Microorganisms in Plant Conservation and Biodiversity

MICROORGANISMS IN PLANT CONSERVATION AND BIODIVERSITY

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Microorganisms in Plant Conservation and Biodiversity Edited by

K. Sivasithamparam The University of Western Australia, Perth

K.W. Dixon Kings Park & Botanic Garden, Western Australia and The University of Western Australia, Perth and

R.L. Barrett Kings Park & Botanic Garden, Western Australia

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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0-306-48099-9 1-4020-0780-9

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v

CONTENTS

Contributors

vii

Foreword

ix

Preface

xi

Acknowledgements

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1. Plant conservation and biodiversity: the place of microorganisms

DR Given, KW Dixon, RL Barrett and K Sivasithamparam

1

2. Conservation of mycorrhizal fungal communities under elevated atmospheric and anthropogenic nitrogen deposition

LM Egerton-Warburton, EB Allen and MF Allen

19

3. Symbiotic nitrogen fixation between microorganisms

and higher plants of natural ecosystems

JS Pate

45

4. Bacterial associations with plants: beneficial, non N-fixing interactions

B Gerhardson and S Wright

79

5. Ectomycorrhizas in plant communities

MC Brundrett and JWG Cairney

105

6. Arbuscular mycorrhizas in plant communities

MC Brundrett and LK Abbott

151

7. Orchid conservation and mycorrhizal associations

AL Batty, KW Dixon, MC Brundrett and K Sivasithamparam

195

8. Ericoid mycorrhizas in plant communities

KW Dixon, K Sivasithamparam and DJ Read

227

9. The diversity of plant pathogens and conservation: bacteria and fungi sensu lato

DS Ingram

241

10. Ex situ conservation of microbial diversity

W Gams

269

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Contents

11. Impact of fungal pathogens in natural forest ecosystems: a focus on Eucalypts T Burgess and MJ Wingfield

285

12. Microbial contaminants in plant tissue culture propagation E Bunn and B Tan

307

13. Phytosanitary considerations in species recovery programs GEStJ Hardy and K Sivasithamparam

337

Index

369

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CONTRIBUTORS Lynette K. Abbott Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia. Edith B. Allen Department of Botany and Plant Sciences, The University of California, Riverside CA, 92521, USA.

Michael F. Allen Centre for Conservation Biology, The University of California, Riverside CA, 92521, USA.

Russell L. Barrett Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia.

Andrew J. Batty Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia; Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

Mark C. Brundrett CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural Research, Private Bag No 5, Wembley, 6913, Western Australia; Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia (current address).

Eric Bunn Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia.

Treena Burgess Forestry and Agriculture Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, RSA.

John W.G. Cairney Mycorrhiza Research Group, Centre for Horticulture and Plant Sciences, Parramatta Campus, University of Western Sydney, Locked Bag 1797, Penrith South DC, 1797, New South Wales, Australia.

Kingsley W. Dixon Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia; Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

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Contributors

Louise M. Egerton-Warburton Department of Botany and P l a n t Sciences, The U n i v e r s i t y of California, Riverside CA, 92521, USA; Chicago Botanic Garden, Lake Cook Rd, Glencoe IL 60022, USA (current address).

Walter Gams Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD, Utrecht, Netherlands.

Berndt Gerhardson Plant Pathology and Biocontrol Unit, P.O. Box 7035, S-750 07 Uppsala, Sweden.

David R. Given Isaac Centre for Nature Conservation, P.O. 84, Lincoln University, Canterbury 8150, New Zealand. Giles E. St J. Hardy School of Biological Sciences and Biotechnology, Murdoch University, Perth 6150, Western Australia.

David S. Ingram

St Catharine’s College, Cambridge University, Cambridge, CB2 1RL, UK.

John S. Pate Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, 6009, Western Australia. David J. Read Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK.

K. Sivasithamparam Soil Science and Plant N u t r i t i o n , Faculty of N a t u r a l and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

Beng Tan Department of Biology, Curtin University of Technology, Bentley 6102, Western Australia.

Michael J. Wingfield Forestry and Agriculture Biotechnology I n s t i t u t e (FABI), University of Pretoria, Pretoria, 0002, RSA.

Sandra Wright Plant Pathology and Biocontrol U n i t , P.O. Box 7035, S-750 07 Uppsala, Sweden.

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FOREWORD Plant conservation is increasingly recognised as an outstanding global priority, not only by scientists and committed conservationists but also by the global community and many governments. We know that tens of thousands of plant species throughout the world likely face extinction this century if current trends continue. The most comprehensive global list of endangered plant species produced to date was published by IUCN-The World Conservation Union in 1997 which documented almost 34,000 threatened plant species, and that work acknowledged that this is a considerable underestimation of the true figure of plant species threatened by extinction. Plant species loss is caused by many diverse factors, but primarily through the loss or damage of natural ecosystems and other impacts on wild plant populations and diversity caused by humankind, such as unsustainable collecting and uncontrolled invasions by alien species (plants, animals and microorganisms). Despite considerable efforts made over the last few decades to safeguard the world’s biodiversity in national parks, nature reserves and other forms of protected areas, we are today very much aware that despite our best efforts, the number of threatened species continues to rise. Innovative multi-disciplinary strategies in plant conservation are increasingly recognised as the best option for saving many species. We recognise that not only must we protect plants growing in the wild but also that we must seek recovery for an ever growing number of damaged plant populations and restore their habitats. Unless we gain a comprehensive insight into the factors that sustain these wild populations, our efforts to conserve diversity are ultimately likely to f a i l . The development of practical conservation and restoration methods, based on principles determined from the results of conservation biology research, is therefore an urgent priority. The practice of plant conservation has for too long been a rather hit-or-miss mixture of methods. Species recovery work involving cultivation, reintroduction and restoration has often had to be undertaken without adequate knowledge of the underlying causes of endangerment or of the factors required to successfully recover a threatened species. While we have recognised that microorganisms are often a crucial and essential element in supporting the life-cycles of plant species, we have generally had to hope that our effort undertaken at a macro level will be sufficient to facilitate ecosystem functioning at the micro level. Many of our successful efforts in plant conservation have probably been as a result of good fortune rather than good science. I greatly welcome therefore the preparation of this valuable book. With its focus on both the beneficial and detrimental importance of microorganisms (e.g. as mycorrhizas and pathogens), it provides an important review of the current state of knowledge on the importance and significance of microorganisms for plant conservation. I also hope that it will stimulate many more institutions to recognise that fundamental research on microbiology is an important element of plant conservation programs. The devastating impacts caused by the loss of biodiversity on our global environment and for the future of humanity can only be addressed if our future plant conservation efforts are based on understanding the complex interactions of biodiversity with its environment at all levels, rather than having to

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Foreword

rely on guesswork and good luck. This book should act as an extremely useful contribution to raising awareness of the importance of such aspects of plant conservation and provide an authoritative text to guide many plant conservation practitioners to the importance of microorganisms for successful plant conservation. Peter S. Wyse Jackson Secretary General Botanic Gardens Conservation International Richmond, Surrey, U.K. March 2002

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PREFACE If ‘all grass is flesh’ and the productivity of plant systems is underpinned by the activity of microorganisms, then much of human existence depends upon the present biological diversity of microorganisms. Even the foundations of the industrialised societies of today – fossil oil and iron (the major source of the metal is from banded iron formations which formed when oxygen produced by microorganisms some 3,000 to 2,500 million years ago precipitated oxides of iron (Schopf 1999)) depend upon the past activities of microorganisms. Microorganisms are therefore not only the origin of life itself some 3.5 billion years ago (Shen et al. 2001) but they support much of the biotic, industrial and social fabric of today’s world. As vital and integral components of the engine of existence, the diversity of microorganisms and the biological diversity they support are therefore fundamental in the debate to better manage the processes of conservation and threat abatement. This book addresses the role of microorganisms in conservation – both their support functions and deleterious roles in ecosystem function and species survival. Importantly, a number of contributing authors highlight how microbial diversity is, itself, now under threat from the many and pervasive influences of man. What is clear from this volume is that like many contemporary treatments of plant and animal conservation, the solution to mitigate the erosion of biodiversity is not simple, made all the more complex by the lack of reliable taxonomic information, particularly for the predicted immense diversity of microorganisms. The impacts of human activity touch all parts of the biosphere as highlighted by Egerton-Warburton et al. (this volume) and only now are some of the more advanced economies of the world coming to grips with the scale and inertia of the problem. The fate of an estimated two thirds of the plant species on earth now hangs in the balance (Anon. 2000). As man forges ahead monopolising biodiversity to a mere 100 plant species which represent the major human food and fibre species, another estimated 250,000 other plant species are in peril (Heywood & Watson 1995). With microbial diversity conservatively estimated in the millions of species, the impact of the loss of e q u i l i b r i u m of microbial diversity is daunting and potentially irreversible. Take for example the ‘knock-on’ effects on plant production systems if there is a careless disregard for maintenance of a diverse, healthy and functional microflora. Many agricultural systems do just this and to remain productive, require unsustainably high inputs of energy and chemicals. These inputs themselves further perpetuate the artificiality of the system, ultimately leading to a process of agricultural productivity devoid of natural inputs – essentially broad-scale hydroponics! Egerton-Warburton et al. (this volume) cite 90% of plant species as having some form of symbiotic association with fungi. How surprising is it then that other than a few esoteric examples in forestry, much of our broad-scale agricultural systems pay little or no attention to the role of helper fungi in maintaining soil and plant health? The pivotal role of some microorganisms in maintenance of biodiversity is classically seen in the multifaceted benefits of ectomycorrhizas (Brundrett and Cairney this volume) in supporting a host of other organisms from bacteria and protists to invertebrates and vertebrates – including the elusive hypogeous fruit

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bodies of truffles. If only similar prologues of the level of interaction of more microorganisms could be drafted before the loss of biodiversity eliminates taxa to the point where reconstruction of the intricate and elegant processes of ecological equilibrium is impossible. This book represents an attempt to bring to the fore the ecological underwriting provided by microorganisms. Let us hope that many more volumes will ensue as the value of microorganisms in conservation is recognised as part of the global conservation process.

References Anonymous (2000) ‘Gran Canaria declaration.’ (Botanic Gardens Conservation International: Kew) Brundrett MC, Cairney J (2002) Ectomycorrhizas in plant communities. In ‘Microorganisms in plant conservation and biodiversity’. (Eds K Sivasithamparam, KW Dixon and RL Barrett) pp. 105–150. (Kluwer Academic Publishers: Dordrecht) Egerton-Warburton LM, Allen EB, Allen MF (2002) Mycorrhizal fungal communities under elevated atmospheric and anthropogenic nitrogen deposition. In ‘Microorganisms in plant conservation and biodiversity’. (Eds K Sivasithamparam, KW Dixon and RL Barrett) pp. 19–43. (Kluwer Academic Publishers: Dordrecht) Heywood VH, Watson RT (1995) (eds) ‘Global biodiversity assessment.’ (Cambridge University Press: Cambridge) Shen Y, Buick R, Canfield DE (2001) Isotopic evidence for microbial sulphate reduction in the early Archaean era. Nature 410, 77–81. Schopf JW (1999) ‘Cradle of life. The discovery of earth’s earliest fossils.’ (Princeton University Press: Princeton)

The editors Perth, Western Australia March 2002

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ACKNOWLEDGEMENTS

The editors would like to express their gratitude for the work of all the contributors to this volume. We would like to especially thank those who kindly agreed to review chapters: Craig Atkins. Mark Brundrett, John Cairney, Brett Gaskell, Janet Gorst, Roger Finlay, Stephen Hopper, Maarten Ryder and Sally Smith. We would also like to thank our colleagues at Kings Park & Botanic Garden and The University of Western Australia for their assistance with this project.

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K.Sivasithamparam, K.W.Dixon & R.L.Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 1–18. © Kluwer Academic Publishers.

Chapter 1 PLANT CONSERVATION AND BIODIVERSITY: THE PLACE OF MICROORGANISMS

David R. Given Isaac Centre for Nature Conservation, P.O. 84, Lincoln University, Canterbury 8150, New Zealand.

Kingsley W. Dixon Kings Park & Botanic Garden, Botanic Gardens & Parks Authority, West Perth 6005, Western Australia; Plant Biology, Faculty of Natural & Agricultural Science, The University of Western Australia, Crawley 6009, Western Australia.

Russell L. Barrett K i n g s Park & Botanic Garden, Botanic Gardens & Parks Authority, West Perth 6005, Western Australia.

K. Sivasithamparam Soil Science and Plant N u t r i t i o n , Faculty of Natural & Agricultural Science, The University of Western Australia, Crawley 6009, Western Australia.

1. Introduction The total number of organisms that make up the array of living species that characterise the th in ecosphere on Earth still remains unaccounted. Estimates range from eleven million species (of which 1.5 million have been described) to over a billion (Table 1). Only now is the scientific community beginning to understand the monumental task of cataloguing life on earth. What this exercise is showing is that it is the unseen and forgotten world of invertebrates, f u n g i , non-flowering plants, marine organisms and

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Microorganisms in Plant Conservation and Biodiversity

microorganisms that makes up the vast bulk of the unaccounted species. In any part of the world, the numbers of fungal species are higher than those of green plants (Gams, this volume). Indeed, much of the essential primary production (oceanic algae) and organic recycling to sustain life is a result of microorganisms i.e. life begins and ends with microorganisms. Fungal diversity appears less localised than that of green plants, but much still remains to be explored, particularly in tropical regions, and in a great diversity of ecological niches (Hawksworth 2001). Fungi play many decisive roles in the health and well-being of all ecosystems (Ingram, this volume).

Diversity of bacteria and archaea has recently been recognised as being potentially far greater than that of all Eukaryotes and represents the greatest challenge for scientists documenting the diversity of life (Figure 1; Barns et al. 1996; Whitman et al. 1998; Patterson 1999; Cohan 2001; DeLong and Pace 2001; Dunlap 2001). The task of refining the estimates, by the process of cataloguing biodiversity, will take a great deal of time and expense. Pimm et al. (2001) have estimated that $25 billion annually is required for the protection and management of global biodiversity hotspots identified by Myers et al. (2000) with James et al. (2001) suggesting a similar figure of $21.5 billion, rising to $317 billion for global biodiversity conservation. The All Species Foundation estimate that the description of the remaining biodiversity on earth w i l l take over 650 years at current rates (http://www.all-species.com), by which time a great deal of our biological diversity may have been lost (Myers and Knoll 2001). Microorganisms e x h i b i t a great range of adaptation to extreme environments, from deep ocean trenches, to high temperature and pressure deep w i t h i n the earth, to thermal springs, to ice sheets and exhibit a range of

Plant conservation and biodiversity

3

metabolic processes to survive in this environment (Horikoshi and Grant 1998; Nealson and Conrad 1999). Plant surfaces, from root-tip to shoot-tip, provide a myriad of habitats for microorganisms, many of which sustain healthy growth, while others are pathogenic (Andrews and Harris 2000). In addition to plant surfaces, another suit of microorganisms live within the plant (as endophytes) with a range of antagonistic to mutualistic roles within the host plant (Saikkonen et al. 1998). These mutualistic associations can lead to sometimes startling outcomes such as the spectacular flowering in the holomycotrophic underground orchid (Batty et al., this volume). In a world of explosive scientific discovery and rapidly changing definitions, it is necessary to begin with an explanation of the focus of this book on microorganisms and their role in plant conservation and biodiversity. An extensive review of microbial diversity and ecosystem function is given by Allsopp et al. (1995). Also, since completion of manuscripts for this book, a further volume on fungal conservation has been published (Moore et al. 2001) which is recommended reading as a companion to this volume. This chapter provides an overview of aspects of microbial function affecting plant biodiversity and conservation, i.e. the role of microorganisms in the survival of the earth’s biota. Microorganisms shape plant biodiversity and conservation by either suppressing or enhancing plant development and establishment as expanded in the chapters of this volume. Defining microorganisms is a continually evolving process. As our understanding of microbial ecology and phylogeny grows, so our taxonomic and functional group concepts continue to change. Figure 1 illustrates what are now recognised as three Domains, with the requirement to completely re­ think our concept of Kingdoms, which, if retained in the traditional sense, must increase in number to reflect the genetic diversity now evident though analyses such as rDNA sequencing (Barns et al. 1996; Nealson and Conrad 1999; DeLong and Pace 2001; Stackebrandt 2001). Colwell et al. (1995) and Zavarzin (1995) discuss the issues surrounding the concept and definition of the term ‘microorganisms.’ Bacteria, as a Domain, represent a distinct lineage of microorganisms. Within bacteria, further lineages such as Actinobacteria, Cyanobacteria and Proteobacteria are recognised (more readily from sequence data than physiological characteristics), however definition of bacterial ‘species’, generally accepted as “a collection of strains showing a high degree of overall similarity, compared to other, related groups of stains” (Colwell et al. 1995) is relatively vague. A bacterial ‘species’ may refer to a ‘taxospecies’ (a phenetic cluster), a ‘genospecies’ (a group capable of genetic exchange) or a ‘nomenspecies’ (a group given a binomial name) (Sneath and Sokal 1973; Sneath 1984; Colwell et al. 1995). As evident in figure 1, “the

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Microorganisms in Plant Conservation and Biodiversity

concept of ‘microorganisms’ is not essentially phylogenetic” (Zavarzin 1995), encompassing bacteria, archaea and much of the eucarya. As a term in broad usage (in both application and definition), the concept of ‘microorganisms’ is entrenched in our thinking, and indeed remains a useful term when making general statements. Zavarzin (1995) concludes with a generalistic definition: “Microorganisms are living beings invisible to the naked eye (except when developing in large masses). Most microorganisms are osmotrophic, few are holozoic. Diffusion is the main limitation to

Plant conservation and biodiversity

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dimensions and thus the most essential feature.” This definition captures the essence of microorganisms as those organisms too small to see, and thus usually overlooked or ignored completely.

2. Conservation hypotheses Microorganisms are basal to the very foundations of life on earth. The first evidence of life from 3.5 Byr in the archaean rocks from north western Australia are represented by cyanobacteria (Shen et al. 2001). From these beginnings, microorganisms have focussed the evolution of all life. The integration of microbial components provided the very being for the complex eukaryotes we know today. The chloroplasts and mitochondria which represent both the energy capturing and energy production system of all eukaryotes is a result of microbial integration on a sophisticated and unparalleled scale (Martin et al. 1998; Gray et al. 1999; McFadden 1999). Secondary encounters with microorganisms has resulted in symbioses which may have paved the way for the movement of plants onto land including the role of mycorrhizal agents as defacto roots in early land plants such as Rhynia (Bateman et al. 1998; Brundrett 2002). Symbioses continue to play a role in the evolutionary success of plants in particular as outlined in the chapters of this volume. For example, the Ericales clade is a remarkably ancient and monophyletic group (APG 1998). Since all modern ericads investigated possess ericoid mycorrhiza it is tempting to speculate that this symbiosis provided the necessary resilience for this order to colonise the four corners of the globe and comprise a remarkable level of diversity (Dixon et al., this volume). As pathogens, microorganisms could, conceivably, have provided important drivers for the evolution of adaptive traits and speciation events (Ingram, this volume). 3. Biodiversity conservation The global catalogue of life is also starting to reveal the extent of the extinction crisis for biological diversity. A tragedy being played out against the backdrop of current changes to nature is that future generations may not see certain life forms and landscapes that today we take for granted. “As many as two-thirds of the world’s plant species are in danger of extinction in nature during the course of the 21st century, threatened by population growth, deforestation, habitat loss, destructive development, over consumption of resources, the spread of alien invasive species and agricultural expansion. ” (Anon. 2000). Catastrophic or localised extinctions can occur without human intervention, but in many parts of the world and for vulnerable ecosystems generally, humans are primarily responsible for current extirpations of species and ecosystems. May et al. (1995) have estimated the mean

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Microorganisms in Plant Conservation and Biodiversity

background rate of e x t i n c t i o n in the geological record as about one species per year. The same authors, using three independent analyses based on different scientific approaches, concluded that impending extinction rates are at least four orders-of-magnitude faster than the background rates seen in the fossil record. This figure does not take into account the observation by Hawksworth (1998) that the extinction of an obvious, large organism such as a forest tree probably results in the loss of at least fifteen organisms dependent on or confined to that single species. Do i n d i v i d u a l microorganisms become endangered or extinct in their natural e n v i r o n m e n t s ? There is l i t t l e or no evidence to indicate that disturbance, large or small, could lead to the extinction of specific strains of a microorganism, although several reports exist of numbers of selected groups of culturable soil microorganisms dropping to non-detectable levels (Alexander 1971). From an ecological point of view it is possible to argue that compositional changes may not be very critical if activities of functional groups of organisms are maintained, even at the expense of rare taxa. Raven (quoted in Josephson 2000) cites three principle factors that accelerate the rate of e x t i n c t i o n of p l a n t species: habitat destruction, ecosystem fragmentation and i n v a s i o n of wild habitats by exotic species. There is, moreover, a finality to extinction and the best way to avoid it in the words of Raven is to “save real estate. If a rain forest is destroyed, 19 or 20 species there will remain unknown to science”. A second-best strategy is to reconstruct ecological communities, but few if any examples exist of the success of this process. Species can be restored to a system, but it is much harder and sometimes impossible to restore processes, interspecies relationships and the pre-disturbance microflora. A third-best strategy is to preserve species as germplasm. As Raven concludes, “we would rather have germplasm than not have the species at all.” The spectrum of biological diversity ranges from genetic to ecosystem and biome diversity. The task of conserving as much of the biosphere of Earth as possible, is made more d i f f i c u l t by our poor knowledge of genetic variation w i t h i n species and the significance of these variations on the survival of the threatened taxa. Erosion of biological diversity is occurring throughout the whole spectrum of diversity from small to large, and from the microscopic to landscape level. According to Myers and Knoll (2001) “We have o n l y a r u d i m e n t a r y u n d e r s t a n d i n g of how we are altering the evolutionary future. As a result of our ignorance, conservation policies fail to reflect long-term evolutionary aspects of biodiversity loss”. Should b i o d i v e r s i t y be determined o n l y by classical taxonomy? Literature is m o u n t i n g on the variation within species. Although the recognition of intraspecific differences in the pathogenicity of microbial pathogens of plants and a n i m a l s ( i n c l u d i n g humans) have lead to the creation

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of subspecific taxonomic separations based on their chemistry, such separations of non-pathogenic microorganisms (which greatly outnumber parasitic forms) is not widely accepted. The role of infra-specific variation, particularly in symbiotic relationships, is now being more clearly understood, particularly in orchids (Batty et al., this volume) and ericads (Dixon et al., this volume). Myers (1988) identified ten globally important centres of diversity (mostly areas of tropical moist forest) and later a further eight (mostly mediterranean-climate regions). This work has been followed up by more rigorous research, and the recent publication of a monumental analysis (Myers et al. 2000) identifying 25 ‘hot spots’ – those places on earth where there are concentrations of species with numerous endemics and where there are often considerable threats to biodiversity. Resources cannot be made available everywhere for all species, genes and ecosystems. One suggested approach to prioritisation is to first of all consider the conservation priorities for biological ‘hot spots.’ We cannot escape the fact that these are the places on Earth where there is greatest responsibility for stewardship, protected area systems, environmental education, community conservation initiatives, and sustainable resource use. Recent reassessment of the ‘hot spot’ concept shows that the whole of New Zealand and parts of Australia and South Africa are among the most important focal points on Earth for animals and plants, each with significant threats to biodiversity conservation (Burbidge 1994; Cowling et al. 1996; Craig et al. 2000). Myers et al. (2000) and most other systems for analysis of biological diversity pay scant regard for the collateral diversity of microorganisms that coexist, support, rely upon or are pathogens of plants. Whether global hotspots of vascular plant diversity adequately represent the diversity hotspots for microorganisms remains a virtually unknown yet vital area for research. Wholly unrelated microorganisms may play a critical role in the establishment and survival of native flora. These include mycorrhizal forms (see Egerton-Warburton et al.; Brundrett and Cairney; Brundrett and Abbott; Batty et al. and Dixon et al., this volume), rhizobia (Pate, this volume) and beneficial bacteria that enhance plant growth or suppress parasitic activities of pathogens through their antagonistic activities (Gerhardson and Wright, this volume). This diversity allows the evolution of ecosystems and/or of colonisation of e n v i r o n m e n t s which would otherwise restrict the establishment of the plant taxa that now occupy them (see Cairney 2000).

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Microorganisms in Plant Conservation and Biodiversity

4. Microorganism conservation Human activities have undoubtedly had highly significant impacts on the diversity and efficacy of microorganisms, although the effects are frequently quite subtle and cryptic. Such changes may be manifest as minor fluctuations in species composition through to more extreme shifts that encompass the loss of changes in species dominance and even loss of taxa (e.g. from natural woodland to p l a n t a t i o n forestry as in the case of mycorrhizal taxa). A l t h o u g h s i m i l a r shifts in plant communities are acknowledged as being critical in understanding ecosystem processes the influence of comparable shifts in mycobiont diversity on the mycorrhizal and, in turn, the plant c o m m u n i t y has yet to be fully appreciated (EgertonWarburton et al., this volume). How can we conserve these organisms? The first step is to study the biology, including survival mechanisms in nature, of microorganisms. Our greatest knowledge is generally of the fungi, bacteria and microorganisms which have a significant impact on human life. These are not only the useful microorganisms, but also include such rogues as the nasty “killer bugs”, of which Ebola virus is one of the most notorious. Much research is underway to eradicate such v i r u l e n t pathogens. Although it is desirable to totally eradicate them, it is also important to preserve (of course under strictest of care) live cultures of the pathogens for future research to combat re­ emergence of the same or the evolution of similar parasites (Babiuk et al. 2000). This situation indeed arose recently (Bryan 1999; Ertem et al. 2000) when the World Health Organisation was faced with the dilemma of destroying the last known laboratory cultures of the small-pox virus. Understanding the biology of microorganisms is a challenge because we know less than 5% of the taxonomic diversity and knowledge of their role in ecosystems, whether b e n e f i c i a l or not, is generally rudimentary. In this situation, how do we know what benefits accorded to microorganisms are being lost from ecosystems? Several niches rich in microflora remain under-explored, the most exciting of which are marine environments such as deep-sea troughs (Levin et al. 2001) and deep subsurface environments (Haldeman et al. 1994; Chandler et al. 1998). Recent interest in marine microflora, especially those associated with sponges have resulted not only in the description of new taxa, but the discovery of a whole host of organic metabolites new to science and valuable for pharmaceutical and extractive industries (Grassle 2001; Fenchel 2001; Watson et al. 2001). Ecosystem m a i n t e n a n c e depends h e a v i l y on the f u n c t i o n of microorganisms. Microorganisms play a decisive role in nutrient cycling (McKenzie 2000; de Boer and Kowalchuk 2001; Jobbagy and Jackson 2001), decomposition (HyeongTae 2000; Monreal and Bergstrom 2000;

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Fenchel 2001), mineralisation (Puri and Ashman 1998; Saetre et al. 1999; Fenchel 2001), n u t r i e n t and mineral accession by plants and animals (Marschner 1995; Geeta Singh and Tilak 1998; van Vuuren et al. 1999). Egerton-Warburton et al. (this volume) point out that mycorrhizal fungi are a functional group of organisms that form symbiotic associations with over 90 % of plant species and in most biomes. This indicates that nine out of ten plants we see around us have and use mycorrhizas, not ‘just roots.’ Such associations have been linked to the enhanced growth, survival, drought tolerance, pathogen resistance and nutrient status of the host plant. In return, the mycobiont gains a receptive host and an energy source. By directly utilising C acquired by plants, mycorrhizal fungi process from 10 to 85% of the net primary productivity. Hyphal networks, especially those in roots colonised by two or more fungal links, may provide pathways for the movement of P and N among plants, and C-sharing among fungi. Therefore, mycorrhizas may influence the structure, diversity and productivity of plant communities, and their conservation is critical for maintaining ecosystem stability and function. Gerhardson and Wright (this volume) propose that during evolution, these close contacts between plants and the microorganisms infecting or invariably s u r r o u n d i n g them have developed into various dependencies on both sides. These in turn have in many cases led to specific biological interactions, or symbioses, presumably resulting from a long co-evolutionary process, and in other cases to more or less loose, or even chance associations. We now find these dependencies as host-pathogen interactions, which may be biotrophic or necrotrophic, clear symbiotic interactions (e.g. with certain N-fixing microbes, and the mycorrhiza) and as a variety of looser, probably facultative associations. 5. Ecosystem dynamics Conservation of plants ultimately requires conservation of their associated microorganisms, a task poorly addressed and subject to a far greater degree of complexity and subtlety than most plant conservation initiatives consider. Brundrett (2002) has suggested that plant roots may have evolved as habitats for mycorrhizal fungi, and in any case, there is evidence to suggest that mycorrhiza were a key factor in the colonisation of land by plants (Bateman et al. 1998; Selosse and LeTacon 1998). The interactions between microorganisms and plants are complex, including those related to scale. Whereas an area of high plant diversity may have up to 63 species in a 1 X 1 m quadrant (Kull and Zobel 1991), Torsvik et al. (1990) found that up to 5,000 species of bacteria may occur in a single gram of soil, and Dykhuizen (1998) estimated that a 30 g sample of forest soil may contain over half a million species of microorganisms. Generally

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only 0.1 to 1% of these microorganisms are culturable using current methods and our knowledge of this diversity is reliant on various DNA analyses (Torsvik et al. 1998). If we struggle to understand the dynamics responsible for such plant diversity, how much less do we understand bacterial diversity and population dynamics (which probably exert great influence on dynamics of plant growth). While it is necessary to consider both species diversity and ecosystem function in conservation programs, these two components are not directly related, as functional groups generally consist of multiple taxa of varying abundance (Bengtsson 1998). Changes in species diversity can impact the functional diversity of an ecosystem in that significant changes in abundance or removal of all taxa in a specific functional group can have strong effects on the ecosystem as a whole (Chapin et al. 1997). Species rich heathlands of mediterranean climates, often with over 100 species in a 100 m x 100 m quadrat, depend utterly on the nutrient impoverishment of the site, i.e. add nutrients and the system begins to decline (Egerton-Warburton et al., this volume). Many plant communities are highly dependant on mycorrhizal associations (Hawksworth 1991). Disturbance events such as mining can remove all mycorrhiza from the soil and restoration efforts can be hampered unless sites are inoculated with mycorrhizas, as ericoid mycorrhizas can take up to 12 years to re-colonise, with lesser time for other mycorrhiza types (Hutton et al. 1997; Dixon et al., this volume). The role of microorganisms in the maintenance of the ‘integrity’ of nature has essentially been underestimated as it affects all existing biotic and abiotic components of earth’s environments. For instance, morphology and growth of plant roots appear not to be ‘normal’ when the plants are grown in the absence of microorganisms naturally resident on or around roots (Rovira 1972). Thus all large life forms have evolved with microorganisms and therefore are naturally dependent on them and/or are affected by them. An example of the under-appreciation of microorganism activity is provided by agrarian systems which, as diversity of the crop species is reduced, require increasing reliance on inorganic inputs of nutrients along with increasing amounts of external energy. Feedback results in decline in the diversity of microorganisms w i t h enhancement of the reliance of artificial inputs. This can result in increased disease loading in cropping systems – but increase the overall diversity of species and the organically based nutrient loading, and the ability of non-desirable organisms to dominate is naturally decreased.

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6. The role of pathogens in shaping plant diversity Biodiversity in regional hot spots can often be threatened by natural disasters such as diseases and pests (see Burgess and Wingfield, this volume). Containment of diseases in their environments can be difficult and a bigger challenge is to rehabilitate affected areas (see Hardy and Sivasithamparam, this volume). The acceleration of disease impacts in natural ecosystems is highlighted in the case of the onslaught of Phytophthora spp. in the floristically diverse south-western Australia. Here, introduced pathogens have selectively removed key understorey and overstorey elements on a scale not recorded in any other natural biodiverse ecosystems (Wills 1993). The resulting melt-down in in situ conservation measures has resulted in a range of emergency measures being put in place. Plant pathogens may have played a pivotal role in shaping current plant diversity and ecosystem dynamics (see Ingram, this volume). Growing awareness of plant pathogen effects and observations of changes they cause has provided numerous modern examples of this. Phellinus weirii, a native plant pathogen in western North America has been identified as a key determinant of forest structure and processes (Hansen and Goheen 2000). The effects of Ceratocystis fagacearum on community structure of oak woodlands in Texas and Wisconsin has been monitored, particularly in relation to an epidemic in central Texas which caused mass tree death and subsequent changes in community composition (Collada and Haney 1998; McDonald et al. 1998). Dutch elm disease (caused by Ophiostoma ulmi, O. novo-ulmi and O. himal-ulmi) has a varying effect on trees of the genus Ulmus, in some cases having minimal effect (see Siebrecht 2000), while in others, decimating populations (Hubbes 1999). In ecosystems previously dominated by species susceptible to such devastating pathogens, this can result in the replacement of species communities by less competitive species resistant to the pathogen (Hansen and Goheen 2000). Ristaino and Gumpertz (2000) examine the spatial dynamics of Phytophthora epidemics which can be responsible for significant changes in ecosystem structure and diversity, largely due to the broad host range and multiple distribution mechanisms of propagules. It is highly likely that indigenous pathogens have been key determinants of plant community structure throughout history, and that this should be seen as a natural process. Greater concern has rightly been raised over this potential for change of community structure and composition where the pathogen has been introduced. This is further compounded by the potential for interspecific hybridisation to result in the rapid evolution of introduced pathogenic species resulting in the development of new strains or species of pathogens (Brasier 2001).

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7. Disturbance, conservation and land management Disturbance events can be as obvious as land clearing, or as subtle as pollution of water from upstream flow. In a microcosm experiment investigating the effects of disturbance (freezing and heating) on microbial biomass, and specifically the role of oribatid mites in facilitating recovery of the microbial populations, significant benefits of the mites were evident, including dispersal of fungal spores and stimulation of microbial metabolism (Maraun et al. 1998). Agricultural ecosystems often represent some of the most modified systems where plants are grown with initial cultivation having a significant impact on microbial communities (Calderon et al. 2000). Even in such modified environments, experiments comparing organic and conventional fanning systems consistently show significant benefits of increased organiccontent in the soil (i.e. higher diversity of microorganisms and increase activity) (Fliessbach et al. 2000; Kushwaha et al. 2000). Comparison of tilled fields of varying age w i t h undisturbed native sites in Colorado found decreasing fungal a c t i v i t y with length of time since disturbance was initiated ( K l e i n et al. 1996). Microbial diversity in such environments changes with variations in agricultural practices and is therefore constantly in a state of flux. 8. Social and scientific perceptions of microorganisms Microorganisms permeate all aspects of human life – from the bread we eat to antibiotics. Microorganisms are actively utilised in the mining industry for processes such as acid mine leaching to dissolve ores using acids produced by bacterial processes (Muller et al. 1995; Groudev et al. 1996; Krebs et al. 1997) as well as detoxification of hazardous wastes in soils (McGrath et al. 1998; Fein et al. 1999). A quick scan of relevant journals shows that pharmaceutical and industrial research into the roles and benefits of microorganisms dominates all other microbial research areas. Recent pharmaceutical advances include the potential use of polyhydroxylated alkaloids as anticancer, antidiabetic and antiviral agents (Watson et al. 2001). Industrial advances i n c l u d e biopolymer production by microorganisms (Daniell and Guda 1997) and ethanol production by the bacterium Zymomonas mobils (Gunasekaran and Raj (1999). 9. Conclusions Bacteria and viruses are considered to have constituted the first life-forms to evolve on planet Earth (DeLong and Pace 2001). They were not only the precursors of more complex life forms, but those more complex life forms (including our own species) have evolved from them (figure 1; Bateman et al. 1998). Their fossil remains provide our iron ore and much of our oil, gas

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and breathable atmosphere. Depleting our planet of the rich, cryptic and elusive diversity of microorganisms is therefore at our own peril as we mislay and even deliberately destroy this planet’s unique library of evolution (Myers and Knoll 2001). Is it possible to introduce cultured microorganisms into native or exotic environments? Such introductions into soil environments could be extremely difficult in microbiologically buffered habitats, especially for the establishment of microorganisms that have been cultured in nutrient rich (and unnatural) laboratory media ( B u n n and Tan this volume). Much of the nutrient substrates (e.g. glucose) on which several generations of those microorganisms have been raised in the laboratory are likely to be limiting or absent in the natural e n v i r o n m e n t s . In addition, it is also likely that several enzymes and metabolic pathways required for nutrition in nutrientimpoverished natural environments can be shut-down in laboratory cultures. This d i f f i c u l t y is further complicated by the limitations of labour and methodology involved in tracing the introduced organism and in maintaining the extraordinary complexity of genetic variation found in single species in nature. Plants and microorganisms have evolved together and have in many instances developed a certain level of mutual dependency on each other. Their r e l a t i o n s h i p s range from obligate mycorrhizal associations to opportunistic interactions that may hinder or favour plant growth and establishment. Conservation of either of these partners can therefore be possible only in the presence of each other. This is clearly evident for the associations described in this volume.

Acknowledgements We would like to thank Steve Hopper for comments on the manuscript.

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K.Sivasithamparam, K.W.Dixon & R.L.Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity, pp. 19–43. © Kluwer Academic Publishers.

Chapter 2 CONSERVATION OF MYCORRHIZAL FUNGAL COMMUNITIES UNDER ELEVATED ATMOSPHERIC AND ANTHROPOGENIC NITROGEN DEPOSITION Louise M. Egerton-Warburton Chicago Botanic Garden, Lake Cook Rd, Glencoe IL 60022, USA; Department of Botany and Plant Sciences, The University of California, Riverside CA, 92521, USA.

Edith B. Allen Department of Botany and Plant Sciences, The University of California, Riverside CA, 92521, USA.

Michael F. Allen Centre for Conservation Biology, The University of California, Riverside CA, 92521, USA.

1. Introduction Over the last century, the combination of rapid population growth, consumption of fossil fuels and industrial expansion has resulted in a steady increase in the quantities of pollutants discharged into the atmosphere on a daily basis. During the early 20th century, sulphur dioxide emissions formed the bulk of the pollutants. However, the latter half of the 20th century has seen a shift in the fundamental nature, as well as quantity, of the air pollutants. The most ubiquitous air pollutants are currently carbon dioxide particulate matter, and nitrogenous emissions and their subsequent photochemical oxidants such as ozone. Despite the introduction of legislation and management strategies, monitoring has shown that emissions of and nitrogenous pollutants regularly attain atmospheric levels that exceed acceptable thresholds for damage to the biota. In this review, we address the responses of anthropogenic arbuscular mycorrhizal communities to elevated x N interactions, and the predicted nitrogen (N) eutrophication or global climate change that is correlated with increasing atmospheric

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concentrations of trace gases. Since arbuscular mycorrhiza are a common and widespread phenomenon, the impacts of altered atmospheric components on these organisms is indicative of the wider implications of global pollutants on many other micro-symbiotic associations. Increasing atmospheric concentrations of and nitrogenous emissions are the major determinants of global change (Bazzaz 1990; Galloway et al. 1995; Houghton et al. 1996; Norby 1998; IPCC 2000). Consumption of fossil fuels and deforestation has resulted in an imbalance in the global carbon (C) cycle by increasing the atmospheric loading of In turn, altered C cycling is accompanied by physiological modifications in plants, changes in the rates of C cycling and N transformation in terrestrial ecosystems, and allied alterations in Cdependent systems, such as soil microbes (Zak et al. 1993). Global have risen from 280 ppm in the pre­ atmospheric concentrations of industrial era to ambient levels of almost 370 ppm in the year 2000 (IPCC 2001). Current models predict that atmospheric loads will average 700 ppm by the end of the century (IPCC 1994, 2001; Bazzaz 1990; Indermuhle et al. 1999). Anthropogenic sources also dominate the global N budget. Over 80 percent of oxides of nitrogen and 70% of ammonia emissions worldwide are generated by human activities (Vitousek et al. 1997; Matson et al. 1999). Industrial fixation of N for use as a synthetic fertilizer currently contributes up to 80 Tg of new N into the global cycle each year. Additional new anthropogenic inputs into the N cycles can be traced directly to nitrogenous emissions derived from the combustion of fossil fuels by vehicles and industry (> 20 Tg NO-N per annum; Dignon and the burning of Hameed 1989), domestic animal wastes (32 Tg forests (15 Tg NO- and and nitric oxide emissions from soils (5­ 20 Tg; Vitousek et al. 1997). Such increases in the emissions of airborne N have resulted in enhanced deposition of anthropogenic N into terrestrial ecosystems. Increasing the N input modifies the N cycle both directly, by increasing soil N pool, and indirectly, by altering C and and N deposition phosphorous (P) cycling (Zak et al. 1993). Both can bring about change in terrestrial ecosystems. Because anthropogenic and N are inextricably-linked at the source and the gases release of relate mutually with one another in their reactive forms, the (im)balances of C and N fluxes on the biota need to be considered in concert (Norby 1998). Almost invariably, global change has been evaluated by source-sink analyses of C allocation in plants (Bazzaz 1990; Wedin and Tilman 1996). The contribution of the below-ground biota, especially mycorrhizal fungi, as a feedback mechanism has been largely ignored (Zak et al. 1993; Berntson and Bazzaz 1996; Hu et al. 1999, 2001). Mycorrhizas, plant-fungus mutualisms, are critical for understanding ecosystem dynamics in changing environments. Plant and mycorrhizal growth are tightly coupled due to the reciprocal nature of their C and N cycles (Zak et al. 1993), and thus the symbiosis may impact the rate at which C and N are cycled within terrestrial ecosystems (Allen 1991).

Conservation of mycorrhizas under global change

21

Mycorrhizal fungi are a functional group of organisms that are estimated to form symbiotic associations with over 90 % of plant species and in most biomes (Smith and Read 1997). Such associations have been linked to the enhanced growth, survival, drought tolerance, pathogen resistance and nutrient status of the host plant. In return, the mycobiont gains C (Smith and Read 1997, and references therein). By directly utilizing C acquired by plants, mycorrhizal fungi are responsible for processes that account for 10-85% of the net primary productivity (Vogt el al. 1991; Allen 1991). Hyphal networks (sensu Robinson and Fitter 1999), especially those in roots colonised by two or more fungal links, may provide pathways for the movement of P and N among plants (Eissenstat 1990; Johansen et al. 1992), and C-sharing among fungi (Simard et al. 1997). Therefore, mycorrhizas may influence the structure, diversity and productivity of plant communities (Allen and Allen 1986, 1990; Perry et al. 1989; Hartnett et al. 1993; Allen et al. 1995; van der Heijden et al. 1998), and their conservation is critical for maintaining ecosystem stability and function. Human activities can have important impacts on the diversity and efficacy of mycorrhizal associations. Their effects on mycorrhizae, however, are often chronic and frequently quite subtle (reviewed in Allen et al. 1993). Such changes may be manifest as minor fluctuations in species composition (Allen et al. 2000) through to more extreme shifts which encompass the loss of genera or changes in species dominance (Johnson 1993; Egerton-Warburton and Allen 2000). Although similar shifts in plant communities are acknowledged as being critical in understanding ecosystem processes (Wedin and Tilman 1996; Tilman et al. 1997), the influence of comparable shifts in mycobiont diversity on mycorrhizae and in turn, the plant community, has yet to be fully appreciated. In this review, we focus on the functional diversity and conservation of mycorrhizal communities in the Californian floristic province as indicative of the way in which mycorrhizal relationships in plant communities are affected by increases in anthropogenically-linked or N eutrophication. California is a highly pollution, elevated urbanized region (est. population 40 million) and a recently designated global hotspot of biodiversity and priority for conservation (Myers et al. 2000). Southern California, and in particular the Los Angeles area, has a history of the most extreme air pollution in the contiguous United States. Both elevated and N deposition co-occur within this region. However, N eutrophication may currently constitute the biggest threat to ecosystem stability. Nitrogen eutrophication in southern California corresponds to an increased input of nitrate and between 10-13 % per annum) are returned of all nitrogenous emissions (or 33-38 kg N in southern California. In contrast, N to the soil annually as deposition eutrophication events in Europe are largely the result of from intensive agriculture and animal husbandry (Schulze 1989; Bobbink 1991). Anthropogenic acidification is not a significant outcome of atmospheric pollution in California (Fenn and Bytnerowicz 1993;

22

Microorganisms in Plant Conservation and Biodiversity

Bytnerowicz and Fenn 1996). Regardless, the photo-oxidation of nitrogen oxide emissions in concert with volatile organic compounds frequently contribute to elevated ground level ozone concentrations and severe photochemical smog within much of the Los Angeles Basin and particularly during summer (Miller et al. 1998). The California biota is characteristically termed a high diversity plant community (Sawyer and Keeler-Wolf 1995), and one in which the many plant species are frequently either obligately or facultatively dependent on mycorrhizal fungi, depending on soil nutrient availability, effective precipitation and successional status. In addition, a diversity of mycorrhizal forms occur in the California biota that include, but are not limited to: arbuscular mycorrhizal fungi (AMF) in coastal sage scrub and chaparral, ectomycorrhizal fungi (EM) in chaparral (Adenostoma species), oak woodlands (species of Quercus) and higher elevation forests (species of Pinus), and ericoid, orchidaceous and arbutoid mycorrhizas. We reviewed the responses of the AMF community following exposure to elevated Neutrophication or x N interactions, and the relevance of a trace gas-derived climate change on the symbiosis. Arbuscular mycorrhizal fungi are the most ubiquitous mycorrhizal association and thus shifts or responses in AMF dynamics to human impacts may be particularly widespread and of considerable concern. We use AMF response data to answer the following questions: 1. How does functional diversity and community dynamics of mycorrhizas and efficacy of the symbiosis vary with elevated N deposition, or following x N interactions? 2. How will the downstream effects of elevated and nitrogen oxides, such as ozone and climate warming, influence mycorrhizal functioning? 3. What are the likely outcomes of global change-induced shifts in mycorrhizas on plant community productivity and stability? 4. How may mycorrhizas be best conserved in the climate of global change? 2. Mycorrhizal functioning and responses to elevated atmospheric Current analyses of the global carbon cycle argue in favor of a vital role for terrestrial ecosystems in carbon uptake. Partitioning of the terrestrial enrichment stimulates photosynthesis and sink indicates that subsequently increases plant primary productivity, litter fall and carbon input into terrestrial ecosystems (Bazzaz 1990; Berntson and Bazzaz 1996). As the production of above-ground resources is amplified, the below-ground carbon allocation increases correspondingly and promotes a marked increase in specific root length and the deposition of carbon per unit area within the rhizosphere (Rouhier et al. 1996), and, in turn, improved carbon allocation to mycorrhizas (Hodge 1996). Theoretically, such circumstances should favor mycorrhizal proliferation (Figure 1a). enrichment in California shrublands and grasslands alters carbon allocation to mycorrhizal, but not pathogenic, fungi, and increases the sink strength of the mycobiont (Rillig and Allen 1998, 1999; Rillig et al.

Conservation of mycorrhizas under global change

23

1998a, 1999a,b). With few exceptions, elevated results in an increase in extra-radical hyphal biomass, intra-radical hyphal infection intensity and arbuscular infection (Table 1; Rillig and Allen 1999). In particular, hyphal infection intensity increased significantly in root classes (180-400 µm diameter) where the cortex can readily accommodate fungal proliferation (Rillig and Allen 1998, Rillig et al. 1998b). Rygiewicz et al. (1997) also noted an increased turnover of mycorrhizal hyphae with enrichment. However, there is no apparent change (increase or decrease) in the production of vesicles or intra-radical coils, or the abundance of enrichment (Klironomos et al. 1996, pathogenic fungi in response to 1997; Rillig and Allen 1998; Rillig et al. 1997, 1999a). The development and proliferation of mycorrhizal structures implies an increase in carbon allocation to the mycobiont, especially to the intraradical structures (hyphae and arbuscules but not vesicles), and an increase in the carbon sink strength of the mycobiont (Morgan et al. 1994). Since the arbuscular interface regulates the directional transfer of carbon and nutrients between host and mycobiont, these observations suggest that the potential for nutrient and carbon transfer may be enhanced under elevated conditions. Parallel studies of enrichment in other ecosystems indicate a positive relationship between arbuscular infection, P inflow and content of the host plant with enrichment (Rouhier and Read 1998). In particular, plants grown in conditions failed to access declining soil P reserves (Rouhier ambient and Read 1998). Strong host interspecific differences exist with respect to mycorrhizal response under elevated and habitat; such differences could not be related to life history or phylogeny (Rillig et al. 1998a, 1999b). Responses of AMF, as measured by arbuscular infection, to elevated are inconsistent and can be positive (Linanthus, Plantago, Euphorbia), weakly negative (Lolium), or non-significant (Epilobium, Avena) depending on the study system (Rillig et al. 1999b). Notably, arbuscular infection in Lolium increased in a serpentine soil, but decreased in sandstone annual grasslands (Rillig et al. 1999a,b). The positive response in Euphorbia is of interest since members of the genus may to or physiology or Crassulacean Acid Metabolism (CAM), possess and as atmospheric concentrations rise, will this influence the relative distributions of and CAM plants in general? Experimental data generally demonstrate that elevated levels favour plants due to their compensation of 30-70 ppm at optimal temperatures (versus in plants; Larcher 1995). On the other hand, CAM <10 ppm plants temporarily segregate the processes of carbon dioxide uptake and fixation when grown under arid conditions but follow the and a photosynthetic pathway when water is not limiting. Elevated wetter climate as predicted by global change analysts may thus also favor CAM plants (0-200 ppm compensation in light; Larcher 1995). These differences illustrate that plant responses to may depend on variations among mycorrhizal functional groups and substratum. Correspondingly, mycorrhizal and non-mycorrhizal plant species, and

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Microorganisms in Plant Conservation and Biodiversity

Conservation of mycorrhizas under global change

25

differences in metabolic pathways, such as or CAM, among host plants may also be expected to differ in their responses to elevated Striking differences also exist among mycorrhizal groups with responses (Klironomos et al. 1998). Artemisia tridentata respect to seedlings inoculated with Acaulospora denticulata, Glomus etunicatum, Glomus intraradices or Scutellospora calospora demonstrated that mycorrhizal taxa differ in their growth allocation strategies in a enriched environment. Percent infection of roots by arbuscules and hyphae increased in Artemisia inoculated with either Glomus species. On the contrary, there was no detectable change in infection in plants inoculated with Scutellospora or Acaulospora. Hence, AMF taxa may vary in their ecological specificity and capacity to influence the growth of the host plant. More subtle responses to occur at the AMF community level. At ambient or sub-ambient atmospheric (up to 350 ppm), the AMF community associated with Adenostoma fasciculatum was dominated by the genus Glomus, with respect to spore bio-volume and extra-radical AMF hyphal length. Increasing atmospheric (350-650 ppm) was associated with an increase in hyphal length within soil aggregates, spore bio-volume and subtle shifts within the AMF community composition. Specifically, the abundance and bio-volume of AMF spores increases with this outcome was due to the increasing prevalence of the largespored Scutellospora calospora, and a decline in the abundance of smallspored Glomus species. These findings were paralleled in the AMF hyphal community. An increasing availability of was correlated with a marked increase in the hyphal lengths and abundance of Scutellospora and Acaulospora (Klironomos et al. 1998; L. Egerton-Warburton, unpublished data). Hence, AMF differ in their growth allocation as strategies and capacity to sequester carbon under elevated indicated by the increasing hyphal length and abundance of spores. In concert with the changes in functional diversity of AMF taxa (see above), shifts in the AMF community may therefore impact the structure, nutrient status and productivity of the plant community. We anticipate that such shifts are also influenced by temporal and seasonal changes in both mycorrhizal and host plant root growth and activity (e.g. Zak et al. 1993). Although these responses have yet to be evaluated in California, studies in other biomes demonstrate a distinct phenology of mycorrhizal infection. Little or no mycorrhizal response can be expected in the two months following initiation of enrichment. However, significant increases in mycorrhizal infection enrichment in both AM and EM systems (Rouhier follows prolonged and Read 1998). These phenologies may be driven by alterations in carbon allocation to the mycobiont (Rouhier and Read 1998). The net effect of elevated on AMF appears to be an increase in hyphal biomass or growth, root infection and shifts in community composition and the dominance of AMF taxa. Nevertheless, speciesspecific responses to individual AMF taxa appear to be the rule rather than the exception, and interactions between host plant and mycobiont

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Microorganisms in Plant Conservation and Biodiversity

Conservation of mycorrhizas under global change

27

most likely determine ecosystem feedback to elevated in a bi­ directional feedback loop. Changes in mycorrhizal infection and the extra-radical phase (spores plus hyphae), however, indicate that AMF could substantially alter nutrient availability to the host plant by up- or down-regulating nutrient transfer. Such changes conserve nutrients within ecosystems and result in the downstream effects in increased host plant fecundity and litter production, changes in the quality (C:N) and decomposition of litter and mycorrhizal activity. The magnitude of these responses will dictate long-term ecosystem responses to by altering the dynamics of mycobiont-plant species interaction. 3. Mycorrhizal community dynamics following anthropogenic nitrogen deposition The global upsurge in nitrogen emissions and deposition following urbanization and industrialization is well documented. In many regions, anthropogenic N deposition is increasing at a faster rate than the corresponding rise in atmospheric Nitrogen enrichment dramatically increases aboveground productivity but negatively impacts biological diversity, composition and functioning (e.g. carbon cycling) of ecosystems (Tilman 1988; Berendse 1995). In addition, feedbacks between the below- and above-ground biota can also contribute to this outcome. As ecosystems become increasingly N enriched, plants allocate more C to above- than below-ground structures (Tilman 1988), which results in an intense C sink competition between shoots and roots, and decreased C allocation to mycorrhizas (Smith 1980). Consequently, models exploring the relationship between N enrichment and mycorrhizal structure and function have demonstrated that N eutrophication negatively impacts the mycorrhizal association (Figure 1b; Allen 1991; Smith and Read 1997 and references therein). However, unlike the effects of elevated on AMF dynamics, the effects of N eutrophication on AMF community and hence ecosystem dynamics, has received little attention. Still, the influence of N eutrophication on AMF communities in California can be readily identified by a suite of traits that illustrate a negative response to N enrichment. On a global scale, mycorrhizal diversity is strongly and negatively influenced by chronic N enrichment. Chronic N deposition has been directly linked to a decline in EM diversity and productivity elsewhere (reviewed in Wallenda and Kottke 1998). However, AMF community composition and diversity may shift in response to host plant species alone (Johnson et al. 1992) or combined host plant-nutrient availability interactions (Johnson et al. 1991). In southern California, there was no apparent effect of host plant species (native shrub species) on mycorrhizal diversity (Egerton-Warburton and Allen 2000; Sigüenza 2000; Sigüenza et al. 2000). Arbuscular mycorrhizal communities were comparable among host plant species, and they responded similarly to N enrichment (Egerton-Warburton and Allen 2000). Regardless, an increasing N input in shrublands and grasslands, either via anthropogenic N deposition or N fertilisation, resulted in significant shifts in AMF

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Microorganisms in Plant Conservation and Biodiversity

community dynamics that were identified by alterations in mycorrhizal diversity and productivity. Firstly, N eutrophication results in a rapid and marked shift in AMF richness and diversity (Egerton-Warburton and Allen 2000). Data from an anthropogenic N deposition gradient and N fertilisation studies in California shrublands demonstrate a reduction in AMF species richness and diversity with increasing N input. Such changes were accompanied by displacement of the larger-spored genera Scutellospora and Gigaspora due to a failure to sporulate, and concomitant proliferation of small-spored Glomus species, such as Glomus aggregatum, Glomus occultum and Glomus leptotichum (Egerton-Warburton and Allen 2000), and Glomus tenue (Sigüenza 2000; Sigüenza et al. 2000). These Glomus species are considered indicative of N eutrophication and affiliated with colonization by invasive grasses in southern California. Comparable studies in ecosystems elsewhere indicate that such changes most likely influence plant community dynamics by altering the functional diversity of the AMF community (Johnson 1993). Specifically, these Glomus species tend to be less effective mutualists, particularly in N-enriched soils, and negatively influence host plant productivity and nutrient status. Secondly, N eutrophication may alter the productivity of the AMF community by reducing the carbon sink strength of the mycobiont. An increasing input of N can be correlated with a decline in root infection. In particular, N eutrophication is associated with a marked loss of hyphal and vesicular, but not arbuscular, infection (Egerton-Warburton and Allen 2000). Such data suggests an overall reduction in the carbon expenditure by the mycobiont but that the potential for nutrient and carbon transfer has not been altered by N enrichment. More simply, the carbon sink strength of the mycobiont cannot compete against that of the host at high nutrient loads. Responses of AMF abundance to N eutrophication, as represented by extra-radical hyphal abundance and length, are less consistent. Hyphal responses have been noted as negative and manifest as a significant reduction in hyphal length following prolonged (> 14 months) of N enrichment (Klironomos et al. 1997; Rillig et al. 1998b), or in other cases, non-significant (Rillig et al. 1998b). However, if the abundance of live hyphae declines, then the capacity for plants to acquire water and sparingly available nutrients does so proportionately. In addition, if hyphal turnover is influenced by N enrichment, then soil structure may be altered because microbial decomposition of labile materials is enhanced w i t h increasing N availability (reviewed in Hu et al. 1999). Alterations in the a v a i l a b i l i t y of resources invariably impact the structure of the vegetation (Tilman 1993), either by the direct effect of N on plants or via those mediated by the effects of N on mycorrhizal associations and, in turn, the host plant (Smith and Read 1997). A recent synopsis indicates that coastal sage communities in southern California are currently undergoing a shift from native shrublands to one dominated by exotic annual grasses (Figure 2; Allen et al. 1998; Padgett and Allen 1999; Padgett et al. 1999). Native shrub species frequently demonstrate

Conservation of mycorrhizas under global change

29

poor establishment rates due to competition from exotic grasses coupled with the premature senescence of seedlings. Moreover, native shrub species that are obligately mycorrhizal in the field, such as Salvia mellifera and Artemisia californica, or facultatively mycorrhizal, such as Eriogonum fasciculatum, all demonstrate an inability to regulate growth with N enrichment (Padgett and Allen 1999). Specifically, N fertilisation promotes constant and luxuriant shoot growth and high foliar N content at the expense of root growth and development in native shrub species. These data coupled with the high mortality rates in seedlings grown under N enrichment suggested that high N inputs may contribute to an acceleration in shrub mortality and the decline of shrublands (Allen et al. 1998). Contrary to expectations, exotic grass species (Avena, Bromus) all demonstrated a depression in productivity following N eutrophication (Padgett and Allen 1999). It is possible that plant (grass) growth can be increased by N fertilisation, but at some point, growth may become limited by an insufficiency of other essential resources such as P, calcium or water availability (Tilman 1993). Some of the most critical effects of N enrichment on plant growth can be exerted indirectly via interactions with mycorrhizas (Termorshuizen 1993). Currently, little is known about the interactions between N enrichment, mycorrhizal response and plant productivity in southern California. Yoshida and Allen (2000, 2001) reported that arbuscular mycorrhizas had a more negative influence on the uptake of in Artemisia californica than the invasive annual grass, Bromus madritensis. As a result, Bromus may be a better competitor for than Artemisia, and in vegetation where high anthropogenic deposition is increasing, the encroachment of Bromus may be in part related to N nutrition. Additional studies are currently in progress to determine the positive and negative contributions of host and mycobiont to the plant community with N enrichment. However, we consider that there is sufficient knowledge regarding the effects of N enrichment on each of plant productivity and mycorrhizas in southern California to predict that N eutrophication most likely negatively impacts the functioning of the symbiosis. We can conclude that N eutrophication has profoundly influenced the AMF community. We further suggest that N enrichment may initiate a cascade of negative effects on mycorrhizas that consequently may determine ecosystem responses to N deposition. Nitrogen eutrophication promotes the decline of AMF species richness and diversity that, in turn, initiates a potential loss in plant community diversity, productivity and functioning (van der Heijden et al. 1998). In addition, functional shifts in the mycorrhizal community may be driven by changes in the quality and quantity of plant C allocation to the mycobiont. As a result, such changes may negatively impact host plant productivity, nutrient capture and competitive ability, and potentially alter P cycling and C allocation at the ecosystem level (Figure 1b). Subsequently, changes in C allocation among mycobiont, host and rhizosphere may modify the capacity of terrestrial ecosystems to absorb and sequester C (Figure 1a).

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Microorganisms in Plant Conservation and Biodiversity

4. Interactions between elevated x nitrogen enrichment and influences on mycorrhizas A more variable mycorrhizal response to enrichment occurs in the presence of N, P or NPK enrichment (Table 1), suggesting that interactions between elevated and N deposition may have quite different implications for the terrestrial environment (Figure 3). In Gutierrezia sarothrae (Rillig et al. 1997; Rillig and Allen 1998), a synergistic x N interaction regulates mycorrhizal infection. A greater AMF infection response was noted for the combined treatment than occurred with or N alone. Strong x NPK interactions also occurred in some mycorrhizal type plant species combinations. These may be manifest in the increased production of fine roots and extra­

Conservation of mycorrhizas under global change

31

radical hyphae (e.g. Linanthus, R i l l i g et al. 1998b). In contrast, x NPK enrichment does not elicit the same response in infection intensity in AMF inoculated plants of Bromus hordeaceus (Rillig et al. 1998a). In addition, the level of applied nutrient influences the outcome. For example, at low N availability, a high level of stimulates hyphal with growth in Populus, whereas there was no significant effect of high N availability. These data suggest that AMF communities can respond quite differently to elevated depending on edaphic parameters such as N or N+P availability, and that strong interactions and soil nutrients most likely influence mycorrhizal between functioning (Klironomos et al. 1997). Thus, the combined effects of and N may differ w i t h i n and among ecosystems, and may be driven by local edaphic constraints. 5. Nitrogen oxides, their photochemical conversion to ozone and effects on mycorrhizas Ozone in both the stratosphere and at ground level has become an acts as a important global air quality issue. In the stratosphere, where filter to absorb UV, short- and long-wave infrared radiation, ozone is being depleted. Conversely, levels at ground level are steadily increasing and currently constitute a major component of photochemical smog (UNEP 1988). Ground level ozone is formed by the photo­ oxidation of volatile organic compounds, such as carbon monoxide, methane and non-methane hydrocarbons, in combination with nitrogen oxides (UNEP 1988). The b u r n i n g of fossil fuels is a major anthropogenic cause of nitrogen oxides, while the use of motor vehicles, solvents, and industrial processes in the petrochemical industry constitute the sources of volatile organic compounds. Since the mid-1970s, ground level ozone concentrations have increased globally by 1% on average. However, increases in ozone levels have been considerably higher in the Los Angeles Basin (Miller et al. 1998). Ozone directly affects plant physiology and metabolism. Such changes are manifest in visible foliar injury, reduced reproductive development, annual productivity, increased susceptibility to insects and disease, and altered plant physiology, such as photosynthetic capacity, modification in the partitioning of carbohydrate allocation and particularly the delay of photosynthate allocation to below-ground structures (Davison and Barnes 1998; Tausz et al. 1998). In California, the adverse effects of ozone alone or in combination with other edaphic stressors on plants are well documented (Peterson and Arbaugh 1992) but the influences on the mycorrhizal community have yet to be elucidated. This is surprising given the intensity of the photochemical smog w i t h i n the Los Angeles Basin during summer (Miller et al. 1998). However, many species in the coastal sage and by being dormant and/ or chaparral shrublands avoid the impact of levels tend to be highest (Sawyer drought deciduous in summer when and Keeler-Wolfe 1995). Studies from other ecosystems illustrate the on those summer-active species. Because anticipated to impact of

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Microorganisms in Plant Conservation and Biodiversity

ozone does not penetrate the soil to any extent (Reich et al. 1986), influences mycorrhizas via changes in carbon allocation from the host plant (Andersen and Rygiewicz 1995). From a structural perspective, the frequency of vesicles, hyphal coils and intra-radical hyphae in arbuscular plants increases at heightened ozone levels, while arbuscular colonization decreases. Duckmanton and Widden (1994) suggested that mycorrhizas may respond to stress by increasing the production of less energy demanding and less efficient organs for exchange of nutrients (arbuscules), and by increasing the resources allocated to storage and future growth (vesicles). The increase in internal mycelium appeared to represent an increase in susceptibility of the root to infection by saprophytic fungi. In addition, ozone stress promoted a shift in carbon allocation to the roots of arbuscular mycorrhizal plants that, in turn, reduced the extent and f u n c t i o n i n g of the mycorrhizas (McCool and Menge 1983). Such effects can be further modified, both positively and negatively, by the nature of the mycobiont (McCool and Menge 1984).

In summary, ozone stress appears to be associated with negative changes in the allometric relationship of mycorrhizal occupancy to root volume (reduced mycorrhizal colonization among roots), an alteration in the allocation of carbon sink strength of the mycobiont (reduced abundance of mycorrhizal structures) and a decline in functioning (as

Conservation of mycorrhizas under global change

33

indicated by the loss of arbuscules). In turn, a reduction in functioning may influence the growth and mineral nutrition of the host plants (Smith and Read 1997) and impact the sustainability of ecosystems. 6. Trace gases and the greenhouse effect Escalating concentrations of atmospheric nitrogen oxides and ozone can be correlated with changes in temperature and precipitation patterns that, in turn, have the capacity to modify the global climate (Houghton et al. 1990, 1996; Schiffer and Unninayar 1991). These trace gases can also influence the radiative balance of the atmosphere. This so-called ‘greenhouse effect’ results from “the dirt on the atmospheric infrared window" by trace gases such as that allows incoming solar radiation to reach the surface of the Earth unhindered but restricts the outward progress of long-wave infrared radiation. Such gases can also absorb and re-radiate this outgoing radiation to promote a net warming within the first 10-20 km of the atmosphere; the global mean temperature is expected to increase by 1°C by 2030 (Bolin et al. 1986; IPCC 1994; Barret 1995). Model-predicted maps of temperature and rainfall have forecast a warmer summers (up to 6°C on average) and wetter winters along the Pacific seaboard in the future (Smith and Tirpak 1988). Such changes may alter the migration and distribution of plant species (Vitousek 1994) and subsequently the composition and productivity of plant communities, albeit slowly (King and Tingey 1992). It is reasonable to expect that any changes in plant community composition will directly influence mycorrhizal communities (Johnson et al. 1992; van der Heijden et al. 1998; Klironomos et al. 2000). Because mycorrhizas are carbon sinks and amongst the first soil biota to receive carbon from plants (Rygiewicz and Andersen 1994), climate change may also indirectly influence the dominance and functioning of fungi within a community (Rygiewicz et al. 1997). Microbial communities may respond rapidly to changes (increase or decrease) in soil moisture and temperature, or the combined effects of elevated trace gas concentrations, soil moisture and temperature (Schwartz 1992; King and Tingey 1992; Hu et al. 2001) but not always (see Whitbeck 1994). For example, four cycles of simulated climate change (elevated modified temperature and precipitation regimes) influenced mycorrhizal colonization in a grass (Pascopyrum smithii), but not Bouteloua gracilis (Monz et al. 1994). Specifically, increases effects in Pascopyrum could be in mycorrhizal colonization due to offset by elevated temperatures (4°C above control), and increased precipitation. Hence, any mycorrhizal response may be influenced by the synergy of edaphic and biotic constraints. At first glance, these direct and indirect changes appear modest however any of these shifts will be further amplified during host plant species variation with progressive climate change.

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Microorganisms in Plant Conservation and Biodiversity

7. Scaling up from the fungal to the global community Scaling and modeling from community level to global platform can often predict performance or survival of mycorrhizas, and contribute to an understanding of the functioning of ecosystems and human impact on the terrestrial environment. The studies presented herein indicate that elevated and N deposition were not random in their effects on mycorrhizal community productivity and functioning (Figure 3). In addition, alterations in the mycorrhizal fungal community appear to be a likely and potentially irreversible outcome of global change (but see Oechel et al. 1994). Human impacts are frequently viewed only in terms of the negative impact on the affected AMF communities. However, disturbance may benefit some mycorrhizal species, in, for example, the geographic expansion of some species (so-called ‘winners’), and a concomitant reduction in others (‘losers’) (McKinney and Lockwood 1999). Evidence from the studies on the AMF communities in California illustrates that exposure to anthropogenic change results in both winners and losers. The winners are represented by a small number of rapidly expanding species that benefit from human alteration of the habitat. Under conditions of elevated these appear to be the genera, Scutellospora and Acaulospora. In contrast, chronic N deposition appears to favor small-spored, weedy Glomus species. These Glomus species in particular possess traits that promote survival in disturbed habitats. For example, they are highly dispersable, possess small spores with high fecundity, and display mutualistic to parasitic traits in symbiosis (Johnson 1993; Johnson et al. 1997). The net outcome in both instances is a change in functional diversity and productivity. The replacement of formerly diverse AMF communities by winners may cause a more widespread homogenization of the mycorrhizal community than is currently appreciated, particularly if AMF species are not randomly distributed, such as in species or genets that are specialized in function, or explicit to a particular plant species. Selective extinction may accelerate the loss of (functional) diversity, and denote the removal of unique genetic or morphological diversity, such as Gigaspora on the N deposition gradient (Egerton-Warburton and Allen 2000). The homogenisation may be taxonomically and ecologically depressing to the effect that global change results in simpler AMF communities composed of generalists, the over-representation of some genera and fewer ecological specialists. Based on the causal relationship between mycorrhizal diversity and plant community diversity and productivity (van der Heijden et al. 1998), such shifts may exert a profound impact on ecosystem functioning. Specifically, we suggest these may be manifest as a reduction in plant community diversity at the regional and global scales and changes in plant f u n c t i o n a l response via mycorrhizal-regulated alterations in C and N cycling, the movement of water, and the uptake and/ or exchange of P, N and C. The ecological consequences of AMF homogeneity are obviously multifaceted. However, the question remains as to conservation of

Conservation of mycorrhizas under global change

35

mycorrhizas with global change. Levin (1990) suggested that the persistence of populations depended on heterogeneity. Thus a decline in heterogeneity, or increase in homogeneity, indicates a decline in the potential for persistence of an AMF community. All organisms and communities demonstrate heterogeneity and diversity and this should be selected for in relation to physiological, morphological and demographic responses of the organism (Levin 1990). The key is how to determine the criteria by which to conserve AMF diversity, heterogeneity, and identify the mycorrhizal genera, species or communities at most risk in a climate of global change. The Montréal Process identified seven conceptual criteria and indicators for the conservation and management of ecosystems (http://www.mpci.org/meetings/tac-mexico/tnl-6_e.html#s2). While these criteria were originally intended for forest ecosystems, in a modified form they apply equally well to mycorrhizal communities and their role in ecosystem functioning. We suggest the following seven adapted criteria: 1. 2. 3. 4.

Conservation of mycorrhizal diversity Maintenance of the productivity of mycorrhizas in ecosystems Maintenance of mycorrhizal functioning Conservation of a mycorrhizal role in maintaining soil and water resources 5. Maintenance of the mycorrhizal contribution to global carbon and nitrogen cycles 6. Maintenance and enhancement of the long-term multiple socio­ economic benefits of mycorrhizas 7. Legal, institutional and economic framework for conservation and sustainable management of mycorrhizas Criteria 1-3 Conservation of mycorrhizal diversity, productivity and functioning Diversity, productivity and functioning of a community, whether plant or fungal, drives ecosystem efficacy and the capacity to respond to anthropogenic disturbance. The variability in AMF species richness and range of genotypes of each species within a given area may constitute surrogate descriptors of diversity. However, the full extent of genetic diversity in AMF is poorly understood (Bentivenga et al. 1997). The nucleate state of the AM spore may harbor greater levels of genetic diversity in AMF than is currently appreciated (Sanders et al. 1995; Redecker et al. 1999), and the issue of ecotypes and concepts of plasticity have yet to be fully realized in these fungi (see Bever and Morton 1999). In addition, keystone species, or AMF that are pivotal to community functioning, remain to be fully documented. Clearly, an understanding of the ecological and evolutionary significance of AMF diversity within and among functional groups, the expansion of taxonomic surveys and inventories, and the documentation of endemism among AMF communities is important. Recent and anticipated advances in molecular genetic techniques may ensure that AMF surveys will be productive in

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many contexts. However, it is equally important to perform research on AMF species in symbiosis in order to interpret the significance of genetic diversity on host plant productivity and functioning, particularly in response to anthropogenic change. In the absence of such data, we are forced to assume that AMF species in southern California are phylogenetically similar and exert comparable effects in symbiosis as AMF documented elsewhere. Criteria 4-5 Conservation of the mycorrhizal contribution to maintaining soil and water resources and global carbon and nitrogen cycles Mycorrhizas contribute significantly to terrestrial C and N cycling, constitute sources and sinks in this cycle, and soil structure and aggregation with global change ( R i l l i g and Allen 1999). What is missing is quantitative information for integrating AMF species-based responses into functional group responses. Mycorrhizal functional groups may be based on edaphic criteria, such as the turnover of soil N and C, but guided by quantitative biological descriptions of an individual AMF species and/ or genus. The development of realistic models that link AMF communities to edaphic constraints also requires exploration of the physiological responses of AMF and the interactions that drive alterations in functional diversity with global change. These include, but are not limited to, factors that stimulate or reduce growth rates, nutrient and water acquisition, or C transfer via common mycorrhizal networks (Robinson and Fitter 1999). Criteria 6-7 Maintaining the socioeconomic benefits of mycorrhizas within the frameworks of political and economic institutions A rapidly expanding population with increasing demands for space and land, subsequent changes in consumption patterns, especially of fossil fuels, and consumer-driven access to natural resources has resulted in an exceptional rate of habitat loss and fragmentation of which threatens most native ecosystems as typified by the shrublands in southern California. Historically, the value of the environment in California as elsewhere has been largely ignored until it has been substantially depleted or lost. Attempts to arouse public and governmental support for the prevention of habitat loss often fails due to a lack of understanding of the l i n k between natural resources and the economy. In southern California, the most impacted regions are remnant tracts of coastal sage scrub. Unsullied coastal sage communities tend to support highly diverse AMF communities. However, these remnants are continually sacrificed for urban expansion, or increasingly invaded by plant species that benefit from high soil N loads (Padgett et al. 1999). As the weedy species invade, the community becomes dominated by plant species with lowered or negligible mycorrhizal functioning (Sigüenza 2000; Sigüenza et al. 2000). The remaining tracts of shrublands tend to be adjacent to urban developments and proximity enhances invasion by exotic grass species (Padgett et al. 1999). In this urban environment as in many others in the world, maintenance of the mycorrhizal community has broader

Conservation of mycorrhizas under global change

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environmental values, namely the preservation of remnant shrublands, since many of the native plant species are obligately mycorrhizal. Thus, conservation of the mycorrhizal community requires preservation of remaining tracts of shrublands, and measures to reduce soil N levels via the introduction of legislation to further reduction in vehicular emissions and ameliorative measures, such as mulching, to immobilize inorganic N (Allen et al. 1999). 8. Conclusions Humans have been extraordinarily successful in developing industrialized nations and permitting the scale of human enterprise to develop so rapidly that their consequences are felt at the scale of individual organisms, ecosystems, and the biosphere, and more notably in the human alteration of the global carbon and nitrogen cycles (D’Arrigo et al. 1987; Keeling et al. 1995). How mycorrhizal fungi respond to these changes may well depend on the biogeochemical conditions that occurred during their evolution and their genetic ‘memory’ of these past events. The past four glacial-interglacial cycles (up to 420 thousand years before present; Indermuhle et al. 1999) demonstrate both the extent of (<180 ppm) during glacial periods and climate change and low higher (>300 ppm during interglacial periods (Lorius et al. 1990). Thus, the current and anticipated rise in atmospheric levels is well within those encountered during the evolutionary history of AMF (Allen 1996). Consequently, the capacity of mycorrhizal fungi to respond to is most likely within the scope of their genotypic variability. elevated On the other hand, N is usually a limiting nutrient in many ecosystems (Tilman 1993) and there is substantial evidence that N limits net primary production in most terrestrial biomes (Vitousek and Howarth 1991). An excess of N in geological time scales was most likely associated with major disturbance events whereas human activities have altered the nitrogen cycle deliberately through the production and use of industrial fertilizers, the growth of nitrogen fixing crops and burning fossil fuels (Vitousek and Howarth 1991). The consequences of the anthropogenic changes in N cycle are important because they affect not only the composition of atmosphere, but because N interacts strongly with the carbon cycle to the point of regulating the flux of carbon in many ecosystems. The current convergence of these two major determinants of ecosystem functioning on mycorrhizas appears to push fungal community and ecosystem dynamics in opposite directions, but for the most part the responses to these stressors may depend on the precise mycobiont-host combination and local edaphic constraints, such as soil moisture and temperature. As a corollary, human activities have also impacted upon species diversity at the global scale. While extinction and immigration of species are acknowledged as natural ecological processes, the magnitude and scope of these events is faster than any that have occurred over the past 20,000 years (IPCC 2001). Because emissions of greenhouse gases such

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as and nitrogen oxides are long-lived, they will have a lasting effect on the atmospheric composition, climate and terrestrial ecosystems. From a mycological perspective, this is of particular concern because the current knowledge base of mycorrhizal fungal diversity and functioning is relatively small and the loss of species diversity is irreversible. In addition, humans are also responsible for the introduction of exotic species into communities. These non-native species often change the biotic and abiotic environment sufficiently to promote the decline or loss of mycorrhizas from the community. Synergistically, these changes are occurring so rapidly in both California and on a global basis that an assessment of mycorrhizal fungal diversity and the functional consequences for population, community, and ecosystem processes are now critical for their conservation. Acknowledgments We thank the National Science Foundation and the United States Departments of Agriculture and Energy Competitive Grants Programmes, and the Australian-American Education Foundation (Fulbright) for research funding leading to this synthesis. References Allen EB, Allen MF(1986) Water relations of xeric grasses in the field: interactions of mycorrhizae and competition. New Phytologist 104, 559–571. Allen EB, Allen MF (1990) The mediation of competition by mycorrhizae in successional and patchy environments. In ‘Perspectives on plant competition.’ (Eds JR Grace and D Tilman) pp. 367–389. (Academic Press: New York) Allen EB, Allen MF, Helm DJ, Trappe JM, Molina R, Rincon E (1995) Patterns and regulation of mycorrhizal plant and fungal diversity. Plant and Soil 170, 47–62. Allen EB, Padgett PE, Bytnerowicz A, Minnich RA (1998) Nitrogen deposition effects on coastal sage vegetation of southern California. In ‘Proceedings of the international symposium on air p o l l u t i o n and climate change effects on forest ecosystems.’ Riverside, C a l i f o r n i a . (Eds A Bytnerowicz. MJ Arbaugh and S Schilling) General Technical Report PSW-GTR 164. (Pacific Southwest Research Station-USDA Forest Service: Riverside) Allen EB, Padgett PE, Yoshida L, Sigüenza C (1999) Nitrogen deposition, grassland conversion and r e h a b i l i t a t i o n . In ‘Proceedings of the XVIII international grassland congress.’ Saskatoon, Canada. (Eds JG Buchanan-Smith LD Bailey and P McCaughey) pp. 295–298. (Saskatchewan Agriculture and Food, Saskatoon: Canada) Allen MF ( 1 9 9 1 ) ‘The ecology of mycorrhizae.’ (Cambridge University Press: Cambridge) Allen MF (1996) The ecology of arbuscular mycorrhizas: a look back into the century and a peek into the 21st. Mycological Research 100, 769–782. Allen MF, Allen EB, Dahm C, Edwards FS (1993) Preservation of biological diversity in mycorrhizal fungi: importance and human impacts. In ‘Human impacts on selfrecruiting populations.’ (Ed. G Sundnes) pp. 81–108. (Tapir Press: Trondheim) Allen MF, Zink T, Egerton-Warburton LM, Karen O (2000) Mycorrhizal fungi in coast live oak: diversity and scaling. Bulletin of the Ecological Society of America 81, 241. Andersen CP, Rygiewicz PT (1995) Allocation of carbon in mycorrhizal Pinus ponderosa seedlings exposed to ozone. New Phytologist 131, 472–480. Barret J (1995) The roles of carbon dioxide and water vapor in warming and cooling the Earth’s troposphere. Spectrochimica Acta 51A, 415–417.

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Chapter 3 SYMBIOTIC NITROGEN FIXATION BETWEEN MICROORGANISMS AND HIGHER PLANTS OF NATURAL ECOSYSTEMS

John S. Pate Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia. Crawley, 6009, Western Australia.

1. Introduction Nitrogen (N) is generally considered to rank next in overall importance for plant growth to the elements carbon, hydrogen and oxygen which green plants assimilate photosynthetically from carbon dioxide and water. Unlike other mineral elements essential to life, N is absent from the primary materials of the earth’s crust, resulting in overwhelmingly great dependence of essentially all world fauna and flora on atmospheric molecular nitrogen To a lesser extent, there are also inputs from other atmospheric sources such as nitrate generated during thunderstorms, fallout of ammonia gas from volcanism and and oxides of nitrogen arising from various combustion processes associated with human activity (see Jenkinson 1990, 2001; Galloway et al 1995). Counteracting these inputs, significant net losses of N occur from soils of natural ecosystems, whether as released through denitrification, lost from decomposing organic and released to the atmosphere during fire, or leaching of matter, through surface run-off and deep drainage (Mansfield et al. 1998; Fowler et al. 1998). Inputs of fixed thus remain critical to restoration of nitrogen capital of ecosystems and to the long and short-term functioning of constituent vegetation and dependent biota (Schlesinger and Hartley 1992; Stevenson and Cole 1999). The capacity to fix is largely confined to microorganisms possessing nitrogenase the enzyme system uniquely capable of reducing to

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ammonium A m m o n i u m becomes incorporated by N - f i x i n g organisms into amino acids, protein and other N-containing molecules, thus providing the p r i m a r y source of organically-bound N for growth. in reducing N is costly in terms of energy inputs in the Functioning of form of reductant and adenosine triphosphate (ATP) (see Pate and Layzell 1990), while effective functioning of is also compromised by its high Accordingly, where microorganisms fix in sensitivity to oxygen free-living state, the process occurs most e f f i c i e n t l y under near anoxic conditions - as applies for example, to the obligate anaerobe Clostridium when operating in waterlogged soils or to a range of anaerobic bacteria symbiotically in the guts of various animals such as termites and fixing higher animals. Where high external levels prevail, as in the case of environments supporting the free-living aerobic nitrogen fixers such as Azotobacter, their systems are protected by high rates of cellular respiration in conjunction with a number of other biochemical mechanisms that increase diffusive resistance to This chapter w i l l be dealing exclusively with symbiotic associations in which a particular bacterium (Azospirillum, Bradyrhizobium, Rhizobium), actinomycete (Frankia) or member of the cyanophyta (Nostoc, Anabaena) are housed in specialised nodule-like structures produced on roots or occasionally on stems of a higher plant host. In each of these cases, is overcome by anatomical factors which restrict entry of inhibition by oxygen into infected tissues and strategic forms of internal packaging of the to sites where the microsymbiont which further i n h i b i t diffusion of is functioning. Additionally, the symbiotic organs in question are provided with effective phloem transport systems for delivering photosynthetic products of the host to the enclosed microsymbiont. Readily metabolizable substrates are thereby c o n t i n u o u s l y available for assimilation of the potentially toxic released by the partner to its host. As a general rule, recently assimilated nitrogenous solutes are immediately exported to above ground parts of the host plant through the xylem and transpiration stream of the plant. Different host species employ specific sets of N-rich solutes such as ureides, amides or citrulline to effect such transfer (Pate and Atkins 1983; Pate and Layzell 1990). Symbiotic N fixation collectively results in high overall efficiency, a feature no doubt contributing to the much greater inputs of N made by such fixers (Jenkinson systems on a global basis compared to free-living 2001). As further testimony to the efficiency of operation of symbiotic associations, nodulated a n n u a l grain legumes such as peas, lupins or cowpea grown totally dependent on the atmosphere for their N supply carry only 5­ 10% of their total plant fresh weight as nodules, yet can still generate fixed N from these nodules at rates maintaining near maximum growth over the

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life cycle of the host plant (Layzell et al. 1984; Pate 1999). In so doing, f u l l y functional nodules regularly consume in the order of only 10-15% of the current net photosynthate produced by the shoot system.

2. Biodiversity, distribution and general biology of the associates 2.1 Cycad:Cyanophyta symbioses Cycads are frequently referred to as living fossils since the few species which we see today represent sparsely distributed relics of a group which extended back to the early Permian (Hill 1998b) and clearly comprised a diverse and major component of vegetation during Mesozoic times. The fossil record (see references listed in Jones 1993; Hill 1998b) depicts cycads as a highly distinctive monophyletic group with many widely distributed and probably common genera and species, most of which are not represented in the cycad floras which we see today. Nevertheless, the scattered present day distribution of l i v i n g forms across the tropics and subtropics of all major continents of both hemispheres speaks clearly of a remarkably ancient and fascinating Gondwanan heritage. According to recent accounts (Stevenson 1992; Grobbelaar 1993; Jones 1993; Hill 1998a, b) 10 genera and about 250 species of living cycads are currently known, comprising three families, Cycadaceae, Stangeriaceae and Zamiaceae, whose modern distributions probably indicate disjunctions dating back to fragmentation of Pangaea in the middle of the Mesozoic (Hill 1998b). Cycas, with some 80 species and the only genus of the Cycadaceae, is generally regarded as the most primitive and of Laurasian origin, with major lineages originating in Asia and certain members subsequently migrating to Australia ( H i l l 1998a). The Stangeriaceae consist of the monospecific genus Stangeria (South Africa) and three species of Bowenia (Australia). Zamiaceae contains all remaining living genera of cycads, namely Ceratozamia (11 species), Chigua (2), Dioon (10), Encephalartos (~50), Lepidozamia (2), Macrozamia (~40), Microcycas (1) and the largest genus Zamia (~60 species). Encephalartos is strictly South African, Macrozamia and Lepidozamia are confined to Australia, whereas the remaining genera (Dioon, Ceratozamia, Chigua, Zamia and Microcycas) are all essentially central American. Again, it would appear that Laurasian and Gondwanan elements contribute to present day distribution (Hill 1998a). According to l i s t i n g s provided by Grobbelaar (1993), Lindblad and colleagues (Pate et al. 1988; Paulsrud et al. 1998, 2000) and anecdotal observations made by or given to the author, virtually all cycads are capable of regularly forming symbiotic associations with filamentous cyanobacteria. This applies regardless of whether host plants are in natural habitat or raised in pot culture.

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The structure in which nitrogen fixation occurs in cycads is termed a ‘coralloid root’ in view of the densely packed, dichotomously-branched roots of which it is composed. Coralloid roots form on cycad seedlings (e.g. Macrozamia spp.) by infection of a forerunner structure termed a ‘pre­ coralloid’ root (see Ahern and Staff 1994). A pair of such roots arises spontaneously from just below the cotyledonary node and grows upwards (apogeotropically) to the soil surface (McLuckie 1922). Formation of precoralloid roots can take place under aseptic conditions (Nathanielsz and Staff 1975b; Lamont and Ryan 1977; Webb et al. 1984) and, unless cyanophyte symbionts are present, coralloid root formation fails to take place (J.S. Pate unpublished). The apogeotrophic nature of the roots suggests a pneumatophore-like f u n c t i o n , possibly reflecting an ancestral situation where cycads inhabited swamps and seasonally-waterlogged areas. Acquisition of symbiotic may therefore have arisen secondarily, f o l l o w i n g casual i n v a s i o n of the ‘pneumatophore’ by f r e e - l i v i n g cyanobacteria. Replacement sets of coralloid roots form periodically during the life of a cycad, whether from the base of the main body of the root-stock, or in older plants, at distance along lateral roots (Grobbelaar 1993). In either case, infection w i t h cyanobacteria must take place at the soil surface following emergence of apogeotropic roots. The cyanobacterial symbiont of coralloid roots is intercellular and harboured in specialised, loosely packed cells in the mid-cortex of the root. The densely packed algal filaments are readily recognisable macroscopically by their dark green colour and are surrounded by copious mucilage (see literature reviewed by Grobbelaar 1993; Ahern and Staff 1994). Proliferation of new filaments leads to progressive invasion of newly formed cortical tissue close to the apex of the coralloid roots. Young filaments closest to the apex are typically long and show similar ratios of vegetative thick-walled heterocysts as are encountered in comparable cells to free-living material of the same cyanobacterium. However, ratios of vegetative cells to heterocysts and total number of both types of cells per filament decrease with distance back from the root apex until, in regions where is most active, filaments are relatively few celled and may be composed of almost as many heterocysts as vegetative cells (Nathanielsz and Staff 1975a,b; Lindblad et al. 1991). Growth of cycads is generally recorded as being very slow, with large specimens in nature probably 1000 years of age (see Giddy 1974; Grobbelaar 1993; Jones 1993; Pate 1993). Plants are strictly dioecious with leaf production and subsequent reproduction markedly stimulated by fire in some taxa (Grove et al. 1980; Orndorf 1985; Pate 1993). Where populations of Macrozamia riedlei in jarrah (Eucalyptus marginata) forest are exposed

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to frequent fire, mature females fail to form cones following each burn whereas male counterparts typically cone after even very frequent fires (Pate 1993; J.S. Pate unpublished). Pollination of cycads was initially considered to be wind pollinated (see Hill 1998a) in view of the copious release of pollen (microspores) from male cones and the frequent finding of female cones bearing heavy deposits of pollen presumably originating from adjacent male plants. However, since cone scales fail to open fully and ovules awaiting fertilisation are deeply enclosed, superficial deposits of pollen are unlikely to be effective. It is now increasingly evident that insects may be involved in pollination of cycads (Norstog and Fawcett 1989; Jones 1993; Hill 1998a). For example, a snout weevil has been shown to pollinate Zamia furfuracea (Norstog 1987), species of weevil pollinate Macrozamia communis and Lepidozamia peroffskyana (Jones 1993), and a weevil and beetle pollinate Zamia pumila (Tang 1987). Cycad cones are generally thermogenic, that is, they achieve an intense climacteric in their respiration at their maturity, accompanied by a substantial rise in cone temperature and emission of chemically-distinctive odours. These features may be attractive to specific pollinating agents. Starch reserves within female cones provide a food source for developing larvae or beetles in the case of some of the above cycads. Cycad seeds t y p i c a l l y carry attractively-coloured edible coats surrounding their thick-walled seeds. The starchy kernel of the seed is usually highly poisonous and where indigenous human communities of various geographical regions consume seeds of native cycads, various treatment procedures are traditionally employed. Even so, carcinogenic compounds are unlikely to be removed and long-term effects on health may ensue. A number of cases are on record of poisoning of humans after consumption of untreated seeds as emergency sources of food, e.g. Cycas spp. during the Japanese occupation of Guam. Because of their large size, dispersal of cycad seeds is principally implemented by large megafauna such as hornbills and baboons (Africa) (Grobbelaar 1993), bandicoots and emus (Australia) (Pate 1993) and f r u i t bats (USA) (Tang 1989). Germination typically occurs on the ground surface and may be delayed for a year or more in certain cases, presumably due to an obligate after-ripening period. The large reserves of cycad seeds and prompt formation of coralloid roots on seedlings result in high rates of growth of juvenile plants and early achievement of N sufficiency, whether in native habitat or pot culture (Jones 1993, Pate unpublished data). The cyanobionts recoverable from coralloid roots of cycads are generally classed as Nostoc, or less frequently as Anabaena (Grobbelaar 1993). Usually only one strain of microsymbiont predominates in a specific coralloid root. Recent PCR-based molecular analyses of the genetic

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constitution of freshly isolated symbionts show (UAA) sequences similar to free-living Nostoc and to corresponding cyanobionts of certain lichens (Paulsrud et al. 2000) and two bryophytes (Costa et al. 2001). Similar analyses have revealed diversity in host specificity even among isolates of host genera obtained from botanic garden settings (Paulsrud et al. 1998). Bearing in mind the difficulty of identifying different strains/species of Nostoc purely on the basis of cultural and morphological characteristics, re-examination of earlier work (e.g. Grobbelaar et al. 1988; Marshall et al. 1989) indicating taxonomic diversity of symbionts and specificity or promiscuity of cyanobiont:host relationships is clearly required using molecular-based approaches. Nevertheless, there is already sufficient evidence that fully effective symbioses can be established on a single host using any of a broad range of Nostoc strains or species. Furthermore, coralloid roots form readily on cycads raised from seed using non-sterile soil based pot culture. With inoculation rarely practised in such situations, local rooting substrates must have provided cyanobiont strains which can engage as effectively in symbioses as occurs naturally in soils of the habitats from which the cycads were derived. 2.2 Cyanophycean (Nostoc) symbiosis with Gunnera

This association provides the only known case of an algal-based in Angiosperms. Unlike lichen, liverwort, fern and cycad associations, the cyanobiont of Gunnera is intracellular and the mode of infection and cellular functioning of the symbiotic structures exhibit an interactive complexity equalling that of nodules of actinorhizal and Rhizobium-based symbioses. The reader is referred to the review of Becking (1977) for early literature on the geographical distribution and ecology of Gunnera and more specific studies indicating that symbiotic occurs at high frequency across the 50 or so species of the genus. Gunnera is the sole genus of the Gunneraceae which is distributed across the tropical and subtropical Southern Hemisphere, including regions of South America, New Zealand, SE Asia and South Africa. The genus is associated with wet conditions, sometimes at high altitudes where the plant often comprises a pioneer element of disturbed habitats such as landslides and areas adjacent to volcanic activity. The Gunnera symbiosis is relatively easy to manipulate under laboratory conditions and has resulted in a detailed understanding of the morphological basis of infection (see reviews of Bergman et al. 1992, 1996). The microsymbiont is always a Nostoc sp. Infection occurs through pre-formed pairs of glands at the junction of each leaf with the stem. Carbohydrate-rich mucus secreted by the

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glands appears to attract a number of microorganisms in addition to infective cyanobionts. Proliferating first on the gland surface, the cyanobacterium then enters a motile infective stage. The hormogonia involved invade the gland by an intercellular route and eventually accumulate in great quantity within an extracellular cavity at the base of the gland. Presumably due to some form of mitotic stimulus emanating from the microsymbiont, new host cells are initiated close to the gland and it is these which become specifically invaded by the Nostoc. Achievement of an intracellular location for the microsymbiont is clearly host determined, since the same strain of cyanobacterium will remain intercellular, yet s t i l l capable of when in symbiotic association with non-angiosperms such as Azolla and liverworts. Once inside the cells of the ‘nodule’ of Gunnera, filaments of the cyanobiont divide, their vegetative cells enlarge, and heterocysts differentiate. activity achieves a maximum as intracellular infections spread through the nodule, with up to 65 to 80% of the cells of the filaments of the symbiont found to consist of heterocysts as opposed to vegetative cells. 2.3 Actinomycete (Frankia) symbioses with non-legume Angiosperm taxa Representatives from eight different families encompassing 24 separate genera are currently classed as actinorhizal. The total species resource involved is over 200 in number (Table 1), although presence of actinorhizal nodules has yet to be confirmed for some genera. A number of instances (e.g. in Casuarina, Dryas, Myrica) are on record where nodulation has been observed in certain locations but not others (Bond 1967, 1974; Becking 1977; Torrey 1978). Direct proof of activity by feeding or acetylene reducing capacity, has been confirmed for almost all genera, but only for a relatively small portion of the species known to engage in actinorhizal associations. Actinorhizal species embody a very diverse range of locations and habitats, often associated with natural or other types of disturbance. Of the larger ecologically-important genera, Allocasuarina/Casuarina are found widely and commonly in Australia, Gymnostoma occurs in Malaysia through to the West Pacific, Ceanothus in North America, while species of Elaeagnus are encountered in Europe, Asia and North America, and Alnus and Myrica are broadly spread across the northern hemisphere with some species in South America and South Africa respectively. Various inventories of ecological and physiological features of actinorhizal species (see Becking 1977; Torrey 1978; Dawson 1990; Benson

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and Silvester 1993) suggest a collective global range of habitat associations which may be regarded as r i v a l l i n g in breadth that of the hundred-fold larger species resource of legumes. Herbaceous and annual forms are essentially absent among actinorhizal plants but members of certain genera (e.g. Alnus, Ceanothus, Comptonia, Dryas, Myrica), act as important pioneer components of natural habitats such as mobile sand dunes, deposits left by retreating glaciers and mineral-rich soils exposed following landslides or fire. These attributes, combined with the ability of certain species to perform well in saline and dry or acid permanently waterlogged environments have prompted fairly widespread use of actinorhizal taxa for rehabilitation of mine sites, seaside plantings and reforestation of eroded soils at high altitude (Wheeler and M i l l e r 1990). Some taxa (e.g. Casuarina, Alnus) are also

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exported for timber or fuel alongside woody legumes in agroforestry enterprises. There are a number of reports of substantial amounts of fixed N being returned under both natural or cultivated conditions by taxa such as Casuarina, Alnus, Hypophae and Ceanothus (see Becking 1977; Benson and Silvester 1993). Phylogenetic studies, especially recent molecular-based cladistic analyses, continue to shed light on the possible origin of actinorhizal and other higher plant-based symbioses. The molecular-based (chloroplast gene rbcL) perspective recently provided by Doyle (1998) indicates that capacity probably originated on several for nodulation and associated independent occasions during evolution of angiosperms and that closer relationships between legumes and actinorhizal taxa are now evident than were previously suspected. The scheme presented by Doyle (1998) (incorporated into Figure 1) places all known actinorhizal Angiosperms within the single relatively small ‘Rosid I’ group of families which also includes rhizobial symbioses between legumes and the non-legume genus Parasponia (Ulmaceae). The analysis thus supports a general predisposition towards nodulation within this group (Soltis et al. 1995; Swensen and Mullin 1997). Parallel examination of actinorhizal plants by Swensen (1996), using rbcL sequence data supports a cladistic tree embracing four distinct groupings of actinorhizal taxa. One group comprises three families, Betulaceae, Casuarinaceae and Myricaceae alongside three other nonnodulating families, the second group consists of the family Rosaceae which contains mostly non-nodulating and a few actinorhizal genera, the third group comprises the actinorhizal families Coriariaceae and Datiscaceae together with three n o n - n o d u l a t i n g families. The final group includes Elaeagnaceae and Rhamnaceae, both of which contain nodulating and nonnodulating taxa. Actinorhizal nodules are essentially modified rootlets which typically develop dense aggregate c o r a l l o i d - l i k e structures through repeated dichotomous branching of root apices. Nodules can be long lived and achieve diameters exceeding 5 cm under field conditions (Schwintzer and Tjepkema 1990). In waterlogged habitats (e.g. in cases involving species of Myrica, Casuarina and Alnus) nodules may be located close to the surface or emerge distinctly above the rooting substrate or may even form on trunks when trees are flooded (e.g. Casuarina spp., Duhoux et al. 1993). In sharp contrast, nodules are normally absent from surface soil layers in dry habitats, albeit in some cases recoverable in appreciable amount where excavations are made deep down a soil profile. The early literature (see review by Becking 1977) records that invasion of roots of a c t i n o r h i z a l p l a n t s takes place via root hairs, and that

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characteristic hair curling precedes infection, just as in the case of the

rhizobial infection sequence of many legumes. In Alnus spp., invasion of

root hairs leads to a ‘pre-nodule’ being formed by proliferation of underlying

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pericycle tissues, just as occurs during the initiation of regular lateral roots on uninfected plants. This clearly confirms that the symbiotic organ is essentially a modified root. Intracellular infection of cortical cells of the pre-nodule leads to hyphae of the microsymbiont becoming transformed into thick-walled, internally septate ‘vesicles’ and this is where nitrogen fixation is generally assumed to be located. The same early work (see Bond 1974; Becking 1977) describes two types of nodules, one where constituent infected roots extend slowly and then stop growing (the Alnus type), the second type (Myrica, Casuarina type) where continued attenuated apogeotropic growth of infected roots results in the dense central body of the nodule becoming invested in a sparse outer clothing of thin uninfected roots. The review of Berry (1994) summarises some of the more recent literature on the biology of actinorhizal symbioses, including discussion of the biochemical interactions of host and microsymbiont likely to be involved in the nodulation process. Infection of certain hosts (e.g. species of Elaeagnaceae, Rhamnaceae and Rosaceae) typically takes place by an intercellular route, not via root hairs. The type of infection pathway which occurs is clearly not determined by the microsymbiont, since when one Frankia strain infects two different hosts, invasion may be either intercellular or via root hairs, depending on the host which is involved in consummation of the symbiosis (Miller and Baker 1986; Racette and Torrey 1989). Secretions of extra-cellular polysaccharide materials by hosts may be involved in recognition and sustenance of Frankia where invasion is intercellular. After entering the cytoplasm of host cells, the microsymbiont becomes enveloped in a host-derived membrane while still continuing for a time to containing vesicles. Berry (1994) suggests divide and form further that the multi-lamellate hopanoid external layer of the vesicle protects the system of the Frankia against inactivation by Analogies can thus be drawn between this lipid layer and the thick wall encapsulating heterocysts of cyanobacteria or the peribacterial membranes enveloping bacteroids of legume nodules (although there is no direct evidence that this membrane effects diffusion or protects from inactivation by Berry (1994) identifies onset of N starvation in a host as a causative factor in activity in the initiation of nodules and then in the promotion of the resulting vesicles. Vesicles are absent in actinorhizal nodules of Casuarinaceae so fixation must be accomplished in such cases in unspecialised cells of the endophyte. Interestingly, haemoglobin is encountered at relatively high levels in Casuarina nodules, just as in Rhizobium-based nodules. In the rbcL gene sequence analysis conducted by Swensen (1996), detailed analysis of the four clades of actinorhizal species referred to earlier (Table 1,

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Figure 1) provided some interesting correlations between molecular based attributes and certain morphological and anatomical features of the nodules concerned (e.g. mode of infection, presence or absence of uninfected apogeotropic roots on nodule surfaces, presence or absence of distinct vesicle-like structures and the shape and location of vesicles, if present, in infected tissues). Although not providing information for many actinorhizal taxa, the analysis of Swensen (1996) (see Figure 1) further suggests that actinorhizal-type symbioses probably originated on several independent occasions during the evolution of Angiosperms. The Frankia organism associated with various ‘actinorhizal’ Angiosperms described above is u s u a l l y classified as a filamentous prokaryote (Actinomycete), as indicated initially from morphological and cultural characteristics and more recently in terms of the high guanosine and cytidine content of its DNA and sequence analysis of its 16S ribosomal RNA genes (Berry 1994, Swensen and Mullin 1997). Pure cultures of the organism were not available u n t i l the studies of Callahan et al. (1978) on Comptonia, but now include a broad range of isolates from most genera (e.g. see Kohls et al. 1994; Clawson et al. 1998). Substantial progress is continuing on the extent of cross inoculation specificity between strains of Frankia and various groupings of host taxa. However, interpretation of data is difficult where isolates from a nodule are subsequently shown not to re­ infect the same host (e.g. Racette and Torrey 1989) or where pure isolates fail to re-nodulate a plant host whereas inoculations with crushed nodule suspensions result in normal nodulation (Kohls et al 1994). Cases are also reported where nodules contain several strains of Frankia (Bloom et al. 1989) or where isolating media appear to have selected symbotically ineffective strains, while f a i l i n g to support growth of effective ones. Furthermore, some Frankia strains infect hosts which are ecologically or taxonomically very distant from the hosts of origin and 16s rDNA signatures of certain strains most closely resemble those of strains infecting distantly related families of actinorhizal species. The recently developed phylogenetic tree for Frankia and phyletic neighbours (Normand and Bousqet 1989) indicates four clusters of genomic species based on 16S rDNA. The first is a group effective on Alnus, Casuarina, Allocasuarina and Myrica, the second on Dryas, Coriaria and Datisca, the third on Eleagnaceae and Gymnostoma and the fourth is a group of strains isolated from a diverse range of host plants, but are genetically distinct from the other clusters. Evidence indicates that a number of Frankia strains can cross nodulate within but not outside specific ‘cross inoculation’ groups of hosts (e.g. see studies of Clawson et al 1998; Akimov et al. 1990; Rodriguez-Barrueco et al. 1993). Thus, certain actinorhizal hosts may have co-evolved with

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specific microsymbionts as indicated by the marked compatibility groupings of certain Frankia strains for specific host plant groupings. Genes coding for nitrogenase have now been identified by probing Frankia DNA. In one case (the nif K gene), high sequence homology is evident against the same gene from cyanobacteria. This result and topologies recorded for other nif genes, are regarded by Normand and Bousquet (1989) as indicative of ‘illegitimate’ gene transfers between Frankia, cyanobacteria and other fixers such as Clostridium. Judging from the much more extensive literature on Rhizobium, symbiotic behaviour is likely to become greatly modified by relatively small changes in genomic composition and, if applicable to Frankia, anomalous host patterns of infectivity and effectivity across actinorhizal species might be expected to arise relatively easily during evolution of the various symbioses which we know of today. 2.4 Rhizobial:legume symbioses ‘Rhizobial’ and ‘Rhizobium-type’ are terms used to refer to that broadlybased group of nodule-forming bacteria which can fix nitrogen symbiotically with legumes. In one exceptional case, involving such bacteria extends to the legume-like nodules formed by the non-legume Parasponia (see next section). The bacteria concerned comprise a vast assemblage of forms, some of highly restricted and others of highly promiscuous nodulating capacities in respect of host species. Furthermore, some are capable of in the free-living state others only in the protected near anaerobic environment of the nodule. The Leguminosae (Polhill and Raven 1981) ranks in size with other major Angiosperm families and far exceeds in diversity of life and growth forms and ecological preference similarly large families such as Orchidaceae, Gramineae and Asteraceae. Some 640 genera and 18,000 species are recognised and the family is usually treated taxonomically as three sub-families (Papilionoideae, Mimosoideae and Caesalpinioideae). These are distinguished from one another mostly on the basis of differences in floral morphology. The 31 tribes, approximately 420 genera and 12,000 species of Papilionoideae are predominantly herbaceous and contain virtually all agriculturally important pasture and crop legumes, whereas the five tribes, 64 genera and 2,900 species of Mimosoideae and four tribes, 153 genera and 2,200 species of Caesalpinioideae are mostly woody and mostly not under cultivation. Trees, shrubs and lianas are well represented among the constituent taxa of all subfamilies (Sprent 1999). The current state of knowledge of the extent of nodulation among legumes is summarised by Sutherland and Sprent (1993), Sprent (1994, personal communication and see Table 2) and the reader is referred to the

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classic early work of A l l e n and A l l e n ( 1 9 8 1 ) around which a large proportion of the current database has been b u i l t . Recent studies of nodulation, especially of Caesalpinioideae in Brazil (see Faria et al. 1989) include a number of previously unstudied Caesalpinioideae and have proved particularly useful in clarifying the situation for woody taxa neglected in earlier work. Sprent (2001) has recently provided extensive listings of records for nodulation and in the process, corrected erroneous earlier records and discussed issues relating to groupings where certain members nodulate while others apparently do not. Over one fifth of the total species resource of the Leguminosae has been examined in respect of nodulation. In the Papilionoideae, where almost two thirds of the genera have been examined. 97% of the species examined have proved to be capable of nodulation. Notable exceptions are the Dipterygeae (all species so far studied are non-nodulating), Sophoreae and Swartzieae ( i n c l u d i n g n o d u l a t i n g and n o n - n o d u l a t i n g genera) (Sprent 2000). Nodulation is also very common (90% of species examined) in the Mimosaceae for which two thirds of the genera examined are nodulated, five are non-nodulated and for some other genera, both positive and apparently negative records have been reported. Puzzling cases of non-nodulation have been recorded for Acacia, a genus whose species are mostly prolifically nodulated (Odee and Sprent 1992). Records of the extent of nodulation are still relatively incomplete for Caesalpinioideae. However, all species so far examined in 13 genera of the subfamily were found to be nodulated, while nodulation was apparently absent in a further 38 genera, and in another 20 genera both nodulated and non-nodulated species were encountered. It thus appears that members of the Caesalpinioideae are predominantly not nodulated. From the earliest observations of nodule morphology (e.g. see Spratt 1919; Fred et al. 1932; Allen and Allen 1981), effectively fixing nodules were shown to vary c o n s i s t e n t l y between genera and large taxonomic groupings in terms of shape, pattern of growth and longevity of nodules. Corby (1981) provided the first comprehensive account of such variation in a classification of n o d u l e s on the basis of whether they were branched, essentially non-branched or exhibited an essentially dimorphic morphology. Taxonomic affiliations were implied in designations such as ‘astragaloid’, ‘crotalaroid’ and ‘lupinoid’ among the branched types of nodules and ‘aeschynomenoid’, ‘desmodioid’ and ‘mucunoid’ among the mostly unbranched types of nodules. Correlations between host taxonomy and nodules type have subsequently been borne out to a certain extent where more extensive groupings of legumes in terms of types of nodules have been considered (see Sprent 1981 and more recent treatments of Sprent et al. 1989; Sutherland and Sprent 1993; Doyle 1998; Table 2).

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Sprent (1981) first drew attention to the prevalence of legumes which produce ureides (allantoic acid and allantoin) as exportable products of fixed N among taxa (mostly Phaseoleae) which develop relatively large unbranched, determinate nodules. Conversely, taxa with branched indeterminate nodules and a number with small unbranched nodules are widely known to produce amides (asparagine and to a lesser extent glutamine) when exporting fixed N (Pate and Atkins 1983; Pate 1986). Nodule initiation may also i n v o l v e rhizobia invading root hairs, root epidermis, or sites of emergence of lateral roots. In some unusual cases (e.g. species of Aeschynomene and Sesbania), nodules may form on stems. Again, there is good evidence of taxonomic consistencies in such characteristics (Sprent 2000). The reasonably substantial fossil records of legumes (see Herendeen and Dilcher 1992) suggest an origin in the Cretaceous with the extant sub­ f a m i l i e s already well represented by the early Tertiary. Classic morphologically-based analyses (see review of Polhill and Raven 1981; Crisp and Doyle 1995) delineate most of the orders recognised today, but are not f u l l y supported by recent cladistic analyses based primarily on morphological (see articles in Crisp and Doyle 1995) or molecular based criteria such as rbcL gene analysis (see Doyle et al. 1997). Using rbcL sequence data, Doyle (1998; figure 2) suggests three independent origins for nodulation and the phylogeny proposes unbranched intermediate ‘caesalpinioid’, nodules to be the ancestral nodule type, basal to all three o r i g i n s . M o d i f i c a t i o n of this basic nodule type in the Papilionoideae is then suggested to have given rise to indeterminate

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branched nodules, some of lupinoid type with a peripheral meristem, or to determinate nodules of aeschynomenoid type (e.g. Arachis) or to the desmodioid, ureide-producing nodules of many Phaseoleae (see Table 2). 2.5 The Rhizobium-Parasponia symbiosis This unusual class of symbiosis is the only one so far known between Rhizobium (sens. lat.) and a non-legume (Parasponia). It was first described by Trinick (see reviews of Trinick 1982; Trinick and Hadobas 1988), who i n i t i a l l y referred to the host concerned as a species of Trema. This closely related sister genus to Parasponia is now considered non-symbiotic. Parasponia is a member of the family Ulmaceae and its five species inhabit the Malay / West Pacific region (Mabberley 1997). Species of Parasponia prefer disturbed habitats and nodulate well at low pH (Trinick and Hadobas 1988). Nodules of Parasponia resemble those of actinorhizal species in being essentially modified roots with a central vascular cylinder surrounded by a cortical region of infected tissues (Trinick 1979). Swensen (1996), in an evaluation of actinorhizal symbioses based on rbcL sequence analyses of hosts, places Parasponia in a clade together with the actinorhizal families Elaeagnaceae and Rhamnaceae and a number of non-actinorhizal h a m a m e l i d f a m i l i e s . In a further comparison with actinorhizal genera, Swensen (1996) suggests that the intercellular pattern of bacterial invasion and absence of apogeotrophic root extensions from nodules in Parasponia find counterparts within two of the four clades (I and II) of Frankia-based actinorhizal species. Conversely, the presence in the access to nodule of Parasponia of an outer anatomical barrier restricting the nodule and appreciable levels of haemoglobin in infected tissues of nodules of this symbiosis finds parallels in the actinorhizal taxa Casuarina/Allocasuarina, (Clade IV of actinorhizal family representatives). Trinick (1980) and Trinick and Hadobas (1988) show that both slow and fast growing rhizobia can nodulate Parasponia while also engaging in effective symbioses with certain groups of tropical legumes. Interestingly, the bacterial phylogeny recently proposed by Doyle (1998) provides an example of a single Rhizobium strain (NGR 234) exhibiting a very wide host range, including a diverse assemblage of legumes and Parasponia. 2.6 Rhizobial associates of legumes and Parasponia The literature concerning nodule bacteria isolated from legumes spans more than a century, although studies up to the 1960’s deal almost exclusively with agriculturally-important grain and forage legumes (e.g. see Fred et al. 1932). Incentive for such studies related principally to the introduction of mostly European legumes into farming regions of the world where soils did not u s u a l l y c o n t a i n c o m p a t i b l e i n d i g e n o u s rhizobia. Alongside

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morphological descriptions of isolated bacterial colonies and whether isolates from nodules were fast or slow growing, early descriptions of rhizobia were concentrated particularly on cross inoculation specificity, in the hope of predicting the likelihood of success when inoculating cultivated legumes under f i e l d c o n d i t i o n s . Grading of strain effectiveness when matched with distantly and closely related host genotypes and persistence of introduced strains in competition with indigenous rhizobia were equally important practical research issues, when attempting to fine tune for maximum symbiotic fixation returns under specific situations. Through use of sophisticated means for characterising rhizobia by molecular-based techniques such as mapped restriction site polymorphism (MRSP) analysis of 16s rRNA genes, and PCR/DNA fingerprinting using repetitive sequences (REP-PCR), concepts concerning the genetic diversity of rhizobial symbionts are being c o n t i n u a l l y revised and augmented. Included in such assessments have been an increasing number of studies on symbionts of native legumes (e.g. see Jordan 1984; Dobereiner 1984; Lieberman et al. 1985; Roughley 1987; Galiana et al. 1990; Sutherland and Sprent 1993; Laguerre et al. 1997; Haukka et al. 1998; Lafay and Burdon 1998, Burdon et al. 1999; Thrall et al. 2000). Particularly relevant reviews on these issues are provided by Padmanabhan et al. (1990), Young (1991, 1996), Sprent and Raven (1992), Sutherland and Sprent (1993), Bryan et al. (1996), Doyle (1998) and Young et al. (2001). The key findings of these studies are: Six ‘genera’ of n i t r o g e n - f i x i n g bacteria are currently recognised ( Azorhizobium, Bradyrhizobium, Mesorhizobiu m , Phyllobacterium, Rhizobium and Sinorhizobium) and earlier ‘species’ concepts have been substantially altered (Young et al. 2001). Phylogenetic studies based on 16S ribosomal RNA gene sequences suggest that rhizobia may have arisen from several major lineages of proteo-bacteria, with affinities in some cases with non-symbiotic forms previously recognised as belonging to a separate genus, Agrobacterium but now included in R h i z o b i u m (Young et al. 2001). Although evaluating only a small fraction of the known taxonomic resource of rhizobia, the above studies increasingly suggest a poor level of correlation against earlier-established cross inoculation groupings. This is not surprising when one remembers that in many cases, the host range of a rhizobium is plasmid-borne and therefore potentially interchange­ able with other compatible bacterial strains co-existing in free-living state outside the host legume and classifications must consider this (Young 2001). There is evidence suggesting that characteristics displayed by many tropical rhizobia, including those associated with woody taxa of native

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habitats, correlate poorly in terms of culture morphology and growth rates, host ranges and molecular-based attributes when matched against rhizobia isolated from mostly temperate herbaceous legumes of agricultural significance. Thus, rhizobia of native taxa showing characteristics i n t e r m e d i a t e between those of Rhizobium and Bradyrhizobium, may exhibit complex grades in degrees of effectiveness across host groupings (as shown for example for Acacia spp., Burdon et al. 1999), and may also i n c l u d e slow-growing isolates exhibiting unusually narrow host specificity. Molecular and biochemical evidence suggests that this is most likely due to directed mutagenesis altering many of these ‘specificities’. These studies will clearly prove vital to improving our understanding of the dynamics of bacterial populations and their capacity to co-evolve with hosts. 2.7 Nutrient acquisition strategies of symbiotic associations in respect of limited availability of phosphorus While there is still continued debate world-wide as to which of the elements essential to growth and s u r v i v a l of higher plants are likely to constitute primary limitants to productivity of natural ecosystems, N or P feature most prominently in the literature in this respect. Thus, N is frequently identified as limiting in temperate Northern Hemisphere regions and certain young soils of tropical regions (Vitousek and Howarth 1991), whereas P is more commonly implicated in older heavily leached old landscapes of continents such as Australia, South America and Africa (Reddell 1993; Crews 1993, 1999; Newman 1995). The situation is further complicated by ecosystems appearing to switch in time between phases of limitation by P and N, as reported by Raich et al. (1996) for lava flows in Hawaii and referred to later in respect of post-fire recovery of ecosystems of south west Western Australia. Alongside evidence of P as a regular limitant, one should also consider the confusing literature concerning the basic nutritional role of the element associations. Some accounts (e.g. Robson 1983; in symbiotic Israel 1987; Crews 1993; Reddell 1993) indicate that requirements for P by symbiotically-active legumes are measurably greater than those of fixing counterparts or comparable non-legumes. However, as discussed by Reddell (1993), this may merely denote a catalytic effect of P on nodulation and early growth, with benefits then flowing automatically under N limited situations where companion non-legumes would be specifically disadvantaged. In any event, there should be strong selection pressure in both and non-fixing plants for effectiveness in capture of limiting resources of P, as is indeed regularly practised using specialised mechanisms

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for accessing intractable pools of the element not available to unspecialised root systems of other species. By far the most widely deployed strategy for combating limitation in respect of P involves symbiosis with various forms of mycorrhizae (e.g. see Allen 1992; Smith and Read 1997; Brundrett and Abbott this vol.; EgertonWarburton et al. this vol.). Alternatively, improved access to P can be achieved by non-mycorrhizal ‘proteoid’ or ‘cluster’ roots (Purnell 1960; Lee 1978; Lamont 1982; 1993 and recent reviews of Dinkelaker et al. 1995; Skene 1998; Pate et al. 2001; Pate and Watt 2001). Not surprisingly therefore, there is now overwhelming evidence that the symbioses regularly employ ‘dual’ symbiotic vast majority of systems, namely their regular microorganism and some form of mycorrhizal partner. The mycorrhizal elements most commonly involved among herbaceous and woody legumes are endophytic AM (arbuscular) type mycorrhiza but ectomycorrhizas may feature in certain woody legume taxa (e.g. Aziz and Sylvia 1993; Herrera et al. 1993; Sprent 2001). Among actinorhizal species, certain taxa. (e.g. Gymnostoma, Casuarinaceae) produce mycorrhizal root nodules colonised by arbuscular mycorrhiza (Duhoux et al. nodules. Incidentally, similar 2001) additional to regular mycorrhizal structures are known to occur on certain gymnosperm taxa and were erroneously concluded in the early literature to be capable of fixing (see McLuckie 1923; Bond 1963, 1967; Morrison and English 1967; Becking 1977).

3. Quantification of the likely returns of fixed N by native associations 3.1 Assessing symbiotic competence The underlying premise of this chapter is that symbiotic associations between microorganisms and higher plants constitute key beneficial components by providing fixed atmospheric to themselves and eventually to other organisms of their ecosystems. The first step towards testing this supposition is to demonstrate visually that the putative of a system do indeed bear symbiotic organs and that these appear likely to be currently active in Potential symbiotic activity would be indicated, for example, by presence of green, cyanobacteria-filled in midcortical tissues of the coralloid roots of cycads, by healthy arbuscule­ containing infected tissues in actinorhizal symbioses or by haemoglobinfilled bacterial tissues in nodules of legumes. The second step would be to attempt to determine what proportion of total plant mass is devoted to symbiotic structures during different seasons or over a whole year. For example, if the complement of nodules on a plant were to regularly comprise less than 1% of current plant fresh weight, and

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nodules were recorded as absent or unhealthy for much of the year, one would be safe in concluding that the association was only minimally Conversely, heavy reliance on fixation would dependent on atmospheric be expected if proportional mass of healthy symbiotic organs were regularly in the range 5-15% of p l a n t fresh weight, as observed for example in many a g r i c u l t u r a l l y - i m p o r t a n t legume crops and pasture species under N-limiting conditions (Pate 1977; Sutherland and Sprent 1993). However, it should be remembered that greater proportional weights of symbiotic organs would be expected of rapidly growing host plants than if plant growth was being suppressed by environmental constraints. The third step would be to determine by suitable chemical assay the of symbiotic organs harvested sequentially specific activity in from a population of the species across a season of study. By combining such information on activity with corresponding data for seasonal changes in weight of symbiotic organs, it should then become possible to predict with a reasonable degree of accuracy how much is being fixed over a particular time frame and environmental situation. The final step would be to assess the absolute amounts and proportions of the total biomass of an ecosystem comprised by the symbiotic association under study, and thereby assess the annual inputs of fixed N which it is likely to be making per u n i t habitat area per year. One would thus achieve the ultimate goal of i d e n t i f y i n g the likely quantitative importance of such inputs towards meeting the long-term demands for N by all interacting biota of the system. F u l f i l l i n g this step-wise assessment of returns within native ecosystems is both time consuming and logistically difficult, even in those relatively few cases where u n i f o r m populations of the symbiotic association are present and where r e l a t i v e l y complete recoveries of symbiotic organs can be made from the host plants concerned. Not surprisingly therefore, fully comprehensive evaluations of benefits of symbioses have only been rarely accomplished for native ecosystems (see Sutherland and Sprent 1993), despite many reported successes in respect of crops and pasture herbaceous legumes of agricultural systems (Pate and Unkovich 1998) and for some woody l e g u m i n o u s taxa of plantation and agroforestry systems (see Unkovich et al. 2000; Peoples et al. 2001). Access to reliable and easily applied assay systems for measuring nitrogenase activity is obviously pivotal to precise quantification of Three contrasting methodologies have been employed inputs of fixed for this purpose over the years and the merits and limitations of each will be addressed below.

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3.2 Acetylene reduction assay technique Despite many inadequacies, the reduction assay method remains of considerable value f o r i n i t i a l testing of whether a newly discovered system is currently active symbiotically and, in broader context, for comparing on a activity between semi-quantitative basis the extent of variation in species, habitats and seasons (though errors can be 2-3 fold). For example, studies conducted in mediterranean-type ecosystems of southern Australia involving shrub legumes of genera such as Acacia, Aotus, Bossiaea, Dillwynia, Jacksonia, Kennedia, Platylobium and Viminaria collectively have shown that nodulation cycles are strongly seasonal, with peak specific reducing a c t i v i t y in spring, loss of nodules with onset of the summer drought conditions and initiation of new symbiotic organs is normally delayed until the following wet season (e.g. Grove et al. 1980; Lawrie 1981; Monk et al. 1981; Hingston et al. 1982; Langkamp and Dalling 1982; Walker et al. 1983; Hansen et al. 1987a,b; Sutherland and Sprent 1993; Pate and Unkovich 1998). Where symbiotic performances of short-lived understorey shrub legumes have been followed after recruitment from seed (even up to 70-80%) is after fire, greatest dependence on atmospheric typically evident in j u v e n i l e stages when phosphorus is transiently readily available. Then, as these legume stands approach middle age and resources of the element become c r i t i c a l l y l i m i t i n g , symbiotic dependence tends to decline sharply and may even cease entirely (see case studies reviewed by Pate and Unkovich 1998). In one notable case (Hansen and Pate 1987a,b), mid aged, mostly non-nodulated legume stands in native south west Western Australian eucalypt forest were found to develop prolific, symbioticallyactive sets of nodules when phosphate was applied, indicating that in nutrient impoverished ecosystems of this type, symbiotic performance is normally limited by phosphorus supply. By contrast, growth and fixation continued unabated and at much faster rates in comparably aged stands of the same species sown on phosphate-fertilised rehabilitation plots w i t h i n mined areas of the same forest. Turning to cyanophycean:cycad symbioses, acetylene reduction assays of coralloid roots, combined in certain cases with direct validation of have recorded appreciable nitrogenase activity occurs in fixation by approximately one fifth of the world’s recognised cycad species (Grobbelaar reduction based 1993), albeit mostly in respect of pot grown plants. A study of two naturally growing populations of Macrozamia riedlei in native mixed Banksia, Eucalyptus, Allocasuarina woodland near Perth, Western Australia (Halliday and Pate 1976) provided estimates of 18.8 and 18.6 kg N in situations where the cycads comprised the dominant understorey element. Incorporating i n f o r m a t i o n on population densities, mean biomass and nitrogen contents, these cycad populations were suggested to be fixing

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nitrogen at rates capable of doubling the N content of plant biomass roughly every 10 years (see further discussion by Pate and Unkovich 1998). Finally, the acetylene reduction technique has featured prominently in activity conducted across a diverse qualitative validations of assemblage of genera and angiosperm families known to form actinorhizal nodules (e.g. see reviews of Quispel et al. 1993 and Subba Rao 1993). In were indicated most of these cases appreciable rates of symbiotic under natural conditions or where species were being employed in rehabilitation of degraded areas.

3.3 natural abundance (NA) assays of symbiotic dependence To illustrate some of the failures and successes of the assay method which have been obtained in their studies, Pate and Unkovich (1998) cited a set of equivocal results obtained in their study of two populations of the cycad Macrozamia fraseri and companion woody vegetation in heathlands values for non near Eneabba, Western Australia. Comparisons of fixing reference species and the respective cycads then suggested that substantial levels of fixation appeared to be occurring in one population but not in the other. However, effective symbiosis was strongly suspected for both populations, since each showed prolific sets of healthy coralloid roots on virtually all cycads excavated. In contrast to the above, Pate and Unkovich (1998) also refer to a supposedly well validated result obtained by Tennakoon et al. (1997) in a study of N interrelationships of the xylem-tapping root hemiparasite Santalum acuminatum and its woody hosts in coastal heath near Dongara, data showed close matchings of the Western Australia. The resulting isotopic signatures of the Santalum and the various cohabiting associations (Acacia spp. and Allocasuarina sp.) at the sites studied, whereas other less parasitised or non-parasitised non N-fixing taxa showed values of heavily parasitised significantly more positive values. With legumes and of the matching sets of Santalum close to zero of the atmosphere), compared to +1 to +4 %° for other hosts, dependence of the legumes and the parasite on fixed N was considered highly likely. Excavations examining the intensities of formation of haustoria on the various hosts exploited by the Santalum corroborated the above conclusion by demonstrating highly biased parasitism by Santalum, and indeed other parasitