavenge effectively for nutrients in the absence of the symbiosis. Resistance to herbivory may be a selective factor, along with that of fungal pathogens reported below, contributing to the retention of mycorrhizal colonization in plants such as grasses in temperate ecosystems. It is increasingly recognized that examining responses of plants to mycorrhizal fungi over short periods of their vegetative growth cycle, whether in pots or in the field, may provide misleading results. The extent of this weakness has been highlighted by studies of the bluebell Hyacinthoides non-scripta carried out throughout the life cycle of the plant in a deciduous woodland (Merryweather and Fitter, 1995a,b, 1996). This work clearly demonstrates, apparently for the first time in a natural population of field-grown plants, that the inflows necessary to maintain a positive P budget can only be achieved in the mycorrhizal condition. H. non-scripta is a perennial, vernal geophyte which characteristically dominates the herb layer of deciduous woodland in parts of north-west Europe. It has a coarse root system made up of thick (0.5-1.0 mm) unbranched elements which are produced annually from the base of the bulb. A new bulb and root system are produced every year. By regularly sampling undisturbed plants of H. non-scripta throughout their annual life cycle in a deciduous woodland, Merryweather and Fitter (1995a) explored the relationship between mycorrhizal colonization and inflows of P. There was a rapid increase in the proportion of root length colonized by VA mycorrhizal fungi over the period from root emergence in September (autumn), to reach a maximum value

434

General themes

in excess of 70% in January and February (Fig. 15.8a) even before the shoots appeared above ground. Thereafter, as confirmed by declining numbers of entry points (Fig. 15.8b), new colonization slowed. From the time of root emergence, P inflow increased rapidly at a rate similar to that of colonization, although until December values were negative, indicating a net loss of P. Maximum inflows were reached during the photosynthetic phase (Fig. 15.8c), but these subsequently declined, again at the same rate as that of colonization. The data for P inflow and percentage root length colonized showed a significant correlation (Fig. 15.8d). The individual plants lose significant amounts of P, particularly at the end of the growing season, in seeds, old leaves and roots as they are shed. Glasshouse-grown plants, lacking mycorrhizal colonization, are imable to absorb sufficient P from the soil to balance their P budget and therefore end the season with a large P deficit, which would not permit survival in the field. Soils supporting otherwise undisturbed colonies of H. non-scripta growing in the field were subjected to a benomyl drench at 2-monthly intervals over two years, which greatly reduced mycorrhizal colonization without having any effect on P availability (Merryweather and Fitter, 1996). This led to a large reduction of the P concentration of all vegetative parts of the plant, relative to that in untreated colonies (Fig. 15.9). In contrast, the flowers and seeds of the benomyl-treated plants had the same P concentration as the controls after the first season, with reduction in their P status being observed only after two years (Fig. 15.9). This suggests that when P uptake is reduced, H.

75

162

162

233

162

233

Days from 6th September

233

Days from 6th September

Days from 6th September

20

40

60

80

Mean %rootlength colonized

Figure 15.8 Seasonal pattern of (VA) mycorrhizal colonization and P inflow in Hyacinthoides non-scripta growing in the field, (a) Mean percentage colonization, (b) Mean abundance of entry points, calculated as entry points per intersection. The points on the curves are three harvest running means, (c) Curve of fitted values for P inflow in the field, (d) Correlation between P inflow and mean percentage root length colonized. Linear regression: r^ = 28.8, P = 0.15. From Merryweather and Fitter (1995a), with permission.

The roles of mycorrhizas in ecosystems

435

non-scripta protects its reproductive structures by selectively allocating P to them. Selective allocation of P to reproductive structures by mycorrhizal plants growing in P-deficient soils has also been observed in pot-grown plants of wild oat Avena fatua (Koide et al, 1988b; Bryla and Koide, 1990b). This pattern of allocation in VA mycorrhizal plants may significantly influence fecundity and so played a direct role in determination of fitness in the field. Plants such as bluebell with very coarse root systems can be predicted on theoretical grounds to be responsive to mycorrhizal colonization. However, questions remain as to the role play by the symbiosis in plants such as grasses which, despite the fibrosity of their root systems, retain high levels of colonization in nature. There are many glasshouse experiments with grasses demonstrating that increases in PO^ uptake can lead to increases of yield, but such effects have been difficult to observe in natural communities. Thus Hetrick et al, (1988, 1990), examining the responsiveness of two grass species that dominate the tall grass prairies of the USA, found evidence in the C3 species Bromus inertnis that, despite greater P acquisition in the mycorrhizal condition, there was little or no growth response. In nature, B. inertnis makes most of its growth in the cool seasons of autumn and spring and it is at these times that arbuscule production is at a maximum. Early season growth enables the plant to avoid competition with the other dominant grass species Andropogon gerandii, a C4 plant which grows in the warm season and is extremely responsive to VA colonization. The question arises as to the nature of the mycorrhizal relationship in B. inermis. A subsequent study confirmed that P acquisition by B. inermis was significantly increased by colonization, especially at 18°C compared with 29°C but, again, no growth response followed (Hetrick et al,, 1994). As in the annual grasses described above, benefits of P acquisition may only be expressed late in development in terms of increased fecundity and improved

4 Y 'o> 3 r O)

572 V 1 V

March

April

May

June

Early April

Late April

May

Figure 15.9 Effect of benomyl drench on the concentration of P in the leaves of Hyacinthoides non-schptay measured at intervals over two growing seasons in the field. Shaded columns, control; unshaded columns, benomyl. From Merryweather and Fitter (1996), with permission.

436

General themes

offspring performance. It is possible that luxury accumulation of P early in the growing season increased the competitive ability of the grass by pre-empting availability of the element to other species. The responses of these two grass species to seasonal changes in environmental conditions, as well as to mycorrhizal colonization provides a further example of niche separation, this time a temporal one. An alternative explanation for the apparently beneficial impact of VA mycorrhizal fungal colonization upon the annual grass Vulpia ciliata has recently been proposed (Newsham et al., 1994, 1995). Again using benomyl to control colonization of the roots in the field, no relationship was found between the extent of occurrence of VA fungi and the rates of P uptake, but the biocide also controlled weakly pathogenic fungi such as Fusarium oxysporum, which were known to reduce fecundity of the plant (West et al., 1993a,b). It was therefore suggested by Newsham et al. (1994) that the benefits of VA fungi arose through their ability to protect the plant from pathogens. This possibility was examined further in field-grown populations of V. ciliata exposed to different concentrations of benomyl, so as to control the extent of colonization by VA and pathogenic fungi (Newsham et al, 1995). Fecundity was largely unresponsive to fungicide application, despite the fact that benomyl significantly reduced the abundance of both types of fungi in roots. However, the abundance of root pathogens, especially F. oxysporum, was negatively correlated with fecundity, even though plants displayed no disease symptoms. The poor relationship between fecundity and benomyl application contrasted markedly with the effects of benomyl on VA mycorrhizal and pathogenic fungi, and with the negative effects of root pathogens on fecundity. These effects could be explained if the two groups of fungi interacted, so that when both were greatly reduced by fungicides, the net effect on fecundity was slight. Such a hypothesis was supported by statistical analysis of the data, showing that there was a positive effect of VA mycorrhizal fungi, but only in relation to the negative effect of the pathogens. The interaction between VA mycorrhizal fungi and root pathogens was resolved using a transplant approach. Seedlings of V. ciliata were grown in a growth chamber with a factorial combination of inoculum of F. oxysporum or a Glomus sp., both isolated from V. ciliata at the field site, and then planted into a natural population of the grass in the field. After 62 days of growth, clear evidence was obtained that colonization by Glomus gave a protective effect. Plants inoculated with that fungus performed as well as control plants, even when simultaneously inoculated with F oxysporum, whereas those inoculated with F oxysporum alone grew significantly less well (Table 15.3). The Glomus sp. had a negligible effect on the performance of the plants in the absence of the pathogen. There was no correlation between shoot P concentration and the abundance in roots of either pathogenic or mycorrhizal fungal hyphae. Rather, the differences between treatments seem to have been due to a reduction in the frequency of pathogenic hyphae within roots brought about by VA mycorrhizal colonization. These experiments reveal that the effects of mycorrhizal colonization in the field may involve subtle interactions with other microorganisms that can only be detected by combining sensitive experimental design with careful data analysis. In so far as they deal with single species of plant, however, the studies of Hyacinthoides and Vulpia still provide a very simplified view of the possible impacts of

The roles of mycorrhizas in ecosystems

437

T a b l e 15.3 Effects of a factorial combination of Fusarium oxysporum (F) and a Glomus sp. (G) on shoot biomass and r o o t length of Vulpia ciliata plants g r o w n in the laboratory, transplanted into the field and sampled f r o m the field after 62 days* g r o w t h Variable

Treatment -G -F

Log (In) shoot biomass (mg) 2.4a Root length (cm) 217a

Main effects

Interaction GXF

+G -F

-G +F

+G +F

G

F

2.2a 203a

1.4b I 11 b

2.2a 228a

F = 3.5 F=I7.3*** F = 9.4** F = 9.0**

F = 4.8* F = 9.0**

Means are of 16 replicates; where followed by different letters they differ at P<0.05. Significant main and interaction effects in ANOVA are indicated by: * P<0.05, * * P
mycorrhizas upon stable natural ecosystems in which plants normally coexist in communities of mixed species and uneven ages. Ultimately it is necessary to consider the role of the symbiosis in this more complex situation. The earliest attempts to elucidate such effects examined the responses of plants grown in pots as species pairs, with or without the presence of VA mycorrhizal inoculum (Fitter, 1977; Hall, 1978b; Buwalda, 1980). These revealed (see Chapter 5) that VA fungi had major impacts upon the outcome of competitive interactions between species, in most cases the yield of one being significantly increased at the expense of the other. While it is implicit in such results that the balance between plant species will be influenced by the presence of VA mycorrhizal fungi in the field, the extent of these effects in multi-species communities can again only be judged under more realistic circumstances. In an attempt to achieve greater realism. Grime et ah (1987) reconstructed a plant community representative of that occurring in nutrient-deficient, calcareous soils in north-west Europe. The community consisted of a mixture of grasses and herbs, all but two species of which {Arabis hirsuta and Rutnex acetosa) were known to be heavily colonized by mycorrhizal fungi in the field. The plants were grown from seed for 1 year in a sward of the dominant grass Festuca ovina, seedlings of which had been pre-sown either in the non-mycorrhizal condition or as mycorrhizal individuals which provided a natural source of inoculum for the subordinate plants. Survivorship of all constituents of the community was monitored in the mycorrhizal and non-mycorrhizal microcosms throughout the year and the impact of colonization on their yield and on final structure of the community was determined at a single terminal harvest. Immediately prior to harvesting, ^^C02 was fed to shoots of Festuca in selected microcosms and the pattern of distribution of label was determined. The transfer of radioactivity occurred almost exclusively in the microcosms containing mycorrhizal fungi, and only the plant species which were actually colonized contained high amounts of ^^C (Table 15.4). These observations provide evidence that plants in natural communities are functionally interconnected by their mycorrhizal fungi, but the extent to which net transfer of C occurred between plants was not determined (see above). All the forbs normally colonized by VA mycorrhizal fungi showed significantly higher yields in the mycorrhizal than the non-mycorrhizal microcosms (Table 15.5). In contrast, the crucifer {A. hirsuta) was more productive in the non-mycorrhizal

438

General themes

Table 15.4 Transfer of '^C-labelled assimilate (d.p.m.) from a selected *donor' plant of Festuca which was fed with '^C02 to neighbouring plants grown together as a mixed community of grasses and herbs for one year with (inoculated) or without (uninoculated) VA mycorrhizal fungi Uninoculated

Inoculated

Festuca ovina Briza media Poa pratensis Plantago lanceolata Hieracium pilosella Centaurea nigra Leontodon hispidus Scabiosa columbaria Centaurium erythraea Rumex acetosa

Root length colonized (%)

Shoot radioactivity (dpm)

Root length colonized (%)

Shoot radioactivity (dpm)

89 11 54 67 59 64 70 86 44 0

9276 14 002 6241 18 764 60 719 45 081 15 363 23 912 4213 494

0 0 0 0 0 0 0 0

622 1136 404 730 297 1338 546 786

—

—

0

376

In the inoculated microcosms only Rumex actio^Q (Polygonaceae), which fails to become colonized, shows levels of activity as low as those seen in the uninoculated systems. Data from Grime et o/. (1987).

community, as was the polygonaceous weed R. acetosa, although not significantly. As pointed out earlier, survivorship provides a more direct index of fitness than productivity, especially in nutrient-stressed habitats such as calcareous grasslands. Absence of mycorrhizal inoculum led to major reductions of survivorship in those forbs which are normal constituents of calcareous grassland communities (Table 15.6), while in the cases of A. hirsuta and R, acetosa the reverse was observed, only small numbers of these plants being still alive after six months in mycorrhizal microcosms. Ecologists examining the structure of calcareous grassland communities have recognized the distinction between groups of species that are and are not able to colonize closed turf (Grubb, 1976, 1977; Fenner, 1978). As a result of their inability to establish in small gaps, members of the latter group of 'turf incompatible' plants are inevitably relegated to the ruderal habit. However, no clear mechanisms for their exclusion have been proposed. In the study of Grime et ah (1987), sensitivity to the presence of an established VA mycorrhizal mycelium was a factor in determining survivorship of R. acetosa and A. hirsuta. In experiments designed to examine the basis of these impacts, chambers were designed in which a nylon mesh cylinder with a 37 |Lim pore size enabled separation of root from mycelial effects (Francis and Read, 1994, 1995). Plants known to be hosts to VA mycorrhizal fungi, usually F. ovina and Plantago lanceolata, were grown in the mycorrhizal or non-mycorrhizal condition in the outer compartment of the chambers for sufficient time to enable the mycelium to grow from mycorrhizal plants into the central compartment. The inner compartment was designed to represent a 'gap' or regeneration niche (Grubb, 1977) in established vegetation in which the only major variable was presence or absence of a VA mycelial network. Seeds of a range of test species were sown into the

The roles of mycorrhizas in ecosystems

439

Table 15.5 The effect of mycorrhizal colonization on yield plant"') (mg plant ') of forb species grown together in microcosms for one year

Arabis hirsuta Campanula rotundifolia Centaurea nigra Centauhum erythraea Galium verum Hieracium pilosella Leontondon hispidus Plantago lanceolata Rumex acetosa Sanguisorba minor Scabiosa columbaria Silene nutans

Non-mycorrhizal

Mycorrhizal

0.26* 0.73 1.70 0.23 1.87 0.93 0.83 3.62 9.72 5.06 2.56 16.85

0.13 4.20* 10.90* 7.08* 9.39* 7.63* 3.72* 33.96* 8.77 NS 17.14* 10.19* 44.89*

* Yield significantly different from mycorrhizal or non-mycorrhizal counterpart at P <0.05. NS, Not significant. All species of forb which become colonized by VA fungi have a significantly greater dry weight in the mycorrhizal than in the non-mycorrhizal microcosms. Arabis hirsuta (Cruciferae), which does not become colonized, has a significantly lower yield in the mycorrhizal microcosm, while that of Rumex acetosa (Polygonaceae), also colonized, shows no difference between the treatments. Data from Grime et al. (1987)

Table 15.6 Survivorship (%) of forbs after 6 months In mycorrhizal and non-mycorrhizal microcosms Species

Mycorrhizal

Non-nnycorrhizal

Centauhum erythraea

64 58 49 42 71 53 84 8 II

2 II 6 13 10 6 16 42 60

Galium verum Hieraceum pilosella Leontodon hispidus Plantago lanceolata Sanguisorba minor Scabiosa columbaria Arabis hirsuta Rumex acetosa

Significant increases of survivorship were obtained in most forbs grown in the mycorrhizal condition, the exceptions being Arabis hirsuta and Rumex acetosa, which show the reverse trend.

central compartment at the time of radicle emergence and their subsequent development was followed, so that the impact of the mycorrhizal fungi upon them could be evaluated. The species selected were representative of families considered to be largely non-mycorrhizal, so-called 'non-hosts', or of uncertain status (see Chapters 1 and 3). They included Arabis hirsuta (Cruciferae), Arenaria seripyllifolia (Caryophyllaceae), Echium vulgare (Boraginaceae), Reseda luteola (Resedaceae) and Rumex

440

General themes

acetosella (Polygonaceae). For comparative purposes some species that are both mycorrhizal and turf-compatible were examined. These included P. lanceolata (Plantaginaceae) and Centaureum erythraea (Gentianaceae). The plants showed very different responses to the presence of VA mycorrhizal mycelium around their roots. On the one hand, the non-hosts Arabis hirsuta and Arenaria serpyllifolia grew poorly (Fig. 15.10a) and also showed a reduction in survivorship. On the other hand, normally mycorrhizal species such as P. lanceolata and C. erythraea responded positively in the presence of the mycelium, but grew poorly and survived badly in its absence (Fig. 15.10b). E. vulgare and R. luteola, despite being colonized by the fungus, which produced vesicles but no arbuscules in the roots, showed responses which were sinular to the non-host species. £. vulgare and R. luteola are indeed reported in the literature (see, for example, Harley and Harley, 1987) as having VA mycorrhizas. They provide striking examples of the need for functional analysis of the relationship between fungus and plant, and for caution when extrapolating from 'occurrence' of colonization to pronouncements about the status of the symbiosis. The relationship observed here and in several other species examined by Francis and Read (1995), under conditions which are considered to resemble closely the natural regeneration niche, were definitely of the 'antagonistic' rather than 'mutualistic' type. A further note of caution needs to be sounded here. Large populations of E. plantagineum have been correlated with effective maintenance of high densities of VA mycorrhizal propagules in an arid environment (McGee et al, 1987). It is possible that hyphal and vesicular colonization can actually support the fungus, by providing a route for C transfer from the plant. Absence of arbuscules might, however, preclude any transfer of P to the plant. The basis of the antagonistic effect of VA mycorrhizal fungi upon ruderal species remains to be elucidated. In some cases adverse effects upon root development of non-hosts have been observed in the absence of colonization by the fungus, suggesting that there is a chemical interaction (Francis and Read, 1994; Allen et ah, 1989), whereas in others inhibition is associated with penetration of the root and prolific production of vesicles (Francis and Read, 1995). In the latter case, conventional microscopic analysis would leave many observers to record 'occurrence' of mycorrhizas despite the fact that arbuscules are not seen. This emphasizes the importance of functional analysis of the symbiosis and highlights the dangers associated with any assumption that occurrence of colonization is necessarily equivalent to 'mutualism'. There are two issues. First, we do not know enough about the sites and mechanisms of nutrient transfer within mycorrhizal roots to use structural information on the occurrence and extent of development of hyphae, coils and arbuscules to predict function, although presence of arbuscules may well indicate the potential for P transfer to the plant (Chapter 14). More quantitative and developmental anatomy, coupled with physiology, is required to permit prediction of function based on structural information (Smith and Smith, 1996a). Second, this discussion is based on the community dynamics of the plants. Again, we do not know what advantages there may be to the fungus in excluding the 'turf-incompatible species'. Investigation at the cellular level may be required, as a component in the analysis of this fascinating interaction. In any event, there are clearly circumstances in which VA mycorrhizal fungi are antagonistic to plants which they colonize and it is not unreasonable to suggest that this antagonism is

The roles of mycorrhizas in ecosystems

441

Figure 15.10 Growth of non-host and host plants in simulated gaps in artificial 'swards* of Plantago sp., a potentially mycorrhizal, turf-compatible species. See text for explanation, (a) Representative plant of the *non-host' Arenaria serpyllifolia after 42 days' growth In the presence (M) of VA mycorrhizal mycelium colonizing the soil in the *gap' by growing through 37 jim mesh, or in the absence (NM) of such mycelium. Note the reduction in growth in the presence of the mycelium, (b) Representative plants of the *hosf species Centauhum erythraea in the same system. Note the greater growth in the mycorrhizal (M) treatment. From Francis and Read (1995), with permission.

442

General themes

sufficiently severe in some plants to explain their exclusion from communities where the majority of species are typically mycorrhizal. In such cases VA mycorrhizal fungi can be seen as major determinants both of the structure and biodiversity of plant communities. Ecological studies of the effects of plant density on competition have rarely taken the potential influence of mycorrhizas into account. The general consensus has been that intraspecific competition is highest at high densities. As discussed in Chapter 16, it may be presumed that in the field or in pot experiments using non-sterilized soil, potentially mycorrhizal species would be colonized. Recent experiments directly investigating the consequences of eliminating mycorrhizal fimgi (Koide, 1991b; Allsopp and Stock, 1992; Hetrick et al, 1994; E. Facelli, personal communication) have shown very little competition between non-mycorrhizal species at any density, whereas there was often severe competition between individual plants when they were mycorrhizal (see Fig. 16.2). The bases for the competitive effects have not been worked out, but could include very much improved exploitation of the soil by mycorrhizal as opposed to non-colonized roots. The importance of potential nutritional links between the plants, connected by mycorrhizal mycelium will need careful consideration in the context of both interspecific and intraspecific competition. Mycorrhizas in Tropical and Subtropical Biomes In the tropics, gradients occur from sub-humid Savannah woodlands, which have a pronounced dry season and a considerable susceptibility to fire, towards wet rain forests of low seasonality. These climatic regimes inevitably affect the nutrient balances of the ecosystems. Studies of nutrient composition of litter in tropical ecosystems led Vitousek (1984) to conclude that whereas tropical lowland rain forests were likely to be limited by availability of P, savannah, from which N was lost in recurrent fires, was characteristically N-limited. If real, these deficiencies would be expected to increase selection of adaptations, symbioses amongst them, that optimized access to the limiting nutrient. The occurrence of N2-fixing legumes in the dry savannah, notably Acacia spp. in Africa, Prosopis in America, and Acacia and Casuarina in Australia, can be seen as a response to N limitation of trees in these ecosystems (Hogberg, 1988, 1989). The legumes however, comprise only a proportion of trees in the vegetation (around 2025% in miombo; Hogberg and Pearce, 1986). In any event, alleviation of one deficiency may be expected to lead to its replacement by another. The dependence of N2 fixers upon P supplies is demonstrated by experiments showing that application of P to dry subtropical woodlands increased both the yield and nitrogenase activity of two leguminous understory species. Acacia and Kennedia in Australia (Kingston et al, 1982). The interdependence of N and P supplies should not be overlooked and may help to explain the fact that N-fixing legumes are generally hosts to VA mycorrhizal fungi (Alexander, 1989a,b). The fact that non-nodulated leguminous tree species often successfully coexist in Savannah grasslands alongside those that are nodulated, further suggests, as pointed out by Sprent (1985), that in these arid environments the production of an extensive root system to enable acquisition of water and a diverse range of nutrients will provide the optimum use of resources.

The roles of mycorrhizas in ecosystems

443

This appears to be the strategy of the dominant savannah grasses, the extensive root systems of which are also colonized by VA mycorrhizal fimgi (Newman et al, 1986). The absence of large data sets for nutrient status and turnover of soils in the tropics has led to the use of indirect methods for assessment of the possible role of root symbioses in the nutrient economies of these ecosystems. Amongst these, analyses of tissue nutrient concentration in plants with different types of symbiotic associates and of their characteristic 5^^N signatures have predominated (Hogberg 1990, 1992; Hogberg and Alexander, 1995) examined the leaf N and P concentrations in 98 species and site combinations of Tanzania and north-east Zambia; Nfixing VA mycorrhizal species, non-N-fixing VA mycorrhizal species and non-Nfixing ectomycorrhizal species being recognized as three separate categories. The N and P concentrations expressed in relation to optimal ratio between the elements defined by physiologists, of between 12.5:1 and 10:1 (Table 15.7) are seen to differ between the categories. N-fixing VA mycorrhizal species have supra-optimal N:P ratios suggesting P deficiency, non-N-fixing VA mycorrhizal species show suboptimal N:P ratios indicative of N deficiency, whereas ectomycorrhizal species occupy an intermediate category close to optimal. Such a distribution strengthens the view that VA mycorrhizal colonization selectively favours acquisition of P, while the ectomycorrhizal symbiosis increases access to both elements. Use of nutrient concentrations in tissues is inevitable when investigating complex natural communities composed of plants of large size. However, the data can be misleading because elevated concentrations of, for example P, might be a result of N limitation and consequent poor growth where scavenging for P is more effective than for N. The extent to which the different types of symbiosis seen in tropical plants are functioning to provide access to qualitatively distinct sources of N has been investigated by determining the S^TsT signatures of their foliage (Hogberg, 1990, 1992; Hogberg and Alexander, 1995). The measurements rely on the assumption, which is generally but not universally accepted (Shearer and Kohl, 1986), that atmospheric N has a lower ^^N abundance than those mineral N sources of soil (NO^ and NH4) which are normally considered to be available to plants. In addition, mineralization discriminates against the heavier isotope so that soil organic N has greater ^^N enrichment than inorganic forms. The first analyses of 8^ N signatures of nodulated, VA and ectomycorrhizal species growing in miombo woodlands of Tanzania indicated that there were indeed differences between the groups (Hogberg, 1990; Fig. 15.11). The ectomycorrhizal species had greater 5^^N values than those plants which were nodulated, suggesting that they were using organic N sources. Plants colonized by VA mycorrhizal fungi occupied an intermediate position. Subsequent measurements of plants from the miombo of Zambia and the seasonally wet tropical forest of Korup Cameroon have failed to confirm this pattern (Hogberg and Alexander, 1995). TTie discrepancy highlights the need for caution when interpreting the results of these indirect indices of nutrient use. Differences in the extent of 8 N enrichment of a given N source such as NO^ can occur as a result of events that are localized in space and time. Fire leads to enrichment of ^^N in residual NOs" relative to that of total soil N in dry woodland (Pate et al, 1993), and volatilization of N from patchily distributed termitaria would be expected to lead to increases of ^^N abundance in residual N in soils of the mounds (Hogberg and Alexander, 1995). Similarly, differences in rates of leaching

General themes

Table 15.7 '^N abundance and concentrations of nutrients in foliage of trees from miombo woodland (Misaka, Zambia) and lowland rain forest (Korup, Cameroon) Symbioses 5'^N (%o) ECM VAM N O D + VAM' N (% dry matter) ECM VAM N O D + VAM' P (% dry matter) ECM VAM N O D + VAM' N: P ratio ECM VAM N O D + VAM' Ca (% dry matter) ECM VAM N O D + VAM' Mg (% dry matter) ECM VAM N O D + VAM' K (% dry matter) ECM VAM N O D ± VAM^

Misaka

Korup

2.07 ^ 0.25 (8) 2.87 + 0.80 (6) 0.20 + 0.19(7)

4.88 ^ 0.29 (8) 4.61 + 0.2! (10) 3.30 ± 0.10(2)

1.75 ^ 0.1 1 (8) 1.54 ± 0.12(6) 3.22 ^ 0.07 (7)

2.10 + O.ll (8) 2.11 + 0.20(10) 3.30 ± 0.10(2)

0.14 + 0.01 (8) 0.12 + 0.02 (6) 0.13 + 0.01 (7)

0.13 + 0.01 (8) 0.10 j ^ 0.01 (10) 0.13 + 0.01 (2)

13.1 ± 0.4 (8) 14.4 ^ 2.4 (6) 24.8 + 1.2(7)

16.5 ^ I.I (8) 20.9 ± 1.6(10) 25.8 ^ 0.8 (2)

0.64 + 0.09 (8) 0.77 + O.ll (6) 0.53 + 0.12(7)

0.52 + 0.05 (8) 0.59 + 0.05 (10) 0.32 ^^ 0.15 (2)

(8) (6) (7)

0.27 -»- 0.05 (8) 0.26 + 0.04(10) 0.18 J3 0.02 (2)

0.76 ± 0.09 (8) 0.78 ± 0.14(6) 0.82 + 0.08 (7)

1.24 + 0.19(8) 1.56 •+- 0.22 (10) 0.93 ^^ 0.12(2)

0.32 0.31 0.27

^ 0.04 ^ 0.04 ^^ 0.05

^ Pericopsis angolensis at Misaka may have both ECM and VAM (Hogberg, 1982; Hogberg and Pearce, 1986). ECM, ectomycorrhizal; VAM, VA mycorrhizal; NOD, nodulated. The species are classified into groups according to their root symbioses. Data are means ± SE. Numbers of species are shown in parentheses. Data from Hogberg and Alexander (1995).

and of denitrification (Vitousek et ah, 1989), and of isotope fractionation during uptake and assimilation of N (Handley et al, 1993; Hogberg et a/., 1994), will all tend to confound the interpretation of ^^N enrichment data. A further confounding factor would be introduced if organic N were transferred between plants by linking mycorrhizal mycelium. It is evident from this that if interpretation of foliar 5^^N signatures is to be made more meaningful, detailed measurements of the spatial and temporal changes of ^^N abundance in the various N substrates potentially available to the plant, as well as of changes associated with plant metabolism itself, will all be required. Circumstantial evidence suggests that there is a relationship between mycorrhi-

445

The roles of mycorrhizas in ecosystems

8 Ectomycorrhizas

VA mycorrhizas

4H c o

'^ >

0

m

T

r

o

Nodulated

d

—I

2

4

8

S '^N (%o) F i g u r e 15.11 Frequency diagram showing ' ^ N abundances of foliage of m i o m b o tree species w i t h different r o o t symbioses at different sites: solid bars, Zambia 1995; hatched bars, Zambia 1991; open bars, Tanzania. For discussion see t e x t . From Hogberg and Alexander (1995), w i t h permission.

zal type and soil conditions, especially in the wet and seasonally wet tropical forests. Whereas trees with VA mycorrhizal colonization predominate over large areas of such systems, there are localized occurrences in Amazonian (Singer and Araujo, 1979, 1986) and west African (Newbery et al, 1988, 1996) forests, of communities dominated by ectomycorrhizal species. The communities are characteristically restricted to the most nutrient-poor soils with a surface accumulation of litter and raw humus in which the colonized roots proliferate. Even where legumes occur in such systems they appear to be largely of the non-nodulated type. Along a transect in seasonally wet forest in Korup (Cameroon) within a flora of 200 tree species, nodulated legumes make up only 1% of the total basal area (Hogberg and Alexander, 1995). Such observations suggest on the one hand that N fixation is not an advantage for trees in rain forest, and on the other that ectomycorrhizal

446

General themes

associations are favoured by extreme nutrient deficiency. By analogy with the situation observed in boreal and temperate forests, it has been predicted that these associations are involved in mobilization of organic N from the litter (Alexander, 1989a; Read, 1991a) thus providing advantages over VA mycorrhizas. However, recent analyses of foliage from Korup Forest indicate that ectomycorrhizal species have higher percentage P and lower N:P ratios than their VA mycorrhizal counterparts, suggesting that ectomycorrhizas are more important in P nutrition in the rain forest (Hogberg and Alexander, 1995; Newberry et al, 1996). Positive correlations have been observed between growth, ectomycorrhizal colonization and foliar P concentration in dipterocarp seedlings (Lee and Lim, 1989; Lee and Alexander, 1994). It may be that, as in temperate systems, the ability of the ectomycorrhizal fungi to produce extensive mycelial networks in soil and to selectively exploit localized pockets of organic matter enriched in P or N, provide the selective advantage for this type of symbiosis, but much experimental research remains to be done. In the moist tropics the majority of species probably form VA mycorrhizal associations, with rather non-specific relationships between plants and fungi. Many of the soils are acid, highly leached and P deficient, often because of sequestration as poorly available Al phosphates. Here, VA mycorrhizas are likely to play an important role in rapid cycling of Pi as it becomes available through mineralization. The fact that in these forests ectomycorrhizal plants often occur together in distinct guilds or 'groves' (Singer and Araujo, 1979; Newbery et al, 1988), where their roots proliferate in acidic himiic materials of low quality, is suggestive of specialized functions for this type of mycorrhiza. A factor favouring the maintenance of such guilds, once they are established, is that ectomycorrhizal fungi of tropical, as of temperate and boreal forests, show a greater level of host specificity than do their VA mycorrhizal counterparts (Alexander, 1989a; Thoen and Ba, 1989; Smits, 1992; and see Chapter 6). The greater availability and vigour of the requisite inoculum in and around the guild, relative to that in the surrounding VAdominated systems, will increase the chance of successful establishment of siblings in the vicinity of the guild and promote its integrity. At the same time, host specificity, by favouring the survival of a relatively small number of compatible species, would be expected to lead to reduced diversity within the guild. There is a limited amount of evidence that diversity is, indeed, lower in ectomycorrhizal than in surrounding VA-dominated communities (Alexander et ah, 1989) but the relative importance of nutritional and biological factors in determining these differences remains to be investigated. The Roles of Mycorrhizas in Primary Successions The sequential development of plant communities following major environmental perturbations such as glaciation (Crocker and Major, 1955) and volcanic activity (Simkin and Fiske, 1983) are well documented. It is acknowledged, also, that scarcity of nutrients in the poorly weathered materials exposed by such events may determine the early stages of the primary succession which is initiated on them (Gorham et al, 1979). Under these circumstances it is tempting to suggest a role for mycorrhizal fungi in facilitating the succession, but as yet there is little direct

The roles of mycorrhizas in ecosystems

447

evidence for such a role in nature. Thus, while normally mycorrhizal plants with light seeds such as Dryas (Crocker and Major, 1955) are known to be among the first to colonize recently deglaciated soil, there have been no studies of the pattern of mycorrhizal development on them in situ or, more importantly, of the role of any such development in facilitating establishment. There is evidence that both wind and animals can act as vectors of mycorrhizal inoculum. Warner et ah (1987) demonstrated that spores of VA fungi could be wind blown for up to 2 km, whereas animals were able to transport VA propagules over several miles of sterile pumice on Mount St Helens after its eruption (Allen, 1988). In all likelihood the small spores of ectomycorrhizal fungi will be transported over even greater distances. Trappe (1988) reports the invasion of recently exposed glacial till in the Oregon Cascades by coniferous trees which are abimdantly colonized by ectomycorrhizal fungi. In this case the glacial valley is surrounded by afforested ridges which can provide an abimdance of propagules of both partners. The proximity of established vegetation probably frequently complicates the successional process by providing local sources of reproductive propagules. Allen (1984) found such undisturbed patches of the original plant communities in the centre of the main region of pyroclastic flow on Mount St Helens following its eruption. Evidence from species lists compiled for isolated islands formed by volcanic eruptions does little to clarify the picture, although non-mycorrhizal or facultatively mycorrhizal species, the latter often grasses, figured more prominently as early colonists on Krakatoa. These were succeeded by species likely to be more responsive to mycorrhizal fungi, including orchids and a Casuarina sp. (Simkin and Fiske, 1983). Critical questions concerning the role of mycorrhizal colonization in facilitating establishment of plants in virgin sites can ultimately only be answered by manipulative experimentation, involving addition of plants or fungi to such sites and monitoring over a chronosequence the relationship between colonization, nutrient capture, growth and survival. Primary succession on sand dune ecosystems is a more predictable process in which relationships between soil quality disturbance and mycorrhizal status are apparent (Read, 1989). In the disturbed and nutrient-enriched conditions of the drift line, non-mycorrhizal species predominate, particularly those of the families Chenopodiaceae and Brassicaceae. Under more stable conditions a succession of communities made up of species that are responsive to VA mycorrhizal colonization are found, ranging from open grassland dominated by the grass Ammophila arenaria on the fore-dunes, to herb-rich closed communities on the more stable dimes. The succession from drift line to stable back-dunes typically covers a gradient of decreasing pH and increasing soil organic matter content (Fig. 15.12) over which communities dominated by plants with ectomycorrhizas or ericoid mycorrhizas become increasingly important, with forest or heathland replacing grassland as the climax vegetation type. The Roles of Mycorrhizas in Secondary Successions While primary successions often commence under conditions of nutrient impoverishment, those processes referred to as secondary succession, which follow disturbance of existing vegetation, are normally initiated in an environment of

General themes

448 Occurrence of and Responsiveness to Mycorrhizal Infection

HIGH

LOW HIGHnr~^_

species DRIFT

LINE

(ANNUAL a BIENNIAL

FACULTATIVELY VAMYCORRHIZAC

MOBILE

FACULTATIVELY VA MYCORRHIZAL

O

z

FORE-DUNES

(PERENNIAL

GRASSES)

EARLY

FIXED

DUNES

<

CD

or

OBLIGATELY VA MYCORRHIZAL

Z) t—

FIXED

DUNES

(SPECIES R"CM GRASSLArjD)

Q

ECTOMYCORRHIZAL

SLACKS (SALIX RE PENS SCRUB!

COMPETITORS ASSOCIATED ^^^K LOCAL E U T R O ^ I C A T I O N BY SEA BIRDS ETC/fURTICA. C H E N O P O D S ) ^JON XMYCORRHIZAL

LOW

DUNEFEATHi ERICOID MYCORRHIZAL

(SFtClESPOOR . r^VLJUNETUM)

S-species

MS - species HIGH

LOW BASE STATUS & pH

Figure 15.12 Diagrammatic representation of succession of mycorrhizal communities in a coastal sand dune, along an axis representing decreasing disturbance, pH and availability of mineral nutrients and increasing soil organic matter. From Read (1989), with permission.

enrichment, a pulse of N and P being produced by mineralization of residues left by the previous community (Walker and Syers, 1976). The disturbed soil is characteristically first occupied by weedy annuals, especially of families such as Chenopodiaceae, Brassicaceae and Polygonaceae which are effective colonizers and which, since the early work of Stahl (1900), have been considered to be largely nonmycorrhizal. The conventional interpretation of the basis of the success of these ruderal plants is that as Y strategists they have a high fecundity, short generation time and an ability rapidly to exploit pulses of nutrient availability (Grime, 1979). However, the sensitivity, discussed above, of many such plants to the presence of VA mycelium raises the possibility that reduction of inoculum potential of these fungi, which is known to arise from disturbance (Miller, 1979; Reeves et a/., 1979; Allen and Allen, 1980; Janos, 1980, Jasper et al, 1989, 1992; and see Chapter 2) is a prerequisite for their success. Decline of nutrient availability, as the initial flush of minerals is utilized or lost by leaching, leads progressively to competition between plants for resources, and to a situation in which mycorrhizal colonization could be expected to provide a nutritional advantage to plants. A possible role for mycorrhizas in determining the trajectory of successional processes was acknowledged by Gorham et al. (1979),

The roles of mycorrhizas in ecosystems

449

who proposed that plants characteristic of a particular stage of succession may have a higher 'affinity', through their fungal associates, for nutrients at a particular stage. However, few ecologists have considered the possibility that mycorrhizas play a pivotal role in successional dynamics. The hypothesis that mycorrhizal colonization might provide hosts with a greater competitive ability and that this leads to acceleration of successional processes was tested by Allen and Allen (1988), who introduced inoculum of VA fungi to a high-altitude soil that had been disturbed by open-cast coal mining and was colonized largely by annual 'nonhost' species. The presence of inoculum had the effect of reducing the growth of the ruderals and so in some plots led to increased rates of succession. However, in others, loss of cover provided by the ruderals led to exposure-damage to those species, mostly grasses, that had the potential to respond to colonization. Consequently, the rate of succession declined (Allen, 1989). Experiments of this kind demonstrate the complexity of interacting factors that can influence the successional process and emphasize that above-ground as well as below-ground factors can affect plant response. Much emphasis has been placed by ecologists upon secondary succession in old fields, where progressive decrease of availability of N is believed to be the factor that drives the process (Odum, 1960; GoUey, 1965; Tilman, 1987). Following the ruderal phase, a succession of grass species with increasing ability to compete for N is recognized (Tilman, 1990; Tilman and Wedin 1991). Although these grasses are likely to be colonized by VA mycorrhizal fungi, the role of mycorrhizas in determining the outcome of competitive interactions between them appears not be have been considered. There is much scope for work which includes the natural symbionts of these organisms. On theoretical grounds, because the decline in N availability arises partly through progressive inhibition of nitrification and replacement of mobile NO^ by relatively immobile NHJ ions as the source of mineral N (Robertson and Vitousek, 1981), advantages should increasingly accrue to mycorrhizal plants. Indeed, plants colonized by VA mycorrhizal fungi are likely to have excellent access to P, even when it is in relatively short supply, so this symbiosis will have the effect of increasing the emphasis on P as a factor influencing the sucession. In this context it is worth noting that the prairie grasses discussed above, were typical of habitats limited by N rather than P availability. A response seen in some ecosystems to changing N status is the appearance, as transient occupants, of N-fixing shrubs and trees (van Cleve and Viereck, 1981). In the succession from grassland to boreal or temperate forest, other trees that appear early are members of the genera Salix and Populus which, in addition to having light-weight propagules that enhance their capacity for dispersal into successional environments, are characterized by a plasticity which enables them to form both VA and ectomycorrhizas. Compatibility with VA mycorrhizal fungi may be a factor facilitating their incorporation into a turf dominated by VA mycorrhizal grasses or herbs, while associations with ectomycorrhizal fungi would be advantageous in the situation where a progressively greater proportion of the soil N is present in organic form. It has been suggested (Read, 1993) that the change in N status of an ecosystem from one in which inorganic N predominates, to the later condition in which accumulating plant residues sequester N largely in organic form, may be the key factor selecting in favour of ectomycorrhizal trees in late stages of succession, be

450

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they members of the Fagaceae, as in many temperate forests, or of the Pinaceae in boreal forests in the northern hemisphere. By the same logic, where for reasons of climatic stress, for example at high elevation or latitude, growth of ectomycorrhizal trees is restricted, shrubs with ericoid mycorrhizas that also have the ability to mobilize nutrients from organic sources are favoured (see Chapter 12). As succession towards ectomycorrhizal forest or heathland proceeds there are inevitably stages during which cohorts of species with different types of mycorrhiza coexist. Indeed, even in stable forest communities, conditions of soil and irradiance may permit the persistence of a herbaceous understory of plants with VA mycorrhizas beneath a canopy of predominantly ectomycorrhizal trees. However, here, as in the heathland situation described earlier, different patterns of root distribution can provide niche separation. Merryweather and Fitter (1995b) show that seedlings of the herb Hyacinthoides non-scripta germinating in the organic matter in ectomycorrhizal Quercus woodland are largely non-mycorrhizal. With time, the developing bulb, and the roots produced from it, descend into mineral soil where they develop VA mycorrhizas in isolation from the largely surface-rooting trees. In effect there are two separate commimities, the constituent species of each of which, through their mycorrhizas, are exploiting different resources. Such differentiation can even be seen at the intraspecific level. Reddell and Malajczuk (1984) observed that Eucalyptus marginata plants formed VA mycorrhizal associations when rooted in mineral soil, but ectomycorrhizas if grown in litter. Plasticity of this kind may be of particular value in fire-susceptible ecosystems of the kind in which Eucalyptus spp. occur, these being characterized by a cyclical pattern of accumulation and loss of organic resources due to fire. If, as proposed by some ecologists (e.g. Clements, 1916; Odum, 1971; MacMahon, 1981) succession is a series of predictable processes, the trajectories of which are primarily influenced by nutritional constraints, a potential clearly exists for mycorrhizal colonization to play a significant role in determining both the rate and the direction of the processes. The need, therefore, is for more field-based experiments which investigate the effects of manipulation of mycorrhizal status on the outcome of interaction between species at different stages of the succession. Only by these approaches can the real impact of the symbiosis upon the commimity dynamics be evaluated. From what has been written earlier in this chapter it is evident that some pattern can be recognized in the relationship, eventually established in stable, climax communities, between biome and predominant mycorrhizal type. On this basis. Read proposed (1984, 1991a,b) that the combination of climatic and soil factors found at any position along a gradient of latitude or altitude selects in favour of that mycorrhizal type that has the functional attributes necessary to enable success of both partners in that environment (Fig. 15.13). There is no doubt that on a global scale, in the absence of disturbance, biome-related segregation of predominant mycorrhizal types can be seen even though a given type rarely, if ever, occurs to the exclusion of all others. The extent and nature, if any, of the involvement of the mycorrhizal symbiosis in determining these observed patterns remains to be investigated by experiment.

451

The roles of mycorrhizas in ecosystems ALTITUDINAL RANGE

NIVAL

ALPINE HEATH

LATITUDINAL RANGE

POLAR

ARCTIC TUNDRA

CONIFEROUS FOREST DECIDUOUS FOREST GRASSLAND BOREAL FOREST DECIDUOUS FOREST GRASSLAND HERBACEOUS UNDERSTOREY

SOIL TYPE

LITHOSOL

HEATH UNDERSTOREY PEAT PODSOL BROWN FOREST SOIL GRASSLAND SOIL

HUMUS TYPE

\ ^

PEAT

PREDOMINANT FORM OF NITROGEN

MOR

MOOER

MULL

ORGANIC N COMPOUNDS

CHARACTERISTIC MYCORRIHIZAL TYPE NATURE AND QUANTITY OF EXTERNAL VEGETATIVE MYCELIUM MOST IMPORTANT GROWTH LIMITING NUTRIENT

^ ^ 1 ^ ^ " '

NOj *• VA

FINE INDIVIDUAL HYPHAE NEAR ROOT SURFACE SMALL BIOMASS

EXTENSIVE MYCELIAL SYSTEMS ORGANISED INTO STRANDS HIGH BIOMASS

VA

INDIVIDUAL HYPHAE OF LARGE DIAMETER HIGH BIOMASS CLOSE TO ROOTS ^ . ^ \

INCREASE OF ALTITUDE OR LATITUDE -

KEY M Depth of ^m organic • I matter

Figure 15.13 Diagrammatic representation of the postulated relationship between latitude or altitude, climate, soil and mycorrhizal type, together with the development of vegetative mycelium associated with mycorrhizas. From Read (1984), with permission.

Conclusions A pattern has been recognized in the distribution of the major mycorrhizal types according to biome, which has, in turn, highlighted the distinctive nature, geographical, physicochemical and biotic environment in which each of the major kinds predominate. This pattern provides a conceptual framework within which to investigate the function of mycorrhiza in plant community units that are also recognized by ecologists. Amongst these units, those in which mycorrhizas of a distinctive type can be readily recognized are heathlands, boreal-temperate forests, grasslands and tropical forests, these being characterized by the prevalence of plants with respectively, cricoid, ecto-, VA mycorrhizal, or a mix of VA and ectomycorrhizal, colonization. What is emerging from the increasing number of studies of the function of mycorrhizas in these biomes is that in each the distinctive symbiosis fulfills multi-functional roles some of which appear to have been selected in response to the particular requirements of that system. Increasingly, attention is being turned from studies of individual plants in pots to analyses of communities in microcosms or in the field. Several studies examined in this chapter provide ground-breaking information concerning the roles of mycorrhizas in determining the outcome of interactions between individual plants growing in communities. It is demonstrated that the impacts of colonization differ according to species; there is a range of responses from strongly positive, through neutral to negative, suggesting that mycorrhizas will have a powerful selective influence upon the extent and nature of biodiversity in plant communities. Studies of mycorrhizal function are being extended to natural communities in the field the responses of which are, for the first time, being examined throughout the life cycle. These analyses are revealing that the symbiosis is, indeed, essential for survival of some important species in nature. Further, it is becoming clear that the impacts of

452

General themes

the symbiosis upon those plants that respond positively to colonization may be expressed over short but critical periods of their life cycles. Amongst these, both the regeneration and reproductive phases have been identified, by studies of survivorship and fecundity, respectively, as being of probable importance. As a result, attention is being turned from the question of vegetative growth response over a limited period of the life cycle to analysis of those parameters that have a more direct effect upon fitness of the plant. Importantly, some studies show that improved survivorship and greater fecundity can, in some cases, be a result of non-nutritional effects of the symbiosis. Prominent amongst these effects are enhancements of defence against antagonistic biotrophs, herbivore and soil-borne toxins. One of the main challenges over the next few decades will be to determine the relative importance of nutritional and non-nutritional effects of the symbiosis in plant communities. Much of the progress summarized above has been achieved through experiments with herbaceous plant communities - relatively long-lived assemblages of woody perennials having proved more difficult subjects for analysis. Nonetheless, a combination of microcosm and field-based experiments has enabled some progress towards understanding of the distinctive functions of ectomycorrhizas in boreal and temperate forest biomes dominated by woody plants. Major advances relate inevitably to juvenile stages of development. Field observations have enabled an awareness both of the low host specificity and great diversity of fungal species and genotypes involved in the largely ectomycorrhizal communities that make up these biomes. While recognition of genetic and physiological diversity is a significant advance in itself, evaluation of their importance for ecosystem function has yet to be achieved. Much theorizing about the provision of 'resilience' in ecosystems needs to be converted into critical experimentation. One functional aspect of the specificity-diversity issue which has proved amenable to experimental manipulation is that concerning the provision, by mycelia of both VA and ectomycorrhizal fungi, of interspecific and intraspecific links between plants in natural ecosystems. While it can readily be shown that integration of a seedling root system into the mycelial network facilitates soil exploration, the question of resource transfer between interconnected ectomycorrhizal plants has proved to be more intractable. Recent experiments indicating that net transfer of C can occur between interconnected ectomycorrhizal plants of different species highlight a feature which may be of profound ecological significance. Investigations of the role of mycorrhizas in determining the outcome of interaction between species in natural communities should figure prominently in future research programmes. There is a particular need for more experimentation in tropical ecosystems where the mycorrhizal symbiosis is likely playing a significant role, through its influence upon regeneration in the maintenance of biodiversity.

16 Vesiculat^arbuscular mycorrhizas in agriculture and horticulture

Introduction The broad host range of mycorrhizal fungi, together with their effects on the mineral nutrition and growth of many plant species, particularly in pot experiments (see Chapters 4 and 5), has led to attempts to introduce or manage them for increased crop or pasture production. Most plants used in agriculture and horticulture form vesicular-arbuscular (VA) mycorrhizas and this chapter is devoted to this mycorrhizal type. However, other types are important in particular situations: ectomycorrhizas in temperate forest production and in reafforestation programmes (see Chapter 17); ericoid mycorrhizas in production of fruit crops such as blueberries; and orchid mycorrhizas in plant propagation, particularly for conservation (see Chapter 13). There are many instances where crop productivity is influenced by the VA mycorrhizal symbiosis, but as yet there are few examples where inoculation or management are carried out in normal commercial production. In the main, these attempts have received most publicity in developed and highly mechanized agricultural, horticultural or forestry systems. In these systems, the application of large amounts of fertilizers and pesticides is normal and accepted practice (Menge, 1982, 1983; Abbott and Robson, 1982, 1991; Miller et al, 1984; Hall, 1988) and the potential of making money from sale of inoculum has attracted interest from biotechnology companies (Menge, 1984; Wood and Cummings, 1992). The emphasis has generally been on yields, with relatively less attention being paid to establishment or maintenance of production systems that are 'sustainable' by preserving (or improving) soil resources, as well as economic productivity (Bethlenfalvay and Linderman, 1992; Gianinazzi and Schiiepp, 1994). In these environments and in less developed systems (Sieverding, 1987,1991), naturally occurring mycorrhizal populations may play an important role in crop productivity, a role that is not always appreciated. Wherever costs of production and application of fertilizers are important, or minimum input or organic agriculture is practised, the contribution of

454

General themes

biological processes and organisms (including mycorrhizal fungi) in nutrient dynamics needs to receive more attention (Oberson et ah, 1993). The 'nonnutritional' effects of mycorrhizas in reducing the severity of some plant diseases, and in modifying water relations (see Chapters 5 and 15) or soil structure are also potentially important. These have received less emphasis than increases in production, probably because the economic benefits are less easily quantified or appreciated. The functions and activities of mycorrhizal associations have rarely been included in integrated systems for pest management but there is an argument that this should be considered. There is an even stronger argument for their inclusion in integrated progranmies for the management of nutrient or water use, particularly where soils are P-fixing, fragile or subject to erosion or leaching of nutrients. The possible economic benefits of managing mycorrhizal populations in agriculture and horticulture need to be critically assessed in the context of the ecology of the systems, not simply the growth of the crops (Miller et ah, 1994). In most cases experiments have been carried out with annual crops grown in monoculture. However, tree crops are also important and, particularly in developing countries, are sometimes cultivated in plantations or gardens with considerable species diversity. Examples include coffee and cacao, which are grown with mycorrhizally responsive shade trees (Wibawa et aL, 1995) and many tropical fruits, grown in mixed agroforestry systems (Janos, 1980). It is known that both coffee and cacao, as well as citrus, cashew and many other tree crops of tropical origin, are mycorrhizal and frequently respond to colonization (Janos, 1987; Alexander, 1988; Sieverding, 1991; Snuts, 1992; Smith et aL, 1995). Both cultivation and monoculture appear to change the species composition of the fungal populations and reduce their diversity, but the impact of these changes on crop production has not been adequately evaluated (Black and Tinker, 1979; Allen et al, 1995; Hendrix et al, 1995). Mycorrhizal Involvement in Crop Growth in the Field Unequivocal proof that mycorrhizal colonization contributes to plant growth or yield in the field is difficult to obtain, because roots are normally colonized and appropriate non-mycorrhizal controls are hard to produce (Abbott and Robson, 1982; Fitter, 1985a, 1989; Hall, 1988). Chemical or heat treatments of soil that eliminate the mycorrhizal population may alter the levels of available nutrients or release toxic compounds. They also eliminate other members of the soil biota, which may themselves have direct effects on the plants or interact with mycorrhizal fungi (see Chapter 15). Despite these problems there are many situations where the effects on plant growth are best explained by a mycorrhizal effect, because either fumigation decreased growth or inoculation increased it. McGonigle (1988) evaluated 78 field trials with VA mycorrhizal fungi and found that inoculation (either in sterilized or untreated field soil) resulted in an average yield increase of 37%. He was, however, doubtful that nutrient uptake via mycorrhizas was involved, because the magnitude of the yield increase was not correlated with percentage colonization, a point that will be discussed later. Soil fumigation or steam treatments are often used for high value-crops, to reduce losses caused by plant pathogens. However, 'stunting', poor yields and

Vesicular-arbuscular mycorrhizas in agriculture and horticulture

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variable growth have been recorded for many plant species including Citrus, Persea (avocado). Capsicum, Vitis, Allium, Malus (apple), Prunus (peach), Tamarillo, Liquidambar, Liriodendron, Elaeis (oil palm), Manihot (cassava). Cacao and many woody ornamentals. Sometimes stunting can be reversed or reduced by applications of fertilizer, but many species are unable to make effective use of P and other immobile nutrients unless their roots are colonized by mycorrhizal fungi. This effect is most noticeable in P-fixing soils and varies with plant species (Yost and Fox, 1979; Haas et ah, 1987). Plenchette et al. (1983a,b) investigated the effects of fumigation on the growth of 22 species grown under temperate conditions, in a soil of relatively high P availability (100 |Lig g"^ Bray II). The crops fell into three major groups: those which were mycorrhizal and grew better in non-fumigated soil comprised 16 species, including com {Zea mays), carrot (Daucus carota), tomato {Lycopersicon esculentum), potato (Solatium tuberosum) and a number of legumes; those which were mycorrhizal but whose growth was unaffected by fumigation included oats (Avena sativa) and wheat {Triticum aestivum); and those non-host species, such as cabbage (Brassica olearacea) and garden beet (Beta vulgaris), which actually grew better in fumigated soil. These groups would reasonably have been predicted from the discussion on variations in mycorrhizal responsiveness (see Chapters 4 and 5) and confirm earlier findings (Yost and Fox, 1979) which investigated the mycorrhizal response and P uptake of a number of tropical crops grown in an acid, P-fixing soil. Ten levels of P were applied to establish solution P concentrations in the range 0.0121.0 jiig P ml"^. Fumigation had little effect on bicarbonate-extractable P and caused a small but insignificant increase in inorganic N (NOJ and NH4). Brassica chinensis grew better on fumigated than unfumigated plots and was non-mycorrhizal in both situations. The other crops all formed mycorrhizas in non-fumigated soil, and grew much better in this treatment, although the levels of soil P at which fumigation ceased to exert an effect on P concentration in the tissues differed. The order of responsiveness (together with the critical solution P concentration) was Manihot esculenta and Stylosanthes hamata (>1.6 |ig ml~^), Leucaena leucocephala (1.6 |Lig ml"^). Allium cepa (0.8 jig ml"^), Vigna unguiculata (0.2 |xg ml"^) and Glycine max (0.1 jig ml~^). The influence of soil type, particularly with respect to P supply and P-fixation, is also important (Menge et al, 1982). Fumigation as an experimental technique to eliminate mycorrhizal fungi has been criticized because it also eliminates other soil organisms (including pathogens) and may also release nutrients, with consequent difficulties in interpretation of data simply in terms of mycorrhizal involvement. Jakobsen (1983, 1987) discussed these difficulties in the context of the growth of several temperate field crops, including Pisum and Hordeum, and came to the conclusion that in the cropping system he was investigating, the mycorrhizal contribution to P uptake and growth was important and was underestimated by the fumigation technique. It is worth noting that Hordeum does respond to mycorrhizal colonization and that cultivars vary, with those that are responsive to fertilizer application also being responsive to colonization (Baon et al., 1993). Thus not all cereals fall into the nonresponder group of Plenchette et al. (1983a). Application of more selective biocidal treatments can also reduce mycorrhizal populations, root colonization and cause stunting of plants (Menge, 1982). Although there are few examples of this causing major problems in production.

456

General themes

usually because high levels of fertilizer are used simultaneously, the risk is significant in situations where there is an important mycorrhizal contribution to nutrient uptake. The systemic fungicide benomyl certainly reduces mycorrhizal colonization in both pots and in the field (Fitter, 1986; Fitter and Nichols, 1988; Koide et al, 1988a; Sukarno et al, 1993; West et al, 1993a,b; and see Chapter 16), and has been used experimentally to eliminate or reduce colonization by the fungal symbiont. In controlled conditions and low P soil, the result is much reduced plant growth that can be directly attributed to lack of mycorrhizal P uptake (Sukarno et aL, 1993). However, in field situations the occurrence of fungal pathogens, which would also be eliminated, confuses the picture but has certainly highlighted the possible importance of mycorrhizal fungi in reducing the effects of pathogens and influencing plant growth via this mechanism; (West et aL, 1993b; Fitter and Garbaye, 1994; Hooker et aL, 1994). Severe soil disturbance by tillage, mining or natural causes can also reduce plant nutrient uptake and yield. A major effect in this case is the disruption of the network of mycorrhizal hyphae in soil, with consequent reductions in colonization, nutrient acquisition and growth. In the field this has been shown to be important for growth and nutrition of Zea mays (Evans and Miller, 1988, 1990; McGonigle and Miller, 1993) and a number of species from native vegetation in soil disturbed by mining (Powell, 1980; Jasper et aL, 1989, 1992). Crop rotations involving long periods of bare fallow (1 year or more) have sometimes been adopted, with the aim of accumulating moisture and mineral N in the soil profile. In Queensland, Australia, this practice led to severe stunting and P and Zn deficiency in a wide range of taxonomically unrelated crops (Thompson, 1987). 'Long-fallow disorder' has now been tracked down to a deficiency in mycorrhizal propagules in the soil, with consequent decreases in the rate and extent of mycorrhizal colonization and uptake of nutrients (Thompson, 1990, 1994). The problem can be overcome by eliminating fallow from the rotations and adopting other management practices that maintain mycorrhizal populations. In this case, as in many other examples, the data from soil tests used to determine appropriate rates of P and Zn application, were obtained in situations where the fimgal symbiont made a substantial contribution to nutrient uptake by the plants. Reduction in mycorrhizal colonization reduced or eliminated the fungal contribution to uptake, with the consequence that the response to fertilizer was also reduced and the data from the soil tests were no longer useful (Haas et aL, 1987; Thompson, 1987). Biological contributions to soil fertility, including the mycorrhizal populations, are significant, potentially highly variable and need to be understood when making fertilizer recommendations. These examples show that crop production in the field is frequently dependent on the naturally occurring populations of mycorrhizal fungi. The composition of these populations in terms of species and propagule density is not usually well known and the way in which differences in populations may affect plant productivity is not at all clear. Indeed, the fungal attributes that result in a large contribution to plant nutrient uptake and growth are not fully elucidated, so that selection of efficient fungi still remains largely empirical. It is known that an extensive external mycelium is necessary and that different species of fungi vary in the way that this mycelium develops (see Chapter 2). Rapid and early colonization of the roots and the production of numerous arbuscules are also important and may

Vesicular-arbuscular mycorrhizas in agriculture and horticulture

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be functions both of the innate characteristics of the fungal species, the propagule density and other conditions in the soil. The picture is further complicated by differences in the way in which different plant-fungus combinations function in terms of nutrient transfer and by difficulties in relating physiological effects (e.g. nutrient uptake, growth response) to percentage colonization of the root system, which is frequently measured rather late in the growth period. The density and activity of arbuscules change as a mycorrhizal root system develops (see Chapter 2) and there are reports of VA mycorrhizal roots with few or no arbuscules, particularly in woody species (Smith and Smith, 1996a). In annual plants at least a short lag phase and rapid achievement of maximum colonization are crucial in maximizing a mycorrhizal contribution to P uptake and growth. However, the same plateau value of percentage colonization can finally be achieved in plants where the rate of colonization has been very different. These developmental changes may help to explain why McGonigle (1988) observed a poor relationship between percentage colonization late in the growth phase and growth response to inoculation. Comparisons of different species or isolates of the fungi are difficult. Most have been done without regard to the problems of standardizing inoculum and are therefore difficult to interpret. However, where comparisons have been based on equivalent amounts of inoculum or similar propagule densities, major variations between species and even isolates within species have been found (Daniels et al., 1981; Wilson, 1984; Haas and Krikun, 1985; Wilson and Tommerup, 1992). We have little idea of how the efficiency of particular fungi varies when they are associated with different host species, or whether those fungi which are most efficient with respect to nutrient uptake are the same as those which play a major role in reducing the effects of pathogenic organisms or stabilizing soil. Research into the basis for fungal efficiency is important and may lead to methods of selecting appropriate species and strains for inoculation when this becomes feasible on a broad scale (see below). For the present, management of existing populations is the most attractive option, except where particularly high-value crops are produced under conditions where a mycorrhizal contribution is so important that the high cost of inoculation is small in proportion to the potential gains. Less attention has been paid to the possible advantages of maintaining mycorrhizal populations for disease control or soil structural stability. These aspects will be discussed in separate sections, but the principles of fungal management are likely to be similar for these applications.

Management of VA Mycorrhizal Populations There are essentially two approaches to establishing and maintaining high populations of VA mycorrhizal fungi in soils used for agriculture or horticulture. These are: inoculation (and subsequent management) of selected fungi, and adoption of field practices which increase the inoculum potential of indigenous mycorrhizal fungi. The relative merits of these approaches in different situations have been reviewed many times recently (e.g. Abbott and Robson, 1982, 1991; Menge, 1983,1984; Hall, 1988; Gianinazzi et al, 1989a, 1990; Millner, 1991; Sieverding, 1991; Bethlenfalvay and Linderman, 1992; Wood and Cummings, 1992; Dodd and Thomson, 1994).

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General themes

There is general consensus that before any approach is adopted a number of key factors need to be evaluated. These include: the responsiveness of the crops to be grown (see above and Chapters 4 and 5); the populations of indigenous mycorrhizal fungi present, particularly with respect to their infectivity and effectiveness; the possible effects of soil management (e.g. tillage, P and N application) on these populations; and the characteristics of the soil, as they affect both nutrient availability and fungal survival. To these need to be added information on the incidence of pathogens and methods that are used to reduce their effects (e.g. fumigation, application of fungicides) and an evaluation of whether mycorrhizal mycelium in soil may have a sigruficant effect on the establishment and stabilization of soil structure (Tisdall, 1994). Finally, the economic costs and benefits of any management practices are vital considerations and need to be incorporated into long-term plans for sustainable use of soil resources. Evaluation of the Fungal Populations The most extreme examples of low populations result from soil sterilization or fumigation applied to high-value crops to eliminate pathogens. Here, the greatest potential for successful inoculation with mycorrhizal fimgi exists (Vestberg and Estaun, 1994; Lovato et al., 1995). However, few if any commercial production systems use inoculation because of the difficulties of producing and applying inoculum and of introducing modifications in cultural practices (Menge, 1984; Wood and Cummings, 1992; Lovato et ah, 1995; and see below). In field situations, evaluation of populations requires both accurate identification of the species present and quantification of propagule densities and infectivity (see Chapter 2). At present, bioassays of various types are used to evaluate the infectivity of the soil. These have the advantage that they include all iiifective mycorrhizal propagules but give only linuted information on species composition of the populations. Spore isolation is currently the main method of determining the species present, but it cannot show which fungi are vegetatively active. The limitations are clearly appreciated and a number of different methods are being developed to overcome the problems. These include specific antibodies, DNA probes and specific polymerase chain reaction (PCR) primers for different fungal species, as well as fatty acid methyl ester profiles and isozyme banding patterns (e.g. Rosendahl et al, 1989; Simon et al, 1992, 1993; Bentivenga and Morton, 1994; Clapp et al, 1995; Graham et al, 1995; Hahn et al, 1995). DNA-based methods, in particular, have the potential to be sufficiently precise to distinguish different strains of the same species but will need considerable development to make them satisfactorily quantitative. Until we have such precise and rapid methods of identification and evaluation of populations which are present as vegetative stages and contribute to soil-plant processes, we are unlikely to make much progress in selecting and using the fungi in agroecosystems. If populations are low or ineffective, then inoculation or management to increase propagule densities can be considered. Any fungi introduced need to have a great ability to compete with indigenous populations in colonizing the crops and have reliable and positive effects on yields. This means that they must fit both the soil and the plant, and be economically effective in soil processes (nutrient absorption, growth or soil stabilization).

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Inoculation As Wood and Cummings (1992) point out, the unculturability of the fungi is proving a major barrier to the development of inoculation techniques. Currently, VA mycorrhizal inoculum must be grown in symbiosis with plants. Production costs are high and quality control for exclusion of pathogens is a major concern (Menge, 1982, 1984). However, symbiotic production does have the important advantage of automatically monitoring the ability of the fungi to colonize roots. Assuming that VA mycorrhizal fungi are successfully cultured in the future, it remains to be seen whether the mycelium will be infective and suitable as inoculum. If it is, an infectivity trial will need to be adopted in quality control to guard against changes that may occur during large-scale production and subculturing. The plant-based, trial inoculum formulations that have been tested are diverse and require different methods of application. They include spores and hyphae mixed with a carrier such as expanded clay or pumice, soil pellets and relatively crude and bulky soil-root-spore mixtures. Soil-free inoculum has been produced in aeroponic (Jarstfer and Sylvia, 1992, 1995; Sylvia and Jarstfer, 1992) and nutrientfilm culture (Elmes et ah, 1983; Elmes and Mosse, 1984), as well as in axenic mycorrhizal root organ cultures (Mugnier et ah, 1984; Mugnier and Mosse, 1987). The suitability of these inocula for different applications depends on the identity of the main mycorrhizal propagules, and on their ability to retain infectivity during storage and to persist in soil from year to year, as well as on the methods available for application. At present, routine inoculation in broad-scale, highly developed farming systems is generally not achievable because of the expense of production and bulk of the inoculum itself. Management of indigenous populations is the only currently viable option. However, in relatively small-scale operations, such as nursery production, routine inoculation is certainly feasible and likely to be highly advantageous in increasing growth rates and uniformity of the product. More work in this area is likely to be rewarding (Gianinazzi et ah, 1990; Lovato et al, 1995). The option of making 'home-grown' inoculum of highly colonized roots and soil, that is applied to plots immediately before planting a crop, could make a valuable contribution to food production in many relatively small, low-technology systems. Its potential should not be under-rated because it does not make large profits in monetary terms. It is these situations where the cost and availability of phosphate fertilizers may be very significant. Mycorrhizal fungi may increase the accessibility of relatively cheap fertilizers, such as rock phosphate (RP), when used on acid soils. Again, the response may vary with the plant under consideration. In the work of Wibawa et al, (1995) on shade trees used in coffee and cacao plantations, Sesbania grandiflora appeared to be relatively efficient at acquiring P from deficient soil and did not respond significantly to triple superphosphate (TSP) or RP, regardless of mycorrhizal inoculation. Two other species that grew poorly when unfertilized, responded to TSP but not RP in the absence of inoculation, and responded to RP when inoculated with Gigaspora margarita. Colonization of the roots was not determined, but spore production was significantly influenced by treatment for all species. In general, inoculation increased spore production to a much greater

460

General themes

extent with RP than with TSP, an important consideration in the context of management of populations. Pre-inoculation of seedlings is also a feasible method of introducing inoculant fungi into the field and is appropriate where transplanting is part of the normal production system. A potential advantage is that the inoculant fungus is already established in the root systems and, consequently, will have a competitive advantage over soil-borne species. The method has been tested with a number of different species in the field. Sasa et al. (1987) inoculated Allium porrum in pots, so that at transplanting the roots were about 80% colonized by a mixture of mycorrhizal fimgi. These plants grew better than uninoculated controls after transplanting, with 5.7-fold and 1.5-fold increases in fumigated and unfumigated soil, respectively. Similar increases in growth have been observed for such diverse species as chilli (Bagyaraj and Sreeramulu, 1982), apple (Plenchette et al, 1981) and guayule {Parthenium argentatum; Bloss and Pfeiffer, 1984). Snellgrove and Stribley (1986) adapted normal commercial methods in their work with A. cq^a, but had difficulties establishing colonization in the peat modules used for transplanting. The approach deserves more extensive evaluation, and the results highlight the need to fit inoculation procedures to acceptable production methods, as well as to soil type and plant species. Inoculation with two or more fimgi needs to be considered as it could reduce the variation in response that might be expected with different soils, plant species and growing conditions, following inoculation with single species (Sieverding, 1989; Bethlenfalvay, 1992a). Management of Indigenous Fungal Populations The management of soil to maintain mycorrhizal populations at levels that contribute to plant nutrition is important under many situations, regardless of whether it has been preceded by inoculation. A number of factors influence these populations and the extent to which the fungi colonize plant roots (Table 16.1). Management with the chief aim of enhancing mycorrhizal effects on soil structure and structural stability has not been seriously addressed as yet, but has the potential to make an important contribution to sustainable management of agricultural and horticultural ecosystems. The continuing (although not continuous) presence of host plants is essential for inoculum build-up. Both bare fallow and non-hosts may, sooner or later, reduce mycorrhizal populations, or may delay re-establishment of a pool of infective propagules (Ocampo and Hayman, 1980; Thompson, 1987). In these cases growth responses of plants to colonization are not important; rather, the mycorrhizal root length density in soil and the production of extraradical hyphae and spores will contribute most to the population of propagules. Pasture, which combines production of colonized root and low disturbance, has a high potential for inoculum buildup, as well as production of water-stable soil aggregates, stabilized by hyphae of mycorrhizal fungi (Tisdall, 1980; Tisdall and Oades, 1982). Other management strategies that might maintain fungal populations include sequential cropping, where two or more crops are grown each year, or intercropping, where two crops are grown simultaneously. In either case, if at least one of the crops is potentially mycorrhizal an adequate inoculum level is likely to be maintained (Andrews and Kassam, 1976; Tisdall and Adem, 1990).

Vesicular-arbuscular mycorrhizas in agriculture and horticulture

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Table 16.1 Positive and negative influences on m/corrhizal populations and colonization of subsequent crops by different agricultural management practices Management factor

Positive influence

Negative influence

Plant species

Host species High colonization High spore production High mycorrhizal root length density None Increased propagule densities Minimum tillage Pasture phase Organic-biodynamic Drip-feeding Slow release Rock phosphate None Variable effects None

Non-host species

Bare fallow Pasture Disturbance-tillagerotation Management system Fertilizer application

Fumigation Fungicides Low light (glasshouse)

Reduces populations

Conventional tillage Compaction Conventional High applications of soluble PO^ and N Reduces propagules Variable effects Colonization and growth decreased

Conversely, tillage and other types of disturbance, as w^ell as bare fallow and stockpiling of soil, reduce the populations of viable propagules (Jasper et al., 1987, 1989, 1992; Miller and Jastrow, 1992a,b). The early establishment of mycorrhizal associations plays an important role in regrovvrth of the plant communities in revegetation programmes and a parallel exists with agriculture, where a new community is established each time a crop is sown. The effects of soil compaction on root colonization have not been seriously investigated, although Mulligan et ah (1985) reported reduced percentage colonization as bulk density increased due to traffic of agricultural vehicles and root growth decreased. It might be expected that restricted root growth would result in lower mycorrhizal root length density in soil, even if there were no effects on the ability of the fungi to colonize the roots. Restricted root growth certainly results in lower uptake of nutrients such as P, especially when concentrations in the soil are moderate or high. Although it might be expected that soil compaction and restricted root growth would lead to increased responsiveness to mycorrhizal colonization, in Trifolium suhterraneum there was a decrease in percentage responsiveness from 132 to 105 as soil strength increased from 0.4 to 3.5 MPa. At the same time colonization increased slightly from 38% to 49% (Nadian et al., submitted; and see Fig. 16.1). Biodynamic and organic farm management results in higher percentage colonization of roots of pasture and annual crops than conventional management. Although the concentrations of P in the soil were lower on the biodynamic and organic farms the plants appeared to be using soil P more efficiently (Ryan et ah, 1994). The interactions between application of P and N, growth responses and

General themes

462 1.5

o ex

H

•5 0.5-

0.4

2.3

3.5

Penetiometer resistance (MPa) Figure 16.1 Effect of soil compaction on the shoot dry weight of mycorrhizal {Glomus intraradices; light stipple) and non-mycorrhizal (dense stipple) plants of Thfolium subterraneum. See text for details. From Nadlan et al. (1996), with permission from Kluwer Academic Publishers.

maintenance of soil populations of propagules are of major importance. As far as growth responses are concerned, the form and timing of fertilizer application, as well as the sensitivity of the particular plant-fungus combinations, needs to be taken into account. For most crops P (frequently as superphosphate) is applied once, before sowing, and may have large effects in reducing percentage colonization and growth response. Other forms of P, such as RP, do not have the same effects on colonization and may be much more compatible with maximizing the contribution of mycorrhizas in plant nutrition. Drip-feeding of nutrients in irrigation water (fertigation) is sometimes practised in intensive vegetable production. In one field investigation with Capsicum on a highly P-fixing soil, the practice maintained soil solution P at a relatively low concentration, permitting both extensive mycorrhizal colonization and good growth and yield (Haas et ah, 1987). Application of slow-release fertilizers might be expected to have the same effect. Selection of P-tolerant fungi has also been canvassed, with the possibility of using them in inoculation progranunes where high P application is practised. One aspect of crop management that has not been seriously addressed is the influence of mycorrhizas on plant competition. Harley and Smith (1983) suggested that 'the greatest ecological importance of mycorrhizal colonisation lies especially in the establishment of individuals or species in those habitats where the main conditions to be overcome arise from the environment rather than from competition with other plants'. In other words, low nutrient concentrations in soil are likely to be a more important determinant of responsiveness to colonization than high planting density and hence competition between plants. The large effects of colonization on the growth of plants grown singly in pots shows how mycorrhizas can contribute to the success of plants in the absence of competition. For single species the effects of planting density and pot size have an effect on the

Vesicuiar-arbuscular mycorrhizas in agriculture and horticulture

463

productivity of individuals and the results do bear out the suggestion of Harley and Smith (1983). Many ecological studies indicate that the biomass (or other measure of success) of an individual plant is greatest when it is grown alone, and declines as the planting density of the same species increases. In other words, intraspecific competition is greatest at high densities. Although the mycorrhizal status of the plants was not recorded in many of these investigations, the assumption must be that potential mycorrhizal hosts were colonized in untreated field soil. Recently, a number of experiments investigating the effects of mycorrhizal colonization on these interactions have been reported (e.g. Koide, 1991b; AUsopp and Stock, 1992; Hetrick et ah, 1994; E. Facelli, personal communication; and see Fig 16.2). The results consistently confirm that mycorrhizal plants perform best (as individuals) at the lowest planting densities and apparently compete severely for soil resources at higher densities. In contrast, there was apparently no competition between individuals of the same (potentially mycorrhizal) species when grown without mycorrhizal inoculum. These plants grew relatively poorly (as individuals), even at the lowest planting densities, and their success (again as individuals) was unchanged as density increased (Fig. 16.2). These results carry several implications: 1. The potentially mycorrhizal species are unable to use soil resources effectively in the absence of colonization, so that increased population size increases root length density and the acquisition of nutrients by the population, but not the individuals. 2. Colonized plants have a combined root and hyphal length density which permits effective use of a much greater volume of soil than non-colonized plants, with the consequence that interplant competition is potentially strong. 3. Responses to mycorrhizal colonization will be greatest at the lowest planting densities, both on an individual and a population basis. These findings may help to explain the observations that mycorrhizal growth responses of crops growing in the field are frequently lower than those predicted from pot experiments where plants are grown singly or in small groups for relatively short experimental periods. Such conditions would ensure that the influence of interplant competition in reducing mycorrhizal growth responses was minimized. Data supporting this suggestion are to be found in the work of Bloss and Pfeiffer (1984), who found that the positive response of mycorrhizal guayule {Parthenium argentatum) declined as the plants increased in size in field plots. Crop spacing could very well affect the potential for mycorrhizal colonization to influence growth and yield. Colonization also affects the relative performance of different species in mixtures, altering the species diversity and growth of individuals (see Chapters 4, 5 and 15). For example, the pasture grass Lolium perenne becomes extensively mycorrhizal but, because its roots are relatively efficient at extracting P from soil, it does not respond to colonization. In contrast, Trifolium repens is inefficient in the absence of colonization and in a mixture with L. perenne only performs well when mycorrhizal (Hall, 1978). Similarly, mycorrhizal inoculation changed the relative productivity of a number of grassland species grown in mixtures in low-P soil (Grime et al, 1987; and see Chapter 15). Similar findings are common for groups and pairs of species, indicating how rangeland, grassland and pasture ecosystems can be influenced by their mycorrhizal symbionts (Hetrick et al., 1994). The changes in balance of

General themes

464

mycorrhizal plants

^

de

efigh

non-mycorrhizal plants

ef

«^

14 24 1 6 Density (plants per pot)

e ^

gh

14

24

Figure 16.2 The effects of plant density on biomass of plants of Trifolium subterraneum, inoculated with Gigaspora margarita or not inoculated. Bars with different letters are significantly different Results of E. Facelli (unpublished).

productivity of species of different mycorrhizal responsiveness grown in mixtures will need to be considered in the context of intercropping (see above). There is very little unequivocal evidence that the interactions within and between species are influenced by net transfer of nutrients between the plants. This has been suggested to be important for the movement of organic C from plants growing in full light to shaded neighbours, of N from legume to non-legume species, and of P in both single and multi-species communities. The evidence for and against such movements is discussed in Chapters 4, 5 and 15.

Interactions between VA Mycorrhizas, Pests and Pesticides There are many reports of the interactions between mycorrhizal colonization and the incidence and severity of diseases caused by plant pathogens. The effects are variable and influenced by such things as plant nutrition, relative density of the inoculum of pathogen and mycorrhizal fungus, and whether or not the plants were mycorrhizal before being challenged with propagules of the pathogen (Harley and Smith, 1983; Graham, 1988; Linderman, 1992; Fitter and Garbaye, 1994). The main instances of mycorrhizas reducing disease are for root-infecting fungi and nematodes. Shoot pathogens are usually unaffected or their effects are increased in mycorrhizal plants. A number of mechanisms to explain lower disease losses in mycorrhizal plants have been suggested. These include: competition for colonization sites, so that prior occupancy by a mycorrhizal fungus reduces the opportunities for colonization by pathogens; mobilization of plant defence mechanisms during mycorrhizal colonization, a possibility which now seems relatively unlikely (see Chapter 3); and improved nutrient status, which increases the resis-

Vesicular-arbuscular mycorrhizas in agriculture and horticulture

465

tance of the plants to attack by disease organisms as well as tolerance of disease symptoms, particularly root damage. To these suggestions must now be added the possibility that the mycorrhizal fungi themselves produce antimicrobial compounds, with direct effects on pathogen development. Such compounds have been shown to be produced by some ectomycorrhizal fungi and there is no a priori reason to suppose that other mycorrhizal fungi do not do the same. Indeed, direct negative effects of VA mycorrhizal hyphae on Fusarium, within mycorrhizal transformed carrot roots, have been observed (Benhamou et ah, 1994). Biological control of pathogens is now an accepted component of pest management programmes. Success in this area will be reflected in the reduced use of pesticides, including fungicides. The consequences in terms of mycorrhizas will normally be positive. Damage to non-target mycorrhizal fungi will be minimized and the effects of these on plant nutrition, on soil structure and on root-infecting pathogens themselves will be maximized. The potential for including mycorrhizal fungi in pest control packages as biocontrol agents has not been widely explored, although some potential certainly exists. In the horticultural industry in particular, it is possible to envisage an integrated package in which mycorrhizal fungi (possibly in association with other beneficial microorganisms) are applied with the aim of making most effective use of fertilizer and minimizing losses due to disease. Such packages are likely to become more attractive as the use of chemicals for fumigation and disease control is progressively discouraged and fertilizer becomes a proportionally higher component of the cost of production. The integration of mycorrhizal effects with other soil processes is illustrated by the fact that the effectiveness of the commercial inoculant Provide®, a P-solubilizing fungus Penicillium halajii, is often dependent on the presence of mycorrhizal fungi in the roots of the crop. The symbiont is presumed to be essential for effective capture of released P (Kucey, 1987).

VA Mycorrhizal Hyphae and Soil Structure It has long been recognized that hyphae of mycorrhizal fungi are important binding agents in soil. This was implicit in the use of weight of adhering soil to estimate the length of external hyphae (Graham et ah, 1982), and anyone who has washed soil out of roots knows that the mycorrhizal treatments are much harder work than uninoculated ones! Aggregation of sand grains by VA mycorrhizal hyphae in dunes has been repeatedly confirmed (Koske, 1975; Koske et ah, 1975; Clough and Sutton, 1978; Forster, 1979; Forster and Nicolson, 1979). In these ecosystems, mycorrhizal fungi are particularly important in stabilization because they use recent photosynthate and, unlike soil saprophytes, do not depend on readily available organic substrates from soil, which may be in relatively short supply. In many soils it is also evident that roots and hyphae play a major role, together with other organic components in stabilization of aggregates. Oades (1993) reviewed the contributions of biological processes to the development and stabilization of soil structure, and showed that they vary with soil texture. Aggregate formation and stability depend more on organic matter and the activities of organisms in soils with a relatively low clay content. In many soils there is evidence for the existence of aggregates of different sizes, with the smaller

466

General themes

ones progressively packaged together to form the larger ones. This aggregate hierarchy covers a range of sizes over many orders of magiutude (<2 to >2000 |Lim), with the smaller aggregates held together by stronger forces than the larger aggregates (Oades and Waters, 1991). Roots and fungal hyphae certainly stabilize macroaggregates (>250 [im) acting as temporary binding agents which hold together smaller particles (Tisdall and Oades, 1979, 1982; Tisdall, 1994, 1995). Hyphae are also probably important in stabilizing microaggregates (Oades and Waters, 1991). Clay particles (<2 ^m) adhere to mucilage on the surface of hyphae. The importance of mycorrhizal hyphae as part of this complex binding network was demonstrated by Tisdall and Oades (1979). Figure 16.3a shows that the effectiveness of roots of Lolium perenne (ryegrass) in stabilizing aggregates >2000 |im was related to the length of hyphae in those aggregates. Roots of Trifolium repens had considerably less effect than those of L. perenne, despite the fact that mycorrhizal colonization was 50% and 13%, respectively, after 14 weeks' growth. This illustrates that it is the root and hyphal length density in soil and not the percentage colonization of the roots that are important in this context (Fig. 16.3b). The involvement of roots and hyphae in aggregate formation and stabilization has also been followed in a chronosequence of tall grass prairie restoration (Jastrow, 1987; Cook et ah, 1988; Miller and Jastrow, 1990). In this case the development of mycorrhizas was related to different root-size classes. The analysis showed that fine roots and hyphae had significant direct effects on the geometric mean diameter of water-stable aggregates (GMD), while very fine roots had no direct effects. The indirect effects of both types of root were assumed to be related to their colonized lengths and hence to production of mycorrhizal hyphae. Of the plant species in the communities, prairie grasses and members of the Compositae, both of which produced extensive fine roots, were the best predictors of high GMD, whereas other species were apparently less important. Undoubtedly, both roots and hyphae play a part in the stabilization processes. Thomas et al. (1986) showed that the contribution of roots of Allium cepa to the process was stronger than that of the associated mycorrhizal hyphae, but unfortunately their results were confounded by the difference in sizes of mycorrhizal and non-mycorrhizal root systems. In following up this work, they used a single plant of Glycine max, with a split-root system and mesh dividers to produce four treatments in a single pot: no roots plus saprophytic hyphae, hyphae alone, roots plus hyphae (mycorrhizal roots), and roots plus saprophytic fungi (non-mycorrhizal roots). Although experimental procedures resulted in lower water-stable aggregates at the end of the experiment than in the initial soil, the differences between treatments were significant (in descending order of aggregation): mycorrhizal roots plus hyphae > mycorrhizal hyphae = non-mycorrhizal roots > control. The contribution of roots and hyphae to aggregate stability increases as the concentration of organic matter in soils increases under pasture. The organic binding agents are relatively transient compared with the more persistent agents that cement the smaller particles. Consequently, the larger aggregates, which are so important in determining the occurrence of free-draining pores, are not only relatively temporary but also fragile and subject to disruption by tillage. Miller and Jastrow (1990) emphasized that because of the transitory nature of the bonds, stabilization by roots and hyphae depends on their continued production. This

Vesicular-arbuscular mycorrhizas in agriculture and horticulture

467

Size of particle (^g) E

••

(b)

16

o o CM A CO i z •7? 6 .yi 00

iQ. B O CO

•

12

•

•

• 8

•

0)

4

1

01

• •

1

6

1

1

1

'

8 10 12 14 Hyphal length (m g"^)

*

•

16

18

Figure 16.3 (a) Length of hyphae connbined with water-stable particles in soil after 14 weeks* growth with ryegrass (R), white clover (W) or unplanted (U). Vertical bars, 2X standard errors of the mean, (b) Relationship between hyphal length in whole soil and percentage water-stable aggregates >2000 |im diameter. From Tisdall and Oades (1979), with permission of Australian Journal of Soil Research.

468

General themes

point is obviously relevant for the management of plant-mycorrhizal populations to increase and maintain the structural stability of soil and again highlights the potentially negative effects of bare fallow.

Conclusions Biological activities in soil are widely recognized as playing a vital part in nutrient cycling and availability to plants and in developing and maintaining soil structure, and contributing to 'soil health'. Sustainable land-use requires that soil degradation ceases and that soil management practices are adopted to conserve and augment soil resources. Mycorrhizal fungi comprise just one of the functional groups of organisms that are important in the soil ecosystem, but their position in forming direct links between roots of plants and the soil fabric means that they play key roles in soil-plant interactions. The dependence of mycorrhizal fungi on the photosynthesizing plant means that they do not deplete reserves of soil organic matter as saprophytic microorganisms do, but contribute to its accumulation directly as hyphae and spores and indirectly via their effects on plant growth. The effects of mycorrhizal associations on agricultural and horticultural systems are almost all potentially beneficial, with only a very few reports of growth depressions in field situations that remain imperfectly explained (Modjo and Hendrix, 1986; Modjo et aL, 1987). There is increasing evidence that mycorrhizal fungi contribute to crop productivity and are important in the way in which crops respond to fertilizer applications. It is possible to make estimates of the potential savings in fertilizer if a given crop is mycorrhizal, but real monetary gains from managing mycorrhizal symbioses in broad-scale agriculture remain elusive. Direct inoculation of VA mycorrhizal fungi is currently limited to relatively small-scale, high value systems or subsistence farming. This picture may change when inoculum of VA mycorrhizal fungi can be readily produced in a form that is convenient for wider application. At present, it is important to view mycorrhizal associations as integral coniponents of a complex soil ecosystem and to manage that system in order to maximize the contributions that mycorrhizas most certainly make to soil processes and growth of plants. Research directed towards understanding the activities of mycorrhizal fungi in agricultural and horticultural systems is valuable both in determining appropriate management strategies and as a background against which inoculation techniques will be developed. Less easily quantified benefits of mycorrhizal activity include their effects on soil structure and soil structural stability. Good soil structure is certainly important, both for plant productivity and for control of erosion and the losses that stem from it. The benefits here are in fertility, erosion control and water management, and are inextricably linked to the growth of plants and their fungal symbionts. One aspect of environmental degradation which is receiving increasing attention is the transfer of nutrients, frequently applied as fertilizer, to aquatic systems. The consequence is loss of nutrients for crops, and reduced water quality and eutrophication. The roles played by plants in reducing these nutrient transfers are being recognized in land management and the potential of mycorrhizal fungi to contribute to effective scavenging of nutrients from soil deserves attention. Integrated management of the soil-plant system is clearly important and mycor-

Vesicular-arbuscular mycorrhizas in agriculture and horticulture

469

rhizal symbioses should be viewed in this context. There is no real argument for breeding crops which are more dependent on mycorrhizal symbiosis than current varieties in order to cash in on the sale of inoculum when production and application become economically viable. However, there is an argument for ensuring that levels of colonization are maintained or increased, because this will be important in all aspects of management of mycorrhizas, including nutrient absorption, production of propagules and maintenance of soil structural stability. Furthermore, selection of efficient fungi will require both an understanding of their ecological requirements and their physiological integration with the plants.

17 Mycorrhizas in managed environments: forest production, interactions with other microorganisms and pollutants

Introduction With the realization of the extent of occurence of ectomycorrhizal fungi on trees in many natural ecosystems came the recognition that the symbiosis might be manipulated to enhance productivity in afforestation programmes. Since many commercial practices, particularly those employed in tree nurseries are inimical to the growth of all but a few ruderal species of mycorrhizal fimgi, special techniques have been developed which enable selected fungi to colonize plants prior to outplanting. The application of these techniques has facilitated superior performance in tree crops in many parts of the world, particularly those that lack natural sources of mycorrhizal inoculimi (White, 1941; Wilde, 1944; Shemakhanova, 1962; Stoeckeler and Slabauch, 1965; Mikola, 1969, 1973; Hacskaylo and Vosso, 1971). Over the same period interest has grown in the possibility of harvesting edible fruit bodies of ectomycorrhizal fungi which have been used as commercial inoculum both to supplement diet and revenue. Experience of the use of inoculated seedlings has indicated that responses to mycorrhizal colonization are often greatest under the most extreme conditions, particularly those involving exposure to drought, metal contamination or pathogens. Such observations have led to analysis of the functional basis of the ameliorative effects of mycorrhizal fungi. Considerable advances have been made towards an understanding of the role of the symbiosis in providing resistance to these stresses which, although they also occur in natural ecosystems, are often locally increased by previous land-use practices or by the afforestation process itself. In so far as the atmosphere and climate of the earth are being changed globally as well as locally by the activities of man it is necessary also to consider the extent to which, and the mechanisms whereby, mycorrhizal colonization may respond to

Mycorrhizas in managed environments

471

these larger scale events. Increased direct inputs to soils of N, S and H ions, largely as wet deposition, may have adverse effects upon growth and nutrition of both partners in the symbiosis. Such effects are thought to be contributory factors in the forest decline syndrome experienced in Europe and north-eastern USA. On an even wider scale, progressive global enrichment of atmospheric CO2 can be predicted to influence the C balance of plants and hence, indirectly, that of their fungal symbionts. As a result, there is an emerging interest in the extent to which mycorrhizal systems may act both as sinks for any additional CO2 assimilated by plants, and as pathways through which this C may pass into the soil C pool. This chapter presents an overview of the advances made in the technology of large-scale ectomycorrhiza inoculation programmes, an analysis of present knowledge of the role of this symbiosis in amelioration of selected environmental stresses, and describes some early indications of the responses of both ecto- and vesiculararbuscular (VA) mycorrhizas to elevation of atmospheric CO2. Ectomycorrhizal Inoculum Production and Inoculation Practice The use of defined inoculum consisting of fungi that were physiologically and ecologically appropriate for the planting site, with a view to improving performance of the crop, was pioneered by Moser (1958) in Austria, Takacs (1967) in Argentina, and ITieodorou and Bowen (1973) in Australia. Prerequisites for the widespread use of ectomycorrhizal inoculation programmes are the selection of appropriate fungal symbionts and the development of methods for the large-scale production of inoculum. The two requirements are interrelated because, in addition to providing enhancement of performance of the inoculated crop, the selected fimgus must be able to withstand the physical, chemical and biological stresses involved in the production of the inoculum, as well as those imposed by the soil, usually of a forest nursery, into which it is to be introduced. To date, the most widely used and most successful inoculation programmes have employed Pisolithus tinctorius. Interest in this fungus was prompted by its wide geographic distribution, broad host range (Marx, 1977) and the knowledge that it became prominent on adverse sites, particularly those subject to drought, high temperature or contamination (Schramm, 1966). Various commercial inoculum formulations and inoculation techniques have been developed for use in seedling production systems (Marx and Bryan, 1975; Marx, 1975, 1980; Marx and Kenney, 1982; Marx and Cordell, 1989; Marx et al, 1991). The most successful have involved the growth of vegetative mycelium in vermiculite-peat mixtures moistened with liquid nutrient medium (Marx and Kenney, 1982). Vermiculite provides a well-aerated laminated substratum, within which the mycelium is protected, and addition of peat in different ratios enables adjustment of pH to the required range, usually 4.8-5.5. The recommended nutrient solution has a C: N ratio of between 50 and 60 and is added in volumes sufficient to ensure that all free C is utilized by the fungus in the course of its development in the medium. The presence of available C at the time of inoculation leads to competitive exclusion of the mycorrhizal fungus by saprophytes. The major challenge in the commercial development of inoculation procedures is the scale of the operation required. In the case of pine, for example, 1.5 billion

472

General themes

seedlings are produced per year in nurseries of the southern USA (Marx, 1985). A modification of the practice employed for production of edible mushroom {Agaricus bisporus) spawn has been used to enable scaling-up of inoculum production. The vermiculite, peat-moss and nutrients are mixed and sterilized in a large-volume rotating blender. Starter mycelial inoculum is added, thoroughly mixed, and 10-1 batches of inoculated substrate are aseptically dispensed into sterile plastic bags. The bags are sealed but have 'breather-strips' installed to enable ventilation of the medium during fungal growth. Bags are incubated at room temperature for 5-7 weeks, after which time they can be shipped to nurseries for use. Inoculum of P. tinctorius together with that of two other fungi Hebeloma crustuliniforme and Laccaria laccata, is currently produced and marketed by Mycorr Tech. Inc., University of Pittsburgh Applied Research Center, Pittsburgh, USA. A tractor-drawn nursery seedbed applicator has been developed (Cordell et al, 1981) which places inoculum directly into seed-beds prior to sowing at doses of 0.33 1 m~^ of nursery soils and at a depth of 4-6 cm. At a cost of approximately $7.50 per litre of inoculum, this operation is calculated to represent about 5% of the total cost of establishment of a pine plantation in the southern USA (Marx, 1991). In order to eliminate weeds, pathogens and other symbiotic fungi which are potential competitors, seed-beds are routinely fumigated, most commonly with a methylbromide-chloropicrin mixture, before inoculation. Even so, re-invasion of fumigated soil by spores of naturally occurring mycorrhizal fungi, particularly Thelephora terrestris, normally occurs within days, and it is a requirement of the inoculant fungus that it has the ability to colonize roots quickly. T. terrestris appears to be the dominant mycorrhizal fungus of nursery soils worldwide (Mikola, 1970; Ivory, 1980; Marx et ah, 1984a) and, whether as a result of re-invasion after fumigation, or natural occurrence, its presence as a potential mycorrhizal colonist of roots must be recognized in all nursery studies. Because of the ubiquitous occurrence of T. terrestris, experiments designed to evaluate the influence of an inoculant fungus are complicated by the fact that most of the uninoculated 'control' plants, as well as some of those in the inoculated treatment, are invariably colonized by the local, naturally occurring species. There may also be other 'casual' colonists, amongst which 'E-strain' fungi (see Chapter 10), and Laccaria species are common. Such trials are therefore comparisons of performance between T. terrestris and the inoculated symbiont. Experience with P. tinctorius as the introduced organism strongly suggests that large numbers of mycorrhizas must be produced consistently on the roots of the seedlings if maximum promotion of growth is to be achieved when they are outplanted to reforestation sites. In these situations, Marx et al. (1976,1988) have shown that if less than half of all mycorrhizas are formed by P. tinctorius, no growth promotion relative to that seen in Thelephora-colomzed plants is observed. Outplanting trials in several coimtries (Trotymow and van den Driessche, 1990) have confirmed that the benefits of inoculation increase progressively with extent of colonization by Pisolithus. It is a striking feature of the programme of inoculum production using P. tinctorius that only one vegetative isolate of the fungus has been used throughout. This socalled 'super-strain' was originally obtained from a sporophore found under Pinus taeda in Georgia, USA. Its aggressive traits have apparently been enhanced by annual re-isolation over 30 years from seedlings growing in inoculation trials. Problems with the use of solid substrates for inoculum production include the

Mycorrhizas in managed environments

473

large space required for storage, difficulties in maintaining homogeneity of conditions within and between batches, and the inability to control physicochemical conditions in the medium in the absence of water. Because of these difficulties there have been various attempts to use liquids or gels as culture media. The main advantages of submerged, liquid culture are the homogeneity of the medium and the control which can be obtained over physical and chemical conditions. Vessels suitable for large-scale axenic production of fungal inoculum have been developed for other purposes in the chemical and pharmaceutical industries. They are designed to facilitate careful regulation and optimization of culture conditions for particular organisms, reducing the period of culture compared with solid substrates (Le Tacon et al, 1985; Boyle et ah, 1987). Inoculum produced in this way can be applied directly as a slurry (Boyle et ah, 1987; Gagnon et ah, 1988), requiring some form of fragmentation. Unfortunately, this treatment greatly reduces the vigour of many ectomycorrhizal fungi. Attempts have been made to retain viability of fragmented inoculum by incorporation in a protective carrier medium. Sodium alginate has been successfully used, either applied as a gel directly to the bare roots (Deacon and Fox, 1988), granulated (Kropacek et al., 1989) or as beads (Le Tacon et al, 1985; Mauperin et al, 1987). The susceptibility of many fungi to fragmentation damage, even when protected in this way, has led to a search for alternative culture methods. One approach which has considerable promise involves the production of the inoculum inside hydrogel beads, which can be applied directly, circumventing the fragmentation phase (Jeffries and Dodd, 1991; Kuek et al, 1992). Several ectomycorrhizal fungi, including species of Descocolea, Hebeloma, Laccaria and Pisolithus, have been successfully grown as inoculum in this way, and it has been shown that viability can be retained after storage for up to seven months at low temperature (Kuek et al, 1992). Basidiospore inoculum of P. tinctorius has been used on an experimental basis in the USA and elsewhere. This can yield growth responses, but rarely produces as many mycorrhizas per plant as does the 'super-strain' of vegetative inoculum and so is less effective. Spores can be sprayed onto fumigated plots as a suspension in water, to which a wetting agent such as Tween 20 is added. Marx et al (1991) report that spore doses of 0.5-1.0 g m~^ of soil surface are effective in enhancement of performance of southern pines. Alternatively, spores can be placed in a clay encapsulating mixture (Marx et al, 1984b) to be applied as pellets to the soil or as a coating to the seed. A delay in production of mycorrhizas from spores might be expected because, as described in Chapter 6, colonization would not normally take place from monokaryotic mycelia. Only after hyphal fusion and the formation of dikaryons does mycorrhizal colonization occur. Unfortunately, the extensive work on methods of inoculum production has not been matched by a demonstration of efficacy in forest production. As a result, while there are theoretical studies of the economic advantages to be gained from use of the new inoculation technologies (e.g. Kuek, 1994), prospects for their extensive application are not promising. Furthermore, a consequence of the recognition of the advantages of fungal diversity in ecosystems (Chapters 15 and 16) will be an increasing reluctance to introduce into mixed communities, single, potentially dominant species. A highly competitive fungus, suitable for inoculation programmes, might have considerable influence on the fungal diversity and the gene pool of the resident population.

474

General themes

Although the responses to inoculation with P. tinctorius have been good in warmer and more drought-susceptible parts of the world, this fimgus has proved less successful in cooler climates. In the Pacific north-west of the USA, for example, the 'super-strain' of P. tinctorius performed less well that did local isolates of the fungus (Perry et ah, 1987). In this region, the US Forest Service developed a spore inoculation programme based upon the use of mycorrhizal fungi known to be important in local ecosystems, including species of Laccaria, Heheloma, Rhizopogon and Suillus (Castellano and Molina, 1989). Spores have been applied to seed-beds through the nursery irrigation system, or to container-grown plants using mistpropagation units. Poor colonization was obtained with Rhizopogon and Suillus spp. (Perry et ah, 1987). In contrast, several strains of Laccaria produced abundant mycorrhizas in container-grown plants (Molina, 1982). One strain, subsequently referred to as L. bicolor S238, was found to have particular promise as an inoculant. Some Laccaria and Hebeloma strains have been developed as commercial inoculum, producing high levels of colonization on Pseudotsuga menziesii in containers and imder nursery conditions (Hung and Molina, 1986). It appears, however, that despite success in achieving colonization by vigorous strains of fungi, outplanting performance of the seedlings has improved little (Perry et ah, 1987). This has also been the experience in Europe. Le Tacon et al. (1988,1992) describe a number of experiments in France, Spain and Britain in which the performance of nursery inoculated plants of P. menziesii and Pinus sylvestris has been followed for several years after outplanting. The fungi used were mostly strains of Thelephora, Hebeloma and Laccaria, including the vigorous Oregon strain S238 of L. bicolor, originally isolated by Molina. The extent of success in obtaining colonization by the inoculant fungus varied from nursery to nursery, apparently being determined largely by the rate of re-invasion and vigour of indigenous Thelephora strains. Even where high levels of colonization by inoculant fungi were achieved, improvements in performance of the outplanted trees were rarely observed. Inoculation of P. menziesii with L. bicolor S238 provided significant increases of height growth and a doubling of wood volume at one site in central France 6 years after outplanting, but at the remaining sites differences between control plants colonized by T. terresfris and those that were inoculated were small. Jackson et al (1995) report a similar experience with container-grown P. menziesii and Picea sitchensis which were inoculated with a wider range of fungi and transplanted, after colonization, to six nursery sites across the UK. Few significant effects upon growth were observed and none of the kind that would be useful to a forester in a practical situation. Unfortimately, it appears that many of those fungi selected to achieve optimal colonization in the nursery are poor competitors in the field, especially when outplanting sites contain indigenous populations of mycorrhizal fungi. McAfee and Fortin (1986) preinoculated seedlings of Pinus banksiana with L. bicolor, P. tinctorius and R. rubescens before transplanting them to denuded, burned or natural pine stands. After 2 months in the natural stand, colonization by L. laccata and P. tinctorius had declined significantly whereas that of R. rubescens showed a modest increase. P. tinctorius showed an ability to colonize new roots in a denuded site which lacked competition from an indigenous mycorrhizal population. These are the circumstances in which the greatest successes have been achieved in inoculation programmes involving this fungus. The failure of L.

Mycorrhizas in managed environments

475

bicolor to compete with indigenous fungi is in line with the observation of Bledsoe et al. (1982) that the closely related L. laccata failed to persist on seedlings of P. menziesii when challenged by native fungi on outplanting sites in Washington. There are a number of possible explanations for the failure of inoculation to produce beneficial effects at outplanting sites. Probably amongst the most important of these is the inability of introduced inoculum to persist on the roots of planting stock after transfer from the nursery to the field. In addition to the fact that soil conditions experienced by nursery and container grown plants are very different from those in most outplanting sites, the lifting, storage and transport of seedlings, especially those raised in bare-root nurseries, can be expected to reduce the vigour of fine roots and their fungal associates. These are circumstances under which replacement of introduced fungi by those resident in soil of the replant site are likely to occur most readily. It is noteworthy in this context that the most strongly beneficial effects of inoculation have been observed where plants are transferred to disturbed or treeless sites in which inoculum potential of any indigenous fungi is likely to be low. Here, in contrast to the situation so often reported in soils with a pre-existing vegetation cover, responses to inoculation can be quite dramatic (Table 17.1), involving improvements in survival as well as increases in yield (Marx, 1991), and they appear to be most marked where the soil is contaminated with metal ions (see below). In this context, the ability of ectomycorrhizal Betula spp. to colonize mine spoils spontaneously is widely recognized.

Table 17.1 Percentage increase in survival and volume growth of pine seedlings after 2-4 years with Pisolithus tinctohus ectomycorrhizas over controls with naturally occurring ectomycorrhizas on various adverse sites Pinus species

P. resinosa P. echinata P. virginiana P. taedataeda P. rigida P taeda

P. virginiana P taeda

Site

Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Coal spoil Kaolin spoil Fullers' earth Copper Basin Copper Basin Borrow Pit

Data from Marx (1991) and Marx et al. (1989).

Adversity

pH 3.0 pH4.l pH 3.1 pH 3.8 pH 3.8 pH 3.4 pH4.3 pH3.3 pH 3.4 pH4.l pH3.4 pH4.3 Low fertility Low fertility Eroded Eroded Droughty

% Increase in seedling Survival

Volume

214 5 87 480 0 57 8 20 14 41 96 16 0 0 0 0 17

60 400 444 422 420 215 180 415 750 400 800 380 MOO 47 45 88 412

476

General themes

The Use of Ectomycorrhizal Inoculation Programmes to Produce Edible Fungi While emphasis in applied research on ectomycorrhizas has so far concentrated on improvement of tree production, there is an increasing awareness of the potential to exploit the commercial value of the fruit bodies produced by ectomycorrhizal fimgi. At present, a small number of mycorrhizal species (Table 17.2) are prized for their gastronomic quality and are hence of high value. They are collected mostly from natural stands and constitute only a small fraction of the total global production of edible fungi (Fig. 17.1), most of which are saprophytes grown under controlled conditions. In order to increase supply of mycorrhizal fruit bodies, the current demands for which far outstrip supply, numerous commercial organizations are involved in planting trees which have been pre-colonized by inoculation with appropriate fimgi. Particular emphasis has been placed on truffles {Tuber spp.) because of their extremely high economic value, the most important of these being the black truffle, T. melanosporum. Techniques for the germination of the ascospores of this fungus and for the aseptic production of mycorrhizas by a number of Tuber Table 17.2 Some high-priced mycorrhizal truffle and edible mushrooms Botanical name

Common names

Markets

Tuber melanosporum Perigord black truffle Worldwide Vitt truffe due Perigord (Fr) tartufo nero pregiato (It)1 schwarze Truffel (G)

Tuber magr)atum Pico

Italian white truffle truffe d'Alba tartufo bianco pregiato weisse Truffel Boletus edulis cep, penny bun Bull, ex Fr. cepe de Bordeaux porcino steinpilz Car)tharellus dbarius chanterelle Fr. girolle gallinaccio pfifferlinge Tricholoma matsutake matsutake (S. Ito et Imai) Sing.

Worldwide

Approximate recent prices

Fresh 550 (wholesale, London) Fresh 3250 (retail, London) Fresh IOO--430 (picker, France) Bottled 860-1800 (retail, London) Canned 500 (wholesale, Cahors) Bottled 1000 (retail, London) Fresh 800 (wholesale. Bologna)

Europe North America

Fresh 45 (retail, Hamburg) Fresh 10 (retail, Bologna) Dried 60 (retail, Zurich)

Europe North America

Fresh 10 (retail, Hannover)

Japan

72-720 (wholesale, domestic produce) 75 (wholesale, China) 72 (wholesale. South Korea) 36 (wholesale, from North Korea)

Mycorrhizas in managed environments

477

Wood ear 465 Shiitake 526

.....i^S^^Ws^.^*^^^

^^\, ,

,,,

^..^mmKimmmMm^m,^^j.Amm>^

EnokltaKO

187

Silver ear 140 Nameko 40 Others 155 Oy^t^'' 917 ^ ^ ^ ^ ^ ^ T - ^ ^ ^ - - - . - - s i j g | H ^ Mycorrhizal >200

Button 1590 Figure 17.1 The contribution of mycorrhizal fungi to the approximate world production of edible mushrooms in 1991. Values are tonnes XI000. From Hall et al. (1994), with permission.

spp. were pioneered in France (Grente et ah, 1972; Chevalier and Desmas, 1975; Chevalier and Grente, 1978) and Italy (Palenzona, 1969; Fontana and BonfanteFasolo, 1971). T. melanosporum has a broad host range and can be successfully grown on calcareous soils with the hardwood genera Corylus, Quercus, Carpinus and Castanea, as well as softwoods such as Pinus. Commercial production of colonized seedlings, particularly of Quercus and Corylus, now takes place in a number of centres in both the northern and southern hemispheres. In France alone about 160000 plants colonized by T. melanosporum are produced annually, mostly by Agri-Truffe of St Maixant; some are exported to the USA (Hall et al, 1994). On a smaller scale, colonized plants are being produced in New Zealand, by the New Zealand Institute for Crop and Food Research, in a programme pioneered by Hall, involving the introduction of both fungus and plant as exotic species. Truffieres have been established on both North and South Islands, usually as mixed plantings of Quercus and Corylus on potentially favourable sites, at some distance from any other ectomycorrhizal communities to reduce competition between pre-existing and introduced fungi. Truffles were collected from the introduced, inoculated plants within 5 years of establishment of a truffiere at Gisboume, New Zealand (Hall et ah, 1994). Since in other parts of the world (e.g. Europe and California) the first truffles are usually produced only after 7-10 years, the prospects for production of truffles in New Zealand and development of an export industry appear bright. Despite advances in science and technology which provide the prospect of largescale production of a number of edible mycorrhizal fungi, the commercial success of any venture is not assured. To a large extent the value of the commodities (especially of truffles) is based upon its limited availability, so that prices will certainly drop if large-scale production is achieved. However, especially in crops that can be used for timber, harvesting of edible fruit bodies could be an additional source of revenue or food, especially in developing countries. The large-scale establishment of eucalypt plantations in China, using planting stock pre-inoculated with edible fungi, has considerable potential to provide an important dietary supplement (Dell and Malajczuk, personal communication).

478

General themes

Improvement of Drought Resistance and W a t e r Balance In Ectomycorrhizal Systems There has long been an interest in the possibility that ectomycorrhizal fungi might improve the water relations of trees. Such effects would be of particular significance in an applied context where seedlings were being transplanted from nursery beds, especially if the site being afforested was subject to drought. There are reports that mycorrhizal colonization can be of some benefit to trees planted into such environments. Cromer (1935) believed that colonization increased the drought resistance of Pinus radiata by protecting the roots from shrinkage and providing increased uptake of water from soil at low water potentials (\|/). Some support for this view was supplied by Pigott (1982), who showed that roots of Tilia cordata colonized by Cenococcum geophilum were able to survive soil \|/ as low as - 5 . 5 MPa. While Cenococcum mycorrhizas could not store sufficient water to sustain transpiration for more than a short period when soil was dry, their protective effect enabled the colonized roots to recover their absorptive functions rapidly after soil was rewetted. In studies of P. ponderosa, Goss (1960) showed that mycorrhizal colonization improved the survival of plants after short periods of exposure to drought but had little effect over longer periods. Under conditions of extreme drought in the arid steppes, Zerova (1955) found that mycorrhizal colonization afforded little protection to oak seedlings, although colonization improved their vigour at intermediate levels of soil moisture. It must be emphasized, however, that in the absence of direct measurements of tissue water balance, such benefits could be attributable to nutritional rather than hydrological effects. Dixon et al. (1983) studied the water balance of bare-root and container-grown seedlings of Quercus velutina which had been inoculated with Pisolithus tinctorius or left uninoculated. They found that container-grown plants with extensive mycorrhiza development had a significantly improved water balance following transplantation, pre-dawn shootwater potential (\|/shoot) values being significantly higher in the mycorrhizal plants during mild drought. Similar responses to colonization by this fungus have been observed in Pinus virginiana (Walker et ah, 1982) and P. taeda (Walker et al., 1989). In the latter case, the plants had been grown on a mine spoil for 7 years with or without colonization by P. tinctorius. Colonized plants had a midday ¥xyiem 0.2 MPa higher than that of the uninoculated controls. These results are at variance with those of some other studies (Sands and Theodorou, 1978; Sands et al, 1982; Lehto, 1989; Coleman et al, 1990) which report either no impact of ectomycorrhizal colonization upon \|/piant or a negative effect. Sands and Theodorou (1978) found greater resistance to liquid flow in the soilplant pathway when seedlings of Pinus radiata were colonized by Rhizopogon roseolus than when they were uncolonized. However, these authors made the important observation that the potential benefits of mycorrhizal colonization were likely to have been reduced in their experiment because the extraradical mycelial system, and in particular its rhizmorphs, had failed to develop. The potential of the mycelial system to provide conduits for the transport of water was indicated by Boyd et al (1986) who showed that if rhizomorphs of Suillus bovinus connecting mycorrhizal seedlings of Pinus sylvestris to moist soil were cut, transpiration declined markedly within minutes. The importance of the extraradical

479

Mycorrhizas in managed environments

phase for water absorption was further emphasized by Lamhamedi et al. (1992) who examined the ability of a number of genetically distinct dikaryons of Pisolithus tinctorius to influence V|/xyiem of Pinus pinaster seedlings growing under moderate drought. They found significant correlations between \|/piant/ total root system resistance, and both the extension growth and the rhizomorph diameters of the different strains (Fig. 17.2); those genotypes that produced the most extensive system of thick rhizomorphs enabled plants to sustain the highest \|/xyiem ^t low Vsoil.

It is likely that there are also considerable intra- and inter-specific differences in the ability of ectomycorrhizal mycelial networks to resist exposure to drought. Rapid resumption of absorptive activity following a prolonged dry period would clearly be important for the plant. Experiments which determine the resilience of ectomycorrhizal mycelia, perhaps along the lines of those described by Jasper et al. (1989) examining VA systems, would be valuable. These workers demonstrated that, provided that the mycelial networks were intact, they could retain viability for at least 36 days, even when \|/soii was as low as - 2 1 MPa. A complete evaluation of the role of mycorrhizas in the water relations of plants cannot be achieved simply by measurement of tissue \j/ (see also Chapter 5). Colonized plants are likely to be larger than their non-mycorrhizal counterparts and, as a result, in pot studies at least, they may use available water resources more quickly, possibly even developing lower \|/xyiem- Thus measurements of \|/piant provide an indication of the impact of mycorrhizal colonization on

O

3x28

•

37x34

A

34x25

•

2x36

a

8x28

•

11x15

O

34x20

•

9x22

*

27x34

•

17x20

25

extension oU.m.P^ase^

Figure 17.2 Effects of the dianneter of rhizomorphs (|Lim) and of extension rate of the extraradicle mycelial system (cm^) of a range of dikaryotic isolates (designated by number on left of figure) of Pisolithus tinctorius, upon the xylem water potential (vj/xyiem) of P"^^^ pinaster seedlings. From Lamhamedi et al. (1992), with permission.

480

General themes

water relations only when effects of plant size, transpiration rate and tissue nutrient status are taken into account. Despite these complexities there is sufficient evidence to suggest that the extraradical mycelia of ectomycorrhizal roots, by playing a role in absorption and transport of water as \|/soii declines, may be of direct benefit to plants. There is a need for experiments to determine the extent and nature of the involvement of this mycelium, especially its rhizomorphs, in transfer of water, and to evaluate the importance of mycorrhiza for the water relations of plants grown under controlled nutrient regimens.

Improvement of Metal Resistance by Ectomycorrhizal Fungi A number of groups have investigated, in the laboratory, the basis of the apparent alleviation of metal toxicity arising from mycorrhizal colonization. The acidity typical of most substrates of ectomycorrhizal plants, and in particular of many of the man-made substrates such as mine-spoils which they colonize, is conducive to the solubilization of potentially toxic metal ions. Amongst these, Al and Fe are quantitatively the most significant in natural soil, while mine-spoils may be polluted by Ni, Pb, Zn and Cd, either separately or in combination. Colonization of clonal cuttings of Betula pubescens by Paxillus involutus provides significant increase in resistance to Zn toxicity and the growth enhancement associated with mycorrhiza formation is related to reduced transfer to the leaves (Brown and Wilkins, 1985; Denny and Wilkins, 1987). Jones and Hutchinson (1986), using Ni, obtained a similar result in seedlings of B. papyrifera which were colonized by Scleroderma flavidum, which is widely present on spoil heaps. This associate was far more effective in providing resistance than was Lactarius rufus. Ni was preferentially concentrated in the roots of the plants where it appeared to be sequestered with polyphosphate (Jones and Hutchinson, 1988). In contrast to the pattern of distribution of Zn, there was some evidence that Ni was accumulated in senescent leaves so that avoidance of exposure to the metal was largely a feature restricted to stem tissue. Large differences occur between ectomycorrhizal symbionts in their effectiveness in providing resistance to metal toxicity. There is evidence both at the species and strain levels that exposure to metals in the soil can lead to selection for resistance. Colpaert and van Assche (1987b) isolated strains of Suillus luteus from Zn-contaminated soil that were able to grow in the presence of 1000 l^ig g~^ Zn. Strains of the same fungus obtained from fruit bodies growing on uncontaminated soil showed little or no growth above 100 jig g~^ Zn. Zn-resistant strains of S. bovinus conferred significantly more Zn tolerance upon plants of Pinus sylvestris than did non-resistant strains, and tolerance was most probably attributable to binding of the metal in the extraradical mycelium of the fungus (Colpaert and van Assche, 1987a,b).

Disease Suppression by Ectomycorrhizal Fungi When crowded together as dense monocultures in nursery beds, seedlings are particularly susceptible to attack by fungal pathogens. As a result, there is much

Mycorrhizas in managed environments

481

interest in the ability of ectomycorrhizal fungi to act as biological control agents. It appears that ectomycorrhizal fungi can provide their hosts with enhanced resistance to attack by fungal pathogens. Both Pisolithus tinctorius and Thelephora terrestris have been shown to reduce the impacts of the root pathogen Phytophthora cinnamomi on Pinus spp. (Marx, 1969, 1973), while inoculation with Laccaria laccata has been shown to reduce the incidence of disease caused by the pathogen Fusarium oxysporum in Pseudotsuga menziesii (Sylvia and Sinclair, 1983a), Picea abies (Sampangi and Perrin, 1985) and P. sylvestris (Chakravarty and Unestam, 1987a,b). It has been suggested that protection against fungal pathogens is achieved as a result of the physical barriers imposed by the hyphal mantle (Marx, 1973) or by the production of phenolic compounds in the plant tissues in response to the presence of the mycorrhizal fungus (Sylvia and Sinclair, 1983b). While both of these effects may indeed be involved in contributing to defence in the adult plant, there is evidence that ectomycorrhizal fungi exert direct antibiotic effects upon would-be pathogens. Duchesne et al. (1988a,b) observed that inoculation of seedlings of Pinus resinosa with the fungus Paxillus involutus significantly reduced pathogenicity of Fusarium oxysporum before mycorrhizal colonization took place. Increases of seedling survival were associated with a sixfold decrease in sporulation of F. oxysporum in the rhizosphere of the plant (Duchesne et al, 1987). Ethanol extracts of the rhizosphere indicated that fungitoxic effects of P. involutus were present within 3 days of inoculation of seedlings with P. involutus (Duchesne et ah, 1989). Disease suppression at this critical stage of plant development prior to formation of the ectomycorrhizal symbiosis may be of particular significance both in the nursery situation and on the natural regeneration niche. Little is known about the chemical basis of the observed antibiotic activity, although Kope et al. (1991) isolated two antifungal compounds from a liquid medium in which Pisolithus tinctorius had been growing. These were identified as hydroxy forms of benzoylformic and mandelic acid and given the names pisolithin A and B, respectively. It is regrettable that most of the experimeni;s on antibiotic effects of ectomycorrhizal fungi have been carried out under rathl^r unrealistic conditions. Epidemiological studies under natural conditions are necessary to determine if, or at what stage, colonization of roots by ectomycorrhizal fungi can enhance disease resistance. The recent recognition of the stimulatory effects of some classes of bacteria upon the processes of mycorrhizal colonization of roots has heightened awareness of the complexity of microbial interaction in soil, as is shown below.

Mycorrhiza Helper Bacteria Transmission electron microscopy of ectomycorrhizal roots collected from the field has revealed the presence of bacteria in the mantle (Foster and Marks, 1966), and the population of bacteria isolated from mycorrhizal roots of Pinus is distinct from those growing in association with uncolonized roots (Rambelli, 1973). The presence of such bacteria as casual associates df the mycorrhizosphere is to be expected. However, the possibility that they are directly involved in the dynamics of mycorrhiza formation was suggested by studies of Bowen and Theodorou (1979) which showed, in vitro, that the ability of the fungus Rhizopogon luteolus to colonize roots of

482

General themes

P. radiata was enhanced in the presence of some bacterial isolates but inhibited by others. Subsequently, Garbaye and Bowen (1987), using three fungal symbionts, examined the process of mycorrhiza formation in different steam-sterilized soils inoculated with a population of bacteria obtained from one of the soils. The effects of addition of bacteria were different in each of the soils and each of the fungi responded in a distinctive manner to the inoculum. Positive effects upon mycorrhizal colonization outnumbered those that were negative. When bacteria growing in the mantle of surface-sterilized roots of P. radiata mycorrhizal with Rhizopogon luteolus were isolated and characterized, up to 10^ colony forming units were obtained, most of which were fluorescent pseudomonads (Garbaye and Bowen, 1989). Significant positive effects upon mycorrhiza formation were produced by 80% of these isolates on re-inoculation of this plant-fungus combination. Stimulatory effects of the presence of fluorescent pseudomonads of the Pseudomonas putida group have also been reported in the case of VA (Mosse, 1962; Meyer and Linderman, 1986) and orchid (Wilkinson et aL, 1989) mycorrhizas. This suggests that the effects of the bacteria may be rather general in nature. However, a number of recent experiments have indicated that relationships between fungi and bacteria in the ectomycorrhizal symbiosis may be more specific, and on the basis of these, Garbaye (1994) has proposed that a special category of 'mycorrhization helper bacteria' (MHB) should be recognized. The ability of 47 strains, mostly of fluorescent pseudomonas, to enhance mycorrhiza formation in the P. menziesiiLaccaria laccata partnership, was compared under nursery, glasshouse and axenic conditions (Garbaye et aL, 1990; Duponnois and Garbaye, 1991). The most efficient isolates increased the amount of mycorrhizal colonization from 67% in the control to 97% and produced the same result in the soil as under sterile conditions, suggesting that the effects occur independently of the influence of the general soil microflora. Further evidence for specificity in the effects of MHBs was gained in experiments showing that, under a wide range of conditions, isolates obtained from mycorrhizas formed by a strain of L. laccata (S238) on P. menziesii consistently stimulated colonization by this strain, and that the closely related L. bicolor had no effect on L. proxima but had negative impacts upon colonization by fungi of other genera (Garbaye and Duponnois, 1992). To date, there is little information on the possible mechanisms of stimulation apparently induced by the bacterium. Garbaye (1994) proposes five different hypotheses (Fig. 17.3) to explain the phenomenon, each of which is amenable to experimental analysis. The possibility that the MHB might enhance the susceptibility of the plant to colonization, either by production of cell wall softening enzymes (Hypothesis 1) or by enhancing the root-fungus recognition process (Hypothesis 2), is considered. Evidence that MHBs produce five enzymes, endoglucanase, cellobiase, hydrolase, pectate lyase and xylanase (Duponnois, 1992), that are known to have wall softening properties, and that cell-free culture filtrates of Pseudomonas spp. containing these enzymes are capable of enhancing colonization of roots by VA fungi (Mosse, 1962) lends support to Hypothesis 1. It is clearly desirable, however, to establish relationships between virulence of the MHBs and enzyme production. Nutritional enhancement of fungal growth by MHBs (Hypothesis 3) is also a possibility but in view of the apparent specificity of the effect it must be of a highly specialized type. Detoxification of compounds present in soil could, indirectly.

483

Mycorrhizas in managed environments

t «

\

'^

• 9

C<^

c

root-fungus cognition and attachment

ROOT I

I

Figure 17.3 Sinnplified representation of the rhizosphere, indicating five hypotheses to explain the way in which a bacterium might promote the establishment of mycorrhizas. From Garbaye (1994), with permission.

provide a nutritional effect and it is noteworthy that many of the responses to MHBs so far reported have been observed in artificial media or nursery soils which have been previously subjected to sterilization treatments. These, whether they involve heat or chemical methods, all have the potential to release toxins and so influence subsequent microbial interactions. Changes in the rhizosphere, perhaps leading to enhancement of production of chelating ligands such as hydroxamate siderophores (Hypothesis 4) or to stimulating of germination of fungal propagules (Hypothesis 5), may also contribute to the effects of MHBs. There is much scope for experimental analysis of the roles of these bacteria. However, it is important to bear in mind that if, indeed, their specificity is as large in nature as is suggested, the mechanisms involved in determining the intimacy of the relationship between fungus and bacterium are likely to be sophisticated and difficult to identify. While it is relatively easy to establish correlations, elucidation of causal effects will be far more difficult. The issues are nonetheless worthy of more thorough investigation, not only because the association between fluorescent pseudomonads and mycorrhizal roots is so widespread, but also because use of MHBs to enhance rates

484

General themes

of colonization, and perhaps persistence, of inoculant fungi would provide important increases in mycorrhizal effectiveness particularly in nurseries infected with indigenous fungi like T. terrestris.

Responses of Mycorrhizas to Atmospheric Pollutants Human activities are changing the environment in which mycorrhizal roots function on a global as well as a local scale. Emissions arising from industrial and vehicular combustion processes, and locally from intensive stock rearing, have led to enrichment of C, N and S levels in the atmosphere and increasing dry or wet deposition of the products H"^, NO^, NH4 and SO^. The ecosystems at greatest risk from such depositions are those developed over slowly weathering rocks, that yield soils of low base status and hence little buffering capacity. Much of the largely ectomycorrhizal boreal forest in the northern hemisphere is developed on soils of this type (Kuylenstiema and Chadwick, 1989) and concern over the impacts of atmospheric pollution, particularly upon forest ecosystems, has increased since widespread forest decline was observed in central Europe during the 1980s. Long-term reductions in soil p H have been documented in areas distant from the main sources of emission, notably in southern Sweden (Falkengren-Grerup, 1987; Hallbacken and Tamm, 1986) where increases in the N:P, N:Ca and N : M g ratios of pine needles relative to those found elsewhere in Sweden are indicative of perturbation in nutrient supply. As a consequence of increasing deposition of N, particularly in the forms of NO^ and NH4, the possibility emerges that northern forest ecosystems which have evolved under circumstances of N limitation (Tamm, 1991) are becoming N saturated (Aber et al, 1989). The impact of this change upon mycorrhizal function and the role, if any, of mycorrhizas in determining ecosystem responses to the perturbation are subjects of great importance, which are beginning to receive attention. Most studies carried out to date treat the effects of acidity separately from those of N deposition. It must be borne in mind, however, that the simultaneous deposition of H"^, NO^ or NH^ ions will lead to strongly interactive effects which can ultimately only be interpreted by a fully integrated approach.

Effects of Soil Acidification on Ectomycorrhizal Occurrence and Function There have been numerous attempts to determine the impact of p H upon growth of ectomycorrhizal fungi in pure culture (see Hung and Trappe, 1983). These show that most grow optimally in the p H range 3-5, which might make them insensitive to soil acidification. However, since the experimental conditions employed usually bear little relation to those prevailing in the field, the results obtained from them may be of limited relevance to the question of acidification in nature. This is evident from results of Erland et al (1990) who compared the growth of a number of ectomycorrhizal fungi over a range of pH values in pure culture on agar with that obtained when the same isolates were associated with Pinus sylvestris in peat. All of the isolates showed more vigour when associated with the plant, several having high growth rates at p H 3.8 in the symbiotic

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condition, but failing or producing very little growth at this pH on agar or peat in the absence of the plant. To counteract soil acidification, extensive application of lime (Ca(OH)2) has been recommended in Swedish forests (Andersson and Nihlgard, 1988). In a laboratory study, Erland and Soderstrom (1990) showed that liming of a forest humus to give a range of p H values from 4.0 to 7.0 led to an increase in colonization of roots of P. sylvestris from 70% at pH 4.0 to nearly 100% at pH 5.0, the value then decreasing linearly to reach a minimum of less then 40% at p H 7.5, Other studies have shown that the colonization process is more sensitive to acidification than is the growth of the seedling root itself (Metzler and Oberwinkler, 1987; Danielson and Visser, 1989). Erland and Soderstrom (1990) identified five different mycorrhizal types on their plants, each of which occurred at all pH values but in different proportions. Parallel effects of lime on the relative proportions of different mycorrhizal types were also observed in seedlings planted in a natural pine forest (Erland and Soderstrom, 1991). In Finland, Lehto (1984) observed that application of lime to adult stands of P. sylvestris, increasing p H from 3.3 to 5.0, failed to alter the amount of colonization but again changed the proportions of the mycorrhizal types present. Experiments involving reduction of soil pH by addition of acidified rain water have produced conflicting results, some showing increases of colonization at the lowest p H (Schaffer et al, 1985; Reich et al, 1987). In this case, the extensive extraradical mycelium of two coralloid types of mycorrhiza was strongly inhibited by the acidification and enhanced availability of Al at low p H was probably responsible for the apparent toxicity. That the outcome of these manipulations is strongly influenced by soil type was demonstrated by Stroo and Alexander (1984), who found that application of acid rain at pH 3.5 had no effect on mycorrhizal colonization of P. strobus in six out of nine soils, but led to an increase in the remaining three. Bearing in mind the complex and interactive effects of changing p H in soil, differences between studies are inevitable. In addition to impacts upon the availability of metals which have the potential to act as toxins, there are effects upon availability of nutrients, in particular N and P. The decreases of soil p H widely reported in Europe might be expected, through their inhibitory effects upon soil microbial activity (Zelles et al, 1987), to reduce N mobilization and availability However, the acidification, while perhaps initiated earlier in the century by SO^ deposition, is increasingly a product of emissions of oxides of N (NO;^)/ which reach the soil as H"*^ and NO3 ions. Soil N enrichment can be further exacerbated in regions such as the Netherlands where intensive stock rearing can lead to NH4 deposition of up to 100 kg ha~^ y~^. A decline in production of sporophores and of species diversity of ectomycorrhizal fungi has been documented for the Netherlands (Arnolds, 1991) and Germany (Derbsch and Schmitt, 1987), and there is evidence for a strong correlation between this decline and increases of N concentration in forest litter horizons (Termorshuizen and Schaffers, 1987, 1991). Both numbers and diversity of sporophores of ectomycorrhizal fungi can be largely restored by removal of the N-enriched surface organic horizons (Baar and De Vries, 1993, 1995; DeVries et al, 1995). Decline of diversity and of total numbers of fruit bodies associated with addition of N in amounts constituting pollution is consistent with the observed responses of the ectomycorrhizal fungi to N fertilization (Menge and Grand, 1978; Ohenoja,

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General themes

1978). However, studies of fertilized plots reveal that fructification of some species, notably Paxillus involutus (Laiho, 1970; Ohenoja, 1988; Shubin, 1988) and Lactarius rufus (Ohenoja, 1988) may increase. Of greater importance for mycorrhizal function is the impact of N-enrichment upon colonization of roots and development of the extraradical mycelium. Although this appears to be less dramatic than effects upon production of fruit bodies, effects are still detectable. Amebrant and Soderstrom (1992) planted 'bait' seedlings of P. sylvestris into plots fertilized with NH4NO3 at 150-300 kg ha~^. They found an initial reduction in the number of colonized root tips, but after several growing seasons there was a recovery. However, when bait seedlings were planted 13 years after N application there was evidence of a changed species composition in the fertilized plots. Transient reduction in numbers of colonized root tips have also been observed in stands of mature trees after N fertilization (Menge et ah, 1977; Tetreault et al, 1978; Laiho et al, 1987). Alexander and Fairley (1983) reported both a shift in composition of mycorrhizal communities and a decline of colonization on spruce roots after N fertilization. Amebrant (1994) observed that addition of N in the form of NH4NO3, (NH4)2S04 or NaNOa to non-sterile peat supporting ectomycorrhizal plants of P. sylvestris and P. contorta in microcosms led to significant inhibition of development of the extraradical mycelium of the fungal associates, irrespective of N source. This, even at N concentrations as low as 1 mg g~^ dry peat, may be of considerable ecological significance. Some fungi were observed to be more sensitive than others. Thus, mycelial growth of one isolate of Paxillus involutus was reduced by approximately 20% whereas growth of a second isolate of the same fungus was only half that of the control treatment which lacked N amendment. Growth of Suillus bovinus was reduced by 70%, and of an unidentified isolate by 97%. Such differences in sensitivity of the mycelium to N enrichment would help to explain the shifts in population structure observed to occur after single fertilizer applications or longterm exposure to pollutant N. A further important observation of Amebrant (1994) is that transfer of colonized plants from N-amended to unamended peat enabled regrowth of the mycelium. The reversible nature of the effect of N will contribute to the ability of the symbiosis to recover from the impact of short-term perturbations. In contrast, continuous N inputs, if they lead to the inhibition of mycelial growth as observed in microcosms, would restrict the potential for C transfer through the soil and hence limit the ability of the fungus to sustain sporophore production and spread of colonization to seedlings. Of greater significance for the nutrition of the tree is the likelihood that inhibition of mycelial development will seriously reduce its ability to capture those other ions, notably P, the deficiencies of which are exacerbated by N sufficiency. This would contribute to the increase in N:P, N.Ca and N:Mg ratios observed earlier to be a product of N pollutant input. The complexity of the influence of pollution on the biology of mycorrhizas is demonstrated by the fact that while some pollutants like N may reduce colonization and mycelial development others, in particular CO2, are predicted to increase them.

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Responses of Mycorrhizas to Elevated Carbon Dioxide Ecologists are fully aware of the fact that atmospheric CO2 concentrations have increased markedly during this century and that they are projected to double again, reaching 700 |xl l~^ by the end of the next century. While the result has been numerous experiments on growth responses to elevated CO2, few have considered the interactions with mycorrhizal fungi/despite the fact that the mycorrhizal condition is normal in nature. As a consequence, the results of the experiments are difficult to extrapolate to the real world and the models of ecosystem response based on these results are inevitably flawed. Some of the earliest studies, while not considering mycorrhizas specifically, noted that the exposure of Quercus alba to double the ambient CO2 concentration leads to a significant increase in production of fine roots (Norby et ah, 1986). Both Q. alha and Liriodendron tulipifera showed increases in growth at this CO2 concentration, even in forest soil of low fertility in which P uptake increased in proportion to C fixed (O'Neill et ah, 1987a,b). It was suggested that this effect was brought about by increased allocation of photosynthate to mycorrhizas. There was no increase in N concentration in L. tulipifera, in line with the expected VA mycorrhizal status of this species. The suggestion that elevated CO2 increases C allocation to roots was later borne out in a study of Pinus echinata and Q. alba, in which both ectomycorrhizal colonization and growth were significantly greater at elevated CO2 concentrations. The increased colonization was apparent at 6 weeks in both species, but only in Quercus did it persist up to 24 weeks (O'Neill et ah, 1987b). The findings indicate both increased C fixation and increased C allocation below ground, which were confirmed in a more detailed study of P. echinata (Norby et al., 1987). Fine root biomass and mycorrhizal colonization were both increased, together with exudation to the rhizosphere. The effects of P supply and mycorrhizal status on the responses of P. taeda to elevated CO2 were examined in a five-month study (Lewis et al, 1994). This relatively long-term exposure to high CO2 levels resulted in reduced photosynthesis in inoculated seedlings when they were grown with limiting P, apparently because they were P deficient. The plants were grown in vermiculite-gravel mixtures and fed with Hoagland's solution, which might well have reduced colonization. In consequence, without estimates of the extent of mycorrhizal colonization, it remains difficult to interpret the results. It is particularly regrettable in view of the increased awareness of the role of the external mycelium, that few measurements of its response to elevated CO2 have as yet been made. In a pioneering study, Ineichen etal. (1995) found that growth of the mycelium of Pisolithus tinctorius was significantly increased under these circumstances. Shoots of Pinus sylvestris seedlings were exposed to 600 |LI1 1~^ or 350 |LI1 1~^ CO2 while their mycorrhizal roots and associated mycelia developed, in microcosms, over cardboard from which they could be readily harvested. While elevation of CO2 had little effect upon either numbers of ectomycorrhizal roots or extent of mycelial development for the first two months of exposure, after 3 months there was a threefold increase in numbers of colonized roots and the biomass of extramatrical mycelium was double that in the ambient CO2 treatment. This suggests that development of the mycorrhizal system is to some extent C limited. Responses of the kind reported by Ineichen et al. (1995) if occuring also in soil, would provide

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the potential for positive feedback of nutrients to sustain any enhancement of photosynthetic activity or growth. While recognizing the importance of mycorrhizal fungi to our understanding of plant and ecosystem function, those attempting to produce models of the possible effects of elevated CO2 have been frustrated by an inability to define the possible contributions of the symbioses to plant responses even in environments with ambient CO2 levels (Thomley, 1991; Andersen ei ah, 1992; WuUschleger et al., 1994). Andersen et al, (1992) proposed that mycorrhizal roots should be recognized in models as discrete plant 'organs', because of their effects on C allocation, root growth and nutrient absorption. They adapted the TREGRO model (Weinstein et aL, 1991) by incorporating data on respiration and hyphal dimensions of a mycorrhizal fungus obtained from short-term experimental studies (Andersen and Rygiewicz, 1991). The model predicted that growth of Pinus colonized by Hebeloma crustulinifortne would be reduced by 65% after 3 years, relative to non-mycorrhizal plants. Andersen et al. (1992) themselves concluded that these findings were unrealistic. It seems probable that data obtained from short-term studies may be a very poor reflection of what occurs in the long-term life of plants. As discussed in Chapter 6, seasonality of C-allocation to ectomycorrhizas must be recognized and it will be important to improve the data that is available to modellers. Only if this is done, will it be possible to construct models that are able to predict the responses of plants growing under natural conditions to elevated CO2 and other perturbations in the environment. At the ecosytem level there is an even greater lack of hard information. Debate centres on the issue of long-term responses to high CO2 levels. Some models predict a short-term increase in the productivity in tundra and temperate forests, followed by a decline (Rastetter et al., 1991), whereas others suggest that long-term productivity will be sustained above current values for up to 500 years (Post et al., 1992). At the heart of this debate is the question of the influence of elevated CO2 on litter quality. Here, knowledge of mycorrhizal function is likely to be critical. Implicit in the assumptions of Rastetter et al. (1991) is that the litter quality will decline in twice-ambient CO2 concentrations, because plants will produce residues with increased C:N ratio and the soils will have a lower rate of N mineralization. Post et al. (1992) make no such assumption. In both models the ecosystem response to CO2 availability is seen to be determined by resource quality, but neither includes the role that mycorrhizal fungi play in determining nutrient uptake by plants or mobilizing nutrients in the litter. The considerable functional diversity of mycorrhizal associations of timdra and temperate forest ecosystems, and in particular the ability of the fungi to exploit complex sources of N and P (Chapters 6, 8, 9 and 15), can be expected to provide sufficient buffering to influence the predictions of the models. The responses of VA mycorrhizal systems to changing CO2 concentrations have not been much studied. Colonization in the C4 grass Bouteloua gracilis is increased when exposed to elevated CO2 (Monz et al, 1994; Morgan et al., 1994), whereas there is no such response in the C3 grass Pascopyrum smithii (Monz et al., 1994). A comparison of these two species in intact cores highlights the way in which differential responses in an assemblage of mixed species would significantly influence the outcome of competitive interactions and so determine community structure (Monz et al., 1994). Increased colonization has also been observed in

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Liriodendron and Citrus (O'Neill et al, 1991, Dickson and Vellen, personal communication). In the work with Liriodendron, the percentage root length colonized was the same whether the plant was grown at ambient or twice ambient CO2, but because the total root production was very much greater under elevated CO2, the total mycorrhizal colonization per plant was much increased (O'Neill et al, 1991). Such increases in fungal biomass, as well as root tissue, would contribute to the observed increases in C allocation below ground. We need to know much more about the consequences of changing colonization patterns and of mycelial development in all mycorrhizal types, as they are likely to influence photosynthesis and subsequent C allocation, as well as nutrient capture. Conclusions Mycorrhizal inocula have been successfully introduced to enable commercial afforestation in many countries of the world. At the same time it has often been the experience, particularly in sites with a well established resident flora of mycorrhizal fungi, that inoculant species have had little impact. It thus appears that natural selection, operating over many generations in natural and semi-natural forests has produced a stable population of symbionts that is resistant to invasion by alien organisms. This may, in fact, be a desirable feature otherwise inoculant fungi, artificially selected by man for their vegetative vigour and now introduced with little legislative restriction to so many parts of the world, would come to dominate natural forests. The consequences of the loss of biodiversity which would follow such invasion cannot yet be quantified but, clearly, until the role of an extensive gene pool in sustaining ecosystems is evaluated it would be prudent to protect and retain as much diversity as possible. There remains enormous scope for investigation of both fundamental and applied aspects of the mycorrhizal symbiosis. Pursuit of these studies at the cellular, whole-plant and community levels will enrich our understanding of the role of mycorrhizas in extant ecosystems both natural and managed. The pressing need for reliable prediction of the impacts of climate change introduces an element of urgency to the quest for knowledge. Innumerable opportunities exist for those investigating mycorrhizal systems to engage in collaborative projects which will provide modellers with information that will make their predictions of environmental change relevant and useful.

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Zengming, Y. and Zhong, H. (1990) A preliminary study on the chitinase and 1,3-glucanase in corms of Gastrodia elata. Acta Botanica Yunnanica 12, 421-426. Zerova, M.Y. (1955) Mykorrhiza formation in forest trees of the Ukrainian SSR. In: Mycotrophy in Plants (ed. A. Imshenetskii). [Translated from Russian by Israel Programme for Scientific Translocations 1967.] USDA and NSF Washington DC, USA. Zettler, L.W. and Mclnnes, T. (1992) Propagation of Platanthera integrilahia (Correll) Luer, an endangered terrestrial orchid, through symbiotic seed germination. Lindleyana 7,154-161. Zeze, A., Dulieu, H. and Gianinazzi-Pearson, V. (1994) DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus, Scutellospora castanea. Mycorrhiza 4, 251-254. Zhu, H., Guo, D.-C. and Dancik, B.P. (1990) Purification and characterisation of an extracellular acid proteinase from the ectomycorrhizal fungus Hebeloma crustuliniforme. Applied and Environmental Microbiology 56, 837-843. Zhu, H., Dancik, B.P. and Higginbotham, K.O. (1994) Regulation of extracellular proteinase production in an ectomycorrhizal fungus Hebeloma crustuliniforme. Mycologia 86, 227-234.

Index

Note: Page references in italics refer to figures; those in bold refer to tables Abies 92, 165, 279 association with Heheloma 383 fruit bodies in 253 glutamine-glutamate shuttle 402-3 mycorrhizal mantle 200 phosphorus absorption 251 transfer between Betula and 406 Acacia 167, 442 Acaulospora 14, 31 effect on phosphorus uptake 137, 137 extraradical mycelium development of 67, 68 germination in 37, 39 phylogeny 21 root fragment colonization by 43 spore infectivity 41 zinc uptake 152, 152 Acer 31, 321, 422 mycorrhizal development in 46, 47-9 Acremonium 433 Afzelia 167 Agaricus 472 Agathis 23 Aglaeophyton (Rhynia) 6, 18, 29, 21, 23 Agriculture / horticulture effect of soil compaction 461, 462 management of VA mycorrhizal populations 457-64, 461 evaluation of fungal populations 458 indigenous populations 460-4 inoculation 459-60 mycorrhizal involvement in crop growth in the field 454-7 see also Soil structure Agrobacterium 41 signal molecules in 90 Agropyron 70 effect of temperature on colonization 75 potassium uptake 153 Alanine uptake Allium 12, 46, 49, 71, 455, 460, 466, 486

arbuscular cycle 62 association with Glomus 51, 70, 72-3, 399, 402 carbon transfer 124 cellular reaction in roots during epidermal penetration 94-5 colonization 50 copper uptake 152 dry weight and phosphorus content changes in 108 early root colonization 54-5 effect of carbohydrate on colonization 78, 79 coloruzation on root growth 79 irradiance and phosphorus on colonization 76, 76 hypodermis development of 52 inoculation of 460 lipid levels in 114 phenolic synthesis 51 phosphorus flux 71, 113 phosphorus uptake 128,129,132,133,134, 140, 387, 388 progress of root colonization in 72-3 resistance responses 100 spatial distribution of colonization 70 transpirational flux 158 yellow pigment 11 Alnus 170, 190, 191, 425 association with Alpova diplophoeus 185 186, 196, 197 epidermal Hartig net in 192, 197 stimulation of hyphal growth by exudates 92 Alpine mycorrhizas 410-12 Alpova association with Alnus 185, 186, 197, 196 Hartig net in 197 Alternaria, signal compounds in 102-3 Aluminium 115, 129, 142 Amanita 170, 222, 223, 263, 269, 270, 271, 272

590 Amanita {continued) association with Picea abies 182, 197 carbohydrate utilization 241-2 endoglucanase activity 198, 200 somatic incompatibility in 179 yield on nitrogen 260 Amaranthus, germination in 90 Amino acids transfer 403 uptake in ectomycorrhiza 259-62 Ammonium assimilation of 337-40 in ectomycorrhiza enzymes of 265-7, 266 pathways of 258-9, 257, 267 uptake 383 Ammonium-assimilating enzymes 265-7, 266,17b, 404 Ammophila 447 seasonal variation in spore density and mycorrhizal colonization 37 Amyelon 24 Anacardium (cashew) 23 Andropogon 435 Antarcticycas 19, 19, 24 Apigenin 41 Apium, mineral nitrogen uptake 148 Apoplast 56, 394-7 carbon acquisition 18 see also Interface; Nutrient transfer Arabidopsis 97 Arabis 437, 438, 439-40 appressorium formation 92 interplant nutrient transfer 155 Araucaria 23 zinc uptake 152 Arbuscules 11,12, 63 cycles 61-^, 62 longevity 61 role in nutrient transfer 60 as site of carbon transfer 15-16 Arbutoid mycorrhizas 301, 302-8 Arbutus 92, 280, 298, 301, 302, 303, 304, 323, 427 mycorrhizas in 302-4 Arctic mycorrhizas 410-12 Arctostaphylos 298, 301, 323, 427 mycorrhizas in 302-4 Arenaria 439^0, 441 appressorium formation 92 Armillaria 226, 333, 352, 353, 370 association with Monotropa uniflora 321

Index

rhizomorphs in 208, 209 trehalose transfer 388 Arsenic 154 Artemisia, colonization of 43 Arum-type mycorrhizas 46-9, 47-9, 86 arbuscules in 393 carbon transfer in 112 coils in 393 infection units in 52-4 Asarum 46 Asparagus 69 Asteroxylon 23 Astroloma 419 Atriplex, appressoriimi formation 94 Auxin theory 226 Auxins, effect on mycorrhizas 228-31 Avena 435, 455 copper uptake 152 responsiveness to colonization 119

Bacteria, helper, mycorrhizal 481-4 Banksia 419 nitrate utilization 150 Bariimi 1 5 3 ^ Basilicum, appressorium formation 92 Benomyl 434-5, 456 Beta 455 Betula 182, 217, 224, 225, 246, 265, 269, 270, 271, 272, 291, 429, 430, 430, 475, 480 association with Hebeloma 224, 225 Lactarius pubescens 224, 225 Pisolithus tinctorius 185, 187, 190, 194-5, 196 effect of nitrogen 269, 272 epidermal Hartig net in 192 Hartig net in 197 transfer between Abies and 406 Biocharun A 41 Biochemical valve 245 Bletilla 366, 367, 371, 374 Boletinus, association with Larix 426 Boletus 222, 319 protein nitrogen use by 259 Boreal forest mycorrhizas 422-31 Boronia 419 Botrytis 370 Bouteloua 433 raised carbon dioxide and 488 stomatal conductance 156 transpiration rates 156

Index

Brachystegia 167 Brassica 41, 455 appressorium formation 92, 94 germination in 90 resistance responses 100 Bromus 435

Cacao 455 Cadmium 153-4 Cajanus, zinc uptake 152 Caladenia 374 Calamagrostis 415 Calcitim 115, 129 Calluna 321, 332, 334, 335, 337, 343, 345, 405, 413 association with Hymenoscyphus ericae 327 Oidiodendron griseum 334, 335 Pezizella ericae 333 colonized mycorrhizal root 325 hair root 324 Capsicum 455, 462 Carbohydrate in ectomycorrhiza effect on fungal colonization 240-7, 241 transfer from plant to fungus 243, 244 orchids and 350 in VA mycorrhiza effect on colonization 78-9 transfer 109, 113 see also Carbon Carbohydrate theory 226 Carbon in ectomycorrhiza 251-2 in ectomycorrhizal fungi 239-53 efflux 400 in orchids 361-9, 388 transfer 124, 429-30 translocation of 388 use 108-14 in VA mycorrhiza 15-16 Carbon dioxide elevated, responses of mycorrhizas to 487-9 VA mycorrhiza sensitivity to 39, 41 Carbon dioxide fixation 402 Carex 412, 414 Carpinus 477 Cassava 455 Castanea 477 Castanopsis, tuberculate mycorrhiza 203

591 Casuarina 442, 447 Cenococcum 170, 208, 220, 284, 286, 287, 291, 302, 427 association with Eucalyptus pilularis 283, 285 Fagus 266 Pinus albicaulis 412 Tilia cordata 478 effect of p H on proteolytic activity 263 endoglucanase activity 200 glutamine synthetase in 258 nitrogen metabolism in 258, 260 polyphosphate in 287, 389, 390 yield of sclerotia 253 Centaurium 46, 440, 441 carbon cost of colonization 123 growth 116 Cephalanthera 352, 353, 372, 374 Cephalozia 418 Ceratobasidium 351, 356, 359, 360, 361, 373, 374, 416, 418 Ceratorhiza 351 Chalcone isomerase (CHI) 99 Chalcone synthase (CHS) 82, 99 Chenopodium chitin content 64 effect of phosphorus on chlamydospores 77 effect of plant exudates on colonization 78 Chitinases 100, 184 Chloridium 297 Chondrogaster 287 Cistus, cortical epidermal Hartig net in 192 Citrus 23, 455 carbon allocation 114 effect of plant exudates on colonization 78 lipid levels in 114 phosphorus levels and 114 raised carbon dioxide and 489 responsiveness to colonization 119 transpiration rates 156 Classification of mycorrhizas 4-8 characteristics 5 Clavaria 336 Cloned probes 65 Cojfea 23 Colchicum 46 Colonization by ectomycorrhizal fungi 185-91 effect on root growth 79 effects of phosphorus 74-5, 75-7

592

Colonization effects of (continued) nitrogen 75 light 76, 76, 77-8 temperature 75-6 soil salinity 74-5 carbohydrate 78-9, 240 by ericoid fungi 326-31 rate of 70-1 of roots 46-64, 71-9 stages in 72-4, 81-2, 83-4 by VA mycorrhizal fungi 18, 23-8, 33-79 Complexipes 14, 294, 295 Conostephium pendulum 419 Contact zone 196 Copper 115, 126, 127, 151-3, 345, 403 Coprosma rohusta, colonization 69 Corallorhiza 352, 353, 372 association with Pinus 374 Coriolus 352 Cortical Hartig net 192 Corticium 351 Cortinarius 220 Corylus 477 Cryptothallus 49 Cucumis 71 association with Glomus fasciculatum 108-9, 111, 112, 113 Gigaspora (Scutellospora) calospora 111 carbon allocation 112, 113 colonization 111 effect of phosphorus on colonization 77 interplant nutrient transfer 155 mineral nitrogen uptake 149, 149 photosynthate used during colonization 111 Cyanotis 28 Cymbidium 355, 367

Dactylis, hyphae in 44r-5 Dactylorchis 351 protocorm 356 Dactylorhiza 364, 365, 365 association with Rhizoctonia 359, 360, 363, 370 carbohydrate in germination 365, 366, 366, 368 phosphorus uptake 367 trehalose in 389 Daucus 455 Dendrobium 366, 367

Index

Deschampsia 410, 414 Descolea 284, 473 association with Eucalyptus diversicolor 234, 235 Dianthus 41 appressorium formation 92 Dicotyledonae, phylogenetic dendrogram 26 Dinitrogen fixation 17-18, 147-8 Disa 355 Disease suppression 480-4 Disperis 355 Diuris 374 Dryas 167, 447 cortical epidermal Hartig net in 192

E-strain fungus 294-7, 472 Echium 439-40 Ectendomycorrhizas 165, 290-8 fungi 293-5 occurrence and structure 290-3 Ectomycorrhiza development 185-91 disease suppression by 480-4 drought resistance and water balance in 478-80 effects of soil acidification on 484-6 extracellular enzymes 417 formation 179-85 contact between fungus and root 180-5 events at root level 183-4 molecular events 180-3 precolonization events 180 genera containing 168-9 Hartig net structure 192-7 cellular interactions 197-200 microscopic structure 196-7 plant-fungus relationships at tissue level 192 improvement of metal resistance 480 molecular studies 222-4 mycelial links between plants 430-4 mycorrhizal mantle 200-3, 204-5 use for classification 201-3 physiological processes regulating formation 226-33 rhizomorph construction 202, 211-17 structure of emanating hyphae and rhizomorphs 208-15 succession of mycorrhizas and fungi 217-24 community level studies 217-22

Index

Ectomycorrhiza (continued) taxonomy and geographic occurrence 167-72 tuberculate mycorrhizas 203-8 types of rhizomorphs 211 see also Ectomycorrhizal fungi; Ectomycorrhizal plants; Ectomycorrhizal roots Ectomycorrhizal fungi 170-3 with broad host range 172-3 carbon supplies for 239-46 genetics 171-81 somatic incompatibility 178-9 succession and replacement on roots and root systems 224-6 interactive replacement 224 replacement on whole root systems 225-6 Ectomycorrhizal plants carbon distribution in intact plant-fungus systems 246-51 community level patterns in carbon allocation 253 growth 233-9 nitrogen nutrition 255-75 growth response of ectomycorrhizal plants to 235 relationship between sugar and, in mycorrhiza formation 226-8, 227, 111 use by mycorrhizal roots and intact plants 265-75 use in pure culture 256-65 non-nutritional effects upon carbon assimilation 251-2 phosphorus nutrition 276-91 absorption in intact plants 281-6 growth response of ectomycorrhizal plants to 234-7 net photosynthesis rate and 251-2, 252 sources in soil 286-8 uptake by excised mycorrhizas and nonmycorrhizal roots 277-83 Ectomycorrhizal roots longevity of 215-19 structure and development 163 Ectomycorrhizins 182 Edible fungi 476-8, 477 Ehrhartia, uptake of toxic elements 153 Eichhomia 28 Elaeis 116, 455 Elaphomyces 272, 273

593 Eleocharis 30 Elythranthera 374 Endogonales, ordinal and family structure 17 Endogone (Glomus) 12, 13, 15, 21, 69, 170 Engelhardtia, tuberculate mycorrhiza 203 Eperua 167 Epiparasitism 321 Erica 343, 413 Ericales, taxonomic, geographic and mycorrhizal relationships 324 Ericoid mycorrhizas colonization process 326-9 enzyme production 339 extracellular enzymes 417 functional aspects 336-46 fungi forming 332-6 intracellular colonization 329-32 penetration of the plant wall 327-9 structure and development 323-32 systemic infection 332 Eriochilus 374 Eriophorum 414 Erodium, effect of phosphorus on colonization 77 Eruca 41 appressoria formation 92 Erysiphe 318 DNA content 39 Erythronium 46 Eucalyptus 24, 165, 169, 171, 185, 270, 273, 273, 284, 423, 450 association with Cenococcum geophilum 283, 285 Descolea maculata 234, 235 Pisolithus tinctorius 185, 187, 190, 187, 194, 197 branching rhizomorphs in 203 carbon distribution in 246 cell wall polypeptide changes in 174 chitinases and peroxidases in 184 effect of mycorrhizal colonization on growth rate 237 entry of celluflor to 396 epidermal Hartig net in 192 experimental manipulation of symbiosis with Pisolithus tinctorius 180-1, 181,181, 182 growth and phosphorus uptake 234, 235 Hartig net formation 179 lateral root primordia in 191 mycorrhizal lateral roots 189

594 Eucalyptus (continued) non-mycorrhizal roots 188 polyphosphate in 280 rootrshoot ratio 236 symbiosis related proteiiis in 178 tuberculate mycorrhiza 203 zones of mycorrhizal root 191-2 Eupatorium 27

Fagirhiza 202 Fagus 240, 241, 268, 269, 270, 312 acid phosphatase in 287 ammonium assimilation 266, 267 association with Cenococcum geophilum 266 Lactarius subdulcis 165, 166, 278 Laccaria 382, 383 carbon metabolism in ectomycorrhizas 240, 241 mycorrhizal mantle 200, 202 rutrogen assimilation in 258, 402 orthophosphate transport 402 phosphorus accumulation 279, 280 polyphosphate in 281, 389 sugar absorption 245, 246 Fatty acid methyl ester (FAME) profiles 15 Favolaschia 352 Festuca 31, 437, 438 carbon transfer 124 interplant nutrient transfer 155 mycelial liiO^ with Plantago 110-11 Flavonoids, stimulatory effect on growth of fungal symbionts 41 Flavonols 90-2, 184 Flavonone 94 Fluoroindole resistance (FIR) 228 Fomes 352 Formononetin 41 Fragaria 40 phosphorus uptake 128 variation in phosphorus demands 122 Frankia, nitrogen fixation and 147 Fraxinus 422 Fungal coils in ericoid mycorrhizas 325 in orchid mycorrhizas 351 in VA mycorrhizas 11 see also Pelotons Fusarium 436, 437, 465, 481

Index

Gadgil effect 424 Gaeumannomyces 97 Galeola 352, 372 Gastrodia 321, 352, 353, 361, 372 Gautieria 287 Gene expression in fungi 171-81 in plants 96-9 Gentiana 49 Gigaspora 14, 69, 459, 464 abnormal mycorrhizal phenotypes 87 ancestral 21 carbon dioxide and mycelial development 39, 40, 41 colonization of Cucumis 111 DNA content 39 DNA replication and nuclear division in 16 effect of flavonoids 41 effect of temperature on colonization 75 extraradical mycelium development of 67, 68 germination in 37, 39, 42 growth enhancement of 39, 40, 41 host dry weight and phosphorus content changes caused by 108 hyphal growth 42 infection 64 maximum germination 35 phosphorus uptake and 40, 133, 142, 381 progress of coloruzation in Allium cepa 72-3 rate of colonization 74-5 seasonal changes in density 35 seasonal variation in spore abundance and germination 37 spore infectivity 41 spore nuclei in 38 spores 15 stimulation of hyphal growth by host exudates 92 tubule systems in 386 zinc uptake 152, 152 zygospore formation in 16 Gigasporinaeae 16 Ginkgo 46, 49, 51 Glaziella 14 Glomales 15, 16 Glominaceae 16 Glomus 12, 14, 15, 27, 31, 116, 143, 411, 433, 436, 437, 462

Index

Glomus {continued) abnormal mycorrhizal phenotypes in mutant plants 87 arbuscule development 56-8, 61, 62, 63 association with Allium 72-3, 399, 402 Cucumis 108-9, 111, 111, 113 cadmium uptake and 154 carbon transfer during colonization by 113 carbon use by 112,113 cellular reaction in roots following epidermal penetration by 94-5 change in gene transcription during colonization 97 chitin in 60 differential hyphal morphogenesis 52-3 DNA content 39 49 early root colonization 54-5 effect of flavonoids 41 light on colonization 78 phosphorus on colonization 78 temperature on colonization 75 effect on hydraulic resistance of root systems 157 phosphorus release 141 phosphorus uptake 128, 132, 133, 136, 137, 140, 388 extraradical mycelium development 67, 68 germination 37, 39-^0, 90 hyphae per gram of soil 68 h)^hal growth 42 network 44 surface area 382 walls 43 infectivity 42 interplant nutrient transfer 155 mineral nitrogen uptake 148, 149, 149 nitrate reductase activity 150 phosphate transporter in 381 plant dry weight and phosphorus content changes caused by 108 potassium uptake 153 RAPDs for spores of 16-17 rate of colonization 74-5 resistance responses to 100 sensitivity to carbon dioxide 39 spore infectivity 41 spore nuclei in 38 sporalation 69

595 stimulation of hyphal growth by host exudates 92 translocation to Trifolium repens 387 variation in responsiveness to colonization by 119, 120 zinc uptake 152, 152 Glossodia 374 p(l,3)glucanases 100 Glutamate dehydrogenase 151 Glutamate synthase 151 Glutamine synthetase 151 Glutamine transfer 402-3 Glutamine translocation 389 Glycine 64, 455 appressorium formation on 92 resistance responses 100 Goodyera 351, 359, 372, 374 carbohydrate use 366, 368 mineral nutrition and mycorrhizal colonization 373 phosphorus use 367 protocorms 360, 361, 365, 365 root length colonized 371 Grid intersect method 72 Griselinia, growth 106 Growth responses 234-7

Hartig net 165,179,190-1,191, 291, 293, 303, 305, 315 cellular interactions 197-200 microscopic structure 196-7 plant-fungus relationships at tissue level 192 structure 192-7 Haustoria in monotropoid mycorrhizas 315 Heathland mycorrhizas 413-22 compartmentation of resource acquisition 414, 414 distribution 413 Hebeloma 170, 171, 173, 174, 179, 217, 217, 225, 266, 302, 427 ammonium assimilation 258 association with Abies 383 Betula 224, 225 Pinus pinaster 228-9, 229, 230, 252 Pinus ponderosa 247 bipolar mating t)qpes in 173 carbohydrate utilization 241-2 carbon allocation 252 endoglucanase activity 200

596 Hebeloma (continued) growth on ammonium in culture 258 indole acetic acid synthesizing activity of 175, 175 inoculum of 472, 473, 474 intraspecific variation in 176 mutants 228 nitrogen metabolism in 258 polyphosphate in 389, 390 raised carbon dioxide and 488 somatic incompatibility in 179 variance in dikaryons of 177, 177 yield on nitrogen 260, 263, 263, 264, 264, 268-73, 271 Hedysarum 27 nitrogen fixation in 150 Helianthemum 167, 170 Helianthus 40, 156 Helichiysum 419 Hesperitin 41 Hihbertia 419 Holcus 31, 432 effect of arsenate uptake on phosphorus transport 154 Hordeum 82, 455 gene determination in 98 mutagenized 87 responsiveness to colonization 119, 120 Horticulture see Agriculture/horticulture Huttonia 355 Hyacinthoides 31, 43, 436 non-mycorrhizal 450 phosphorus uptake and 133, 433-5, 434, 435 variation in phosphorus demands 122 Hydrophobin 183 Hymenochaete 352 Hymenoscyphus 263,272,328,333,334-7,334, 335, 346, 416, 418 ammonium use in 337-42, 337, 342 association with Calluna vulgaris 327 extrafibrillar sheath 327, 327 hypha 328 penetration of Rhododendron roots by 329, 345 phosphorus use in 343, 344, proteinase activity 341 translocation distance 387 vacuole and tubule systems 385 Hyphal length in VA mycorrhiza 65-8, 66 Hysterangium 274, 286, 287, 306

Index

lAA and mycorrhiza formation 228-31 Inununofluorescence 65 in ectomycorrhiza 261-4 Infection see Colonization Inoculation/inoculum 190, 459-60 production of inoculum and practice of 471-5 sources of 33-46 Inocybe 217 mycorrhizal mantle 201 Interface 65, 398 development of 393-4, 395 localization of fungus in an apoplastic exchange compartment 394-7 see also Apoplast Interfacial matrix 56, 305, 308, 309, 310, 330 Intsia 167 Involving layer 197, 198, 395 Iron 115 Isoflavone reductase (IFR) 99-100

Jubemaldia 167 Juniperus 422

K-strategists 219 Kaempferol 92 Kennedia 442 Khaya 23 Kobresia 167, 412

Laccaria 170,171,178, 217, 252, 284, 302, 427, 482 acid phosphatase in 175 ammonium assimilation 267 association with Eucalyptus diversicolor 234, 235 Fagus 382, 383 Pinus strobus 247, 248 Pinus sylvestris 227, 227, 228, 402 P. menziesii 475, 482 carbohydrates and 226 cimiulative growth 213 extracellular fibrillar polymers on 184 glutamine synthetase in 258 growth in media containing ammoniuni 262, 262 inoculum 472, 473, 474 mating systems in 174 mycelial growth in 210

597

Index

Laccaria (continued) nitrogen in 258, 267 rhizomorph growth in 209 somatic incompatibility in 179 yield on nitrogen 263 Lactarius 217, 220, 263, 301, 302, 480, 486 association with Betula 224, 225 association with Fagus sylvatica 165, 166, 278 fruit body yield 253 growth 236, 236 lectins in 184 mycorrhizal characteristics 207 mycorrhizal mantle 200, 201 202, 206 rhizomorphs in 209 Lactuca, mineral nitrogen uptake 149 Laelia, association with Cattleya 350 Larix association with Boletinus 426 ectendomycorrhizas on 165, 170, 171, 291 Lavendula, phosphorus uptake 141 Lead 153 Leccinum 218 rhizomorphs in 215 Leucaena 455 transpiration rates 156, 157 zinc uptake 152 Leucoscypha 165, 166 Lipid levels in VA mycorrhiza 114 Liquidambar 23, 280, 455 growth 106 mineral nitrogen uptake 148 Liriodendron 46, 49, 56-8, 455, 487 raised carbon dioxide and 489 Listronatus 433 Lithocarpus 427 Littorella 28 Lobelia 28 Lolium 71, 433, 463, 466 effect of phosphorus on colonization 77 lipid levels in 114 Long-fallow disorder 456 Loroglossum 370 Lupinus 40, 41, 412 appressorium formation 92 germination in 90 resistance responses 100 Lycopersicon 41, 69, 82, 455 appressorium formation 92 arbuscular colonization of 50 arbuscular cycle 62 mapping populations 97

mutagenized 87 phosphorus uptake 142 responsiveness to colonization 119 Lysinema 326

Magnaporthe, appressorium formation 92 Magnoliales, root systems 24 Malus 23, 455 Manganese uptake 152 Manihot 455 growth 116 phosphorus uptake 142 Mannitol use in ericoid fungi 367, 388, 389 Mantle 165, 200-3, 204-5, 315 use for classification 201-3 see also Sheath Marasmius 352 Mating type 173 Medicago 27, 28, 51, 381 abnormal mycorrhizal phenotypes 87, 88-9 appressorium formation 92 gene transcription change during colonization 97 phenyl alanine ammonia lyase (PAL) and chalcone synthase (CHS) in 82 resistance responses 99, 100 stimulation of hyphal growth by exudates 92 Medicarpin 100 Melaleuca 352, 353 Melilotus, caesium uptake 153 Membrane transport in external mycelium 383 at the interface 393-8 Miltonia 350 Modicella 14 Molinia 414 Monotropa 298, 301, 312, 313, 321, 333 Monotropoid mycorrhizas 301, 308-22 function 319-22 symbionts and development of 312-19 Most probable numbers (MPN) method 34, 65 Mycelium enzyme production by 99-101, 198-200, 239, 370 extraradical 64-8, 67, 132-41, 77, 209-10 functions 336-46 intraradical 94-6 in soil 43-6, 66, 68, 465-8

598 Mycelium (continued) soil structure and 465-8 translocation 384-7 Mycelium radicis atrovirens 412 taxonomic and functional status 296-8 Mycena 273 Myco-heterotrophs 321 nutrient transport 407, 408 Mycorrhiza helper bacteria (MHBs) 180, 482-6 Mycorrhizal association, definition 2 Mycorrhizal dependency 86 Mycorrhizins 96

Narengenin 41 Nasturtium, appressorium formation 92 Neurospora 258, 381 Nickel 126, 153, 480 Nicotiana, resistance responses 100 Nitrate reductase 150, 151, 258-9 Nitrate uptake 151, 383 Nitrogen in ectomycorrhiza 255-75 growth response of ectomycorrhizal plants to 235 relationship between sugar and, in mycorrhiza formation 226-8, 227, 227 use by mycorrhizal roots and intact plants 265-75 inorganic nitrogen sources 265-9 organic nitrogen sources 269-75 use in pure culture 256-65 inorganic nitrogen sources 256-9 organic nitrogen sources 259-65 in ericoid mycorrhizas 338-9, 340-3, 367 in VA mycorrhiza 147-51 mycorrhizal effects on nodulation and nitrogen fixation 147 uptake of mineral nitrogen (nitrate and ammonium ions) 147-51 Non-interactive replacement of ectomycorrhizal fungi 225 Non-protein fungi 263 Nothofagus 170 Nowellia 418 Nutrient transfer between plants 154-5, 406-7 between symbionts 393-405 bidirectional transfer 403-5 mechanisms of transport 398-400

Index

rates 403 t)^es of interface 394 Nutrient uptake efflux from the mycelium 383 by mycorrhizal fungi and mycorrhizas 380-3 see also Carbon; Nitrogen; Phosphorus

Oidiodendron 272, 334, 335, 336, 335, 345, 345, 416, 418 association with Calluna vulgaris 334, 335 Oncimum 41 morphogenetic response 52-3 Onobrychis 27 Onychiurus 431 Ophrys 371 Oryza, arbuscular cycle 62 Orchid mycorrhizas in adult orchids 371-3 fungi of 350-3 isolation and identity 350-3 nutritional characteristics 353 plant/fungus interactions 368-71 seed germination and protocorm development 353-61 asymbiotic 353-5 mycorrhizal colonization of protocorms 355-61 specificity and ecology 373-4 symbiotic development 369 transfer of nutrients from fungus to orchid 361-8 carbon 361-7 mechanisms of 367-9 mineral nutrients 367 transfer of carbohydrate from orchid to fungus 368 ultrastructure 358-9 Orchis 359, 361, 370 Oryzopsis, colonization of 43

Palaeomyces 23 Paris-type mycorrhizas 46-9, 47-9, 86 arbuscules in 393-4 coils in 393-4 Parnassia 46 Parthenium 460, 463 Pascopyrum, raised carbon dioxide and 488 Paspalum 69 Paxillus 265-6, 268-70, 270, 271, 272, 486

Index

Paxillus (continued) association with Betula pendula 182 Betula pubescens 480 Pinus resinosa 481 effects of fungivory on 431 endoglucanase activity 200 life span 216 nitrogen and growth of 259, 261, 263 vacuole and tubule systems 385 PCR primers 65 Pelotons 351, 360 see also Fungal coils Penicillium 465 phosphorus uptake 141 Periarbuscular membrane (PAM) 56, 60, 96 Peribacteroid membrane (PBM) 56, 96 Persea (avocado) 23, 455 Pesticides, interactions between mycorrhizas, pests and 464-5 Petrophile 419 Petroselenium, resistance responses 100 Pezizella 333 association with Calluna vulgaris 333 Phalaenopsis 366-9, 370 stages in development 354 Phanerochaete 226 Phaseolus arbuscular cycle 62 copper uptake 152 phosphorus uptake 141 resistance responses 99 Phenyl alanine ammonia lyase (PAL) 82, 99 Phialocephala 296, 296, 297, 411-14 Phialophora 297, 411 Phleum 31, 70 Phoma 332 Phosphate transporters 381-4 in VA mycorrhizas 382 Phosphoenol pyruvate (PEP) 246 Phosphorus in ectomycorrhiza 276-91 absorption in intact plants 281-6 growth response of ectomycorrhizal plants to 234-7 net photosynthesis rate and 251-2, 252 sources in soil 286-8 uptake by excised mycorrhizas and non-mycorrhizal roots 277-81 efflux from mycelium 383 in ericoid mycorrhizas 341-5, 367 translocation of 387-8

599 uptake 381 in VA mycorrhiza 159-60 carbon production and 118 carbon use 119 effect on colonization 76-8, 127-9 effect on growth 119-21, 122 inflow to hyphae from soil 134-6,138 magnitude of flux 140 mechanisms of acquisition by mycorrhizal roots 132-45 competition between fungal hyphae and soil microorganisms 140-1 hyphae and root uptake kinetics 142 role of extraradical hyphae 132-40 uses of sources unavailable to roots 142-5 mechanisms underlying uptake 131 in non-mycorrhizal roots 115-16 phosphate metabolism in the fungus 145-7 in soil and its availability to plants 129-31 transfer from fungus to plant 139-40, 140 uptake 152-3 Photinia, tuberculate mycorrhiza 203 Photosynthesis 251-2, 252 see also Carbon dioxide fixation Phragmites 30 Phycornyces, transpirational flux 158 Phytophthora 481 resistance responses 100 Picea 2,165,171, 279, 290, 291, 296, 320, 321, 415, 474 acid invertases in 243 ammonium assimilation 266, 267 association with Amanita muscaria 182 Fusarium oxysporum 481 Rhizopogon vinicolor 171 carbon distribution in 246 changes in root with age 217 cumulative growth 213 ectendomycorrhizas 291 ectomycorrhizas on 298 growth 236, 236 Hartig net 196, 198-9 life span 216 life span of fine roots 216, 217 Mycelium radicis atrovirens on 296 mycorrhizal mantle 202

600 Picea (continued) nitrogen uptake 268, 269, 270 penetration of hyphae in 192 rhizomorph growth in 209 Piloderma 302, 303 pectinase activity in 200 Pinus 2, 24, 92, 178, 190, 218, 222, 224, 268, 276, 279, 286, 287, 293, 294, 295, 296, 297, 320, 321, 412, 425, 426, 427, 428, 472, 474, 477, 478, 479, 479, 485, 487 association with clamp-fonning fungus 352 Cenococcum geophilum 282, 283, 284, 284, 412 Corallorhiza 374 Fusarium oxysporum 481 Hebeloma crustuliniforme 247, 268 Hebeloma cylindrosporum 229, 229, 230, 252 Laccaria bicolor 227, 227, 228, 247, 248 Laccaria laccata 247, 248 ATPase activity 244 duration of association 218 hexose absorption in 402 Mycelium radicis atrovirens 296, 297 Paxillus involutus 182, 481 Phytophthora cinnamomi 481 Pleurozium schreberi 424 Rhizopogon luteolus 482 Rhizopogon vinicolor 171 Suillus bovinus 196, 387 Suillus granulatus 269 Wilcoxina mikolae 165, 291, 292-5, 294, 295, 303 carbohydrate and fungal colonization in 240 carbohydrates and 226 carbon distribution in 246, 247 drought resistance 478, 480 ectendomycorrhizas on 290, 291, 293 ectomycorrhizas on 220-2, 221, 298, 302 effect of soil acidification 484-5, 486 ergosterol concentration in roots 227 fossil root 163, 164 Hartig net formation 179 herbivory in 431 lAA in roots of 228 inoculated 474 life span of fine roots 216, 217 mycelial growth in 210, 212, 214 mycorrhiza formation 228, 229, 230

Index

nitrogen levels and 226, 270, 272, 273, 274, 274 phosphorus and 251, 278, 280, 281, 282, 283, 284, 285, 286, 290 potassium storage in tubercles of 203 raised carbon dioxide and 487, 488 suilloid fungi and 301 water transport to 384 Pisolithin A and B 481 Pisolithus 4, 170, 171, 179, 302, 303, 303, 304, 475 absorptive root area caused by 210 association with Betula alleghaniensis 185, 190, 194r-5, 196 Eucalyptus pilularis 185, 187, 190, 187, 194, 197, 246 Pinus 178, 228 bipolar mating types in 173 carbon assimilation 251 carbohydrates and 226, 241-2 cell wall polypeptide changes in 174 cellulytic enzyme production 198 colonization of Eucalyptus diversicolor 234 disease suppression by 481 dolipore septum formation 385 effect on Quercus rubra seedlings 237, 238 effect on water balance 478, 479, 479 experimental manipulation of symbiosis with Eucalyptus lSO-2, 181, 181 extracellular fibrillar polymers on 184 glutamine synthetase in 258 glutamine transport 403 impact of penetration by hyphae of 190 inoculation programmes 471, 473-6 inoculum 472, 473, 474 mycelial growth in 210 nitrogen and 263, 266, 267, 268, 270, 272, 272, 273 phosphate metabolism in 146 phosphorus uptake 251, 282, 283, 284, 285, 286, 381, 383 polyphosphate in 390, 390, 391 potassium in 391 prevention of entry of celluflor 396 probing with hydrophobins 183 raised carbon dioxide and 487 root: shoot ratio 236 superstrain 472, 473, 474 tubular reticulum in 385, 386 use of alanine peptides 262 Pisonia 6, 165, 190 apoplastic impermeability in 396

Index

Pisonia (continued) wall ingrowths 196 Pisum 28, 51, 82, 405, 455 appressorium formation 92 appressorial colonization by Glomus mosseae 96 cellular reaction in roots during epidermal penetration 94-5 intraradical colonization in 94 mutants of abnormal mycorrhizal phenotypes 86, 87, 88-9, 111 PAM and PBM in 96 phosphorus uptake 128 plant proteins during appressorial colonization 96 resistance responses 100 stimulation of hyphal growth by exudates 92 Plantago 28, 31, 438, 440, 441 carbon transfer 124 Glomalean fungi forming VA mycorrhizas with 29 interplant nutrient transfer 155 mycelial links with Festuca 110-11 Pleurozium 424, 425 Podocarpus 23 Pollution, atmospheric, responses of mycorrhizas to 484-89 Polygonum 167, 412 Polyphosphate 287, 389-91 in mycorrhizal sheath 280-3 role in translocation 146-7 Polytrichum, rhizoids and rhizomorphs in 214 Poncirus 40 Populus 23, 169, 449 cortical epidermal Hartig net in 192 peri-epidermal Hartig net in 192 Poria 302 Potassium 153, 154, 403 Prosopis 442 Protein fungi 263 Protein uptake in ectomycorrhiza 262-7 Prunus 455 Pseudomonas 482 Pseudomycorrhiza 165, 290 Pseudotsuga 170, 171, 427, 429, 430, 430 association with Betula 269 Fusarium oxysporum 481 H. crassum 286 H. crustuliniforme 268

601 Laccaria laccata 475, 482 colonization by Rhizopogon vinicolor 219, 252 inoculum 474, 475 mycorrhizal mantle 200, 201 tuberculate mycorrhiza 203 Psilotum 49 Pteridium 424 metal uptake 154 Pterospora 301, 312, 313, 314, 315, 317, 318, 319 Pterostylis 374 Puccinia 318 Pyrola 6, 301, 305, 309 interfacial matrix in 308, 309, 310 intracellular hyphal development 308 mycorrhiza formation 306, 307 mycorrhizas in 304-8 senescence 311

Quercetin 41, 92 Quercetin-3-O-galactoside 92 Quercus 31, 321, 449, 427, 477, 478, 487 effect of Pisolithus tinctorius on growth 237, 238

r-strategists 219 Ranunculus 31, 410, 411 phosphorus uptake and 133 RAPD-PCR polymorphisms 65 Reseda 4 3 9 ^ 0 Resistance responses 99-101 Responsiveness to colonization 86, 118-21, 140, 439-44 Rhizanthella 349, 351-4, 372 association with. Melaleuca 374 Rhizobium 41, 86 signal molecules in 90 Rhizobium-leg^me symbiosis 184 Rhizoctonia 349, 350, 351-4, 363, 364, 366, 370, 374, 389, 411 association with Dactylorhiza purpurella 359, 360, 363, 370 carbon translocation in 388 hyphae 359 pelotons 359 Rhizomorphs 208-17, 287 Rhizophagus 12 Rhizopogon 165, 263, 268, 272, 282, 302, 312, 427, 478, 481

602

Index

Rhizopogon (continued) trehalose transfer 388 association with Seshania 459 fossil root of Pinus 164 Sheath 165, 396, 397 Picea 171 ericoid mycorrhizal 327, 327 Pinus contorta 270 polyphosphate in 280-3 Pinus sylvestris 240 see also Mantle Pseudotsuga menziesii 219, 252 Soil structure 461, 462, 465-8 branching rhizomorphs in 203 Solanum 455 inoculum 474 Sorghum 28, 69 phosphorus and 285, 286, 287 cobalt uptake 154 use of alanine peptides 262 Glomalean fungi forming VA mycorrhizas Rhododendron 308, 328, 329-30, 330, 331, 332, with 29 336, 341, 345 interplant nutrient transfer 155 association with O. maius 334 Spatheglottis, development of 355 colonized root 326 Sphaerosporella, nitrogen metabolism in 260 newly colonized cell 329 Spinacea penetration by H. ericae 329, 343 appressorium formation 92 Rhodothamnus 378 resistance responses 100 Root hydraulic resistance 156 Spiranthes 371, 372, 374 Root-balls 312, 314 Sporocarp 11, 24 Rumex 437, 438, 4 3 9 ^ 0 Stirlingia 419 Russula 218, 220, 222, 301 Stomatal conductance 156-7 mycorrhizal mantle 201, 202 Stylosanthes 455 Subtropical mycorrhizas 442-7 Successions, roles of mycorrhizal Salix 23, 169, 283, 284, 449 colonization in carbon allocation in 247, 250, 250 primary 446-7 mycelial growth in 210 secondary 447-50 phosphorus uptake 250 Sucrose efflux 400-2 Salsola, appressorium formation 92, 94 Suillus 165, 171, 178, 220, 222, 224, 223, 263, Salvinia 30 268, 269, 286, 287, 312, 424, 425, 480 Sarcodes 301, 312, 314, 315 association with fungal peg in 318, 320 Pinus contorta 273, 282, 283 mantle development in 316 Pinus sylvestris 240, 274, 274 Sarcodon 424, 426 bipolar mating types in 173 rhizomorphs in 215 carbon allocation 253 Schizophyllum 178 drought resistance 478, 480 hydrophobins in 183 endoglucanase activity 200 Sderocystis 14, 14 inoculum 474 Scleroderma 285, 480 Sclerogone 15 mycelium growth 225 Scutellospora 31 nitrogen and growth of 259 ancestral 21 pectinase activity in 200 DNA content 39 phosphorus transport in 387 genomic library 97-9 rhizomorphs in 209, 222, 215, 215 germination in 37 soil acidification 486 infection 64 somatic incompatibility in 179 spores 25 use of alanine peptides 262, 262 see also Gigaspora vacuole and tubule systems 385 Scytalidium 333, 334, 334 water transport in 384 Sebacina 351, 374 yield 270, 272 Serpula 226, 384 yield on nitrogen 260 rhizomorphs in 214 Symbiosis, definition 1

603

Index

Tagetes 69 Tamarillo 455 Taxus 46, 422 Temperate forest mycorrhizas 422-31 Temperate mycorrhiza 4 3 2 ^ 2 Temperature, effect on colonization 75-6 Tendril hyphae 214 Thanatephorus 351, 364, 365 Thelephora 170, 179, 220, 247, 249, 285, 302, 352, 374, 427, 472, 474 association with Pinus sylvestris, 174, 274: Salix viminalis 283 cumulative growth 213 disease suppression by 481, 484 mycelial growth in 210 rhizomorph growth in 209, 210, 287 Thelymitra 374 Thuja 429 Tilia, association with Cenococcum geophilum 478 Titanium 1 5 3 ^ Tomentella 222, 352, 374 Translocation 384-93 mechanism of 392-3 pathway and direction in mycorrhizal fungi 384-7 polyphosphate in mycorrhizal systems 389-91 rates of 387-9 solutes involved in 389 in VA mycorrhiza, mechanisms 139,158-9 Transpiration rates 156-8 Trehalose 38, 113-14, 365-7, 389 Tricharina 295 Tricholoma 165, 220 Trifolium 28, 40, 43, 69, 71,135,144, 327, 461, 462, 463, 464, 466 colonization 69 copper uptake 152 early root colonization 54r-5 effect of colonization on growth 107 light on colonization 79 phosphorus depletion 136 phosphorus on colonization 76, 77 extraradical mycelium development of 67 growth from root fragment 42 hydraulic resistance of root systems 157 lipid levels in 114 mineral nitrogen uptake 148 nitrate reductase activity 150

phosphorus requirements 143, 143, 145 uptake 128, 128 potassium uptake 153 primary colonization of 50 progress of colonization in 72-3 radioactive phosphate uptake 137, 138 rate of colonization 43 responsiveness to colonization 121 spatial distribution of colonization 70 transpirational flux 158 translocation from Glomus mossae 387 zinc uptake 152 Trillium 46 Triticum 41, 455 arbuscular cycle 61, 62 phosphorus uptake 141 responsiveness to colonization 121 zinc uptake 152 Tropical mycorrhizas 442-7 Truffles 475, 475 Tsuga 171, 268, 269 Tuber 208, 475-8 Tulasnella 351, 355, 370, 373, 374 Tylospora association with Picea sitchensis 247 fruit bodies in 220 life span 216

Ulocladium 272 Uncina 30

VA mycorrhiza asexual nature 17-18 cellular interactions in hosts and non-hosts 90-101 appressorium formation 92-3 changes in gene transcription during colonization 96-9 effects of colonization on resistance responses 99-101 intraradical colonization 94-6 penetration 93 preinfection growth and branching 90-2 spore germination 90 characteristic structures 11 classification 12-13 control steps in formation of 101-3 copper and zinc uptake 151-3 culture of 13, 34-5, 39

604 VA mycorrhiza (continued) discovery 12 genotypes and mutants with altered phenotypes 86-9 incidence 24-5, 25 interaction of Glomalean fungi with non-hosts 89-90, 4 3 9 ^ 3 interplant transfer of nutrients 123-5, 154-5 nitrogen nutrition 147-51 mycorrhizal effects on nodulation and nitrogen fixation 147-8 uptake of mineral nitrogen 148-51 origins 11 phenotypes modified by mutant and nonhost species 91 phenotypic stages in development 82, 83-4 phosphorus uptake see Phosphorus potassium uptake 1 5 3 ^ soil structure and 465-8 spatial distribution of H^-ATPases in 400-1 spores and sporocarps 13 in temperate ecosystems 432-42 toxic elements uptake 154-5 water relations 155-9 yields 438, 439 see also VA mycorrhizal fungi; VA mycorrhizal plants; VA mycorrhizas, root colonization VA mycorrhizal fungi 30 biomass 64 carbon use 108-14 estimated dates of origin and divergence of 22 Glomalean fossil history and phylogeny 2-3, 18-22 independent growth 103 phylogenetic trees 20 systematics 13-18 VA mycorrhizal plants 30 growth of 105-25 ecological groupings and specificity 28-32 fossil history of mycorrhizal colonization 23-6 molecular-genetic analysis 85 specificity and extent of colonization 26-8 systematics 22-3

Index

mycelial links between plants and carbon allocation 123-5 mycorrhizal responsiveness: costs and benefits of symbiosis 116-23 cost-benefit analysis 116-18 responses in natural ecosystems 122-3 variation in plant breeding progranmies 121-2 variations in extent of colonization and responsiveness 118-21 VA mycorrhizas, root colonization assay 34 distribution and rate of formation of infection units 70-1 external hyphae 64-8 hyphal length in soil 66 morphology and anatomy 46-64 Arum- and Paris-type mycorrhizas 46-9, 47-9 establishment of colonization 49-64 contact and penetration 51-2 development of infection units 52-4 precolonization events 49-51 turnover of arbuscules 54-64 percentage colonization of root system 71-9 influence of environmental factors 74-9 methods of assessment 71-2 progress of colonization in root systems 72-4 sources of inoculum 33-46 spores 35-42 DNA content 38-9 effects of plant exudates 40-1 energy reserves 38 germination and hyphal growth 37-42 hyphal networks 43-6 numbers of nuclei 38, 38 occurrence and infectivity 35-7, 36-7 production 68-70 root fragments 42-3 Vaccinium, 92, 321, 333, 334, 337, 338, 339, 340, 341, 343, 345, 413, 415 association of Hymenoscyphus ericae with 387 nitrogen uptake 338, 339, 340, 342 phosphorus use in 343, 344 Vanilla 349 Vesicle development 64 Vesicular-arbuscular mycorrhizas see VA mycorrhiza Vessel hyphae 215

Index

Vicia 28 abnonnal mycorrhizal phenotypes 86 Vigna 455 Vitis 455 Vulpia 436, 436, 437 phosphorus uptake and 134

Water relations drought resistance 156-9, 478-80, 484-6 transpiration 156-8 waterlogging and VA colonization 28 Waterlogging 28 Wet-sieving techruques 34, 35 Wilcoxina 2, 165, 291, 292-5, 294, 295, 295, 303 Wurmhea 46

Xerotus 352

605 Ypsilonidium 351

Zea 69, 455, 456 arbuscular cycle 62 copper uptake 152 Glomalean hingi forming VA mycorrhizas with 29 importance of inbred lines 121 mineral nitrogen uptake 149 phosphorus uptake 141 responsiveness to colonization 119 transpiration rates 158 yellow pigment 11 zinc uptake 152 Zinc 115, 126, 127, 403, 480 in ericoid mycorrhizas 345 uptake 151-3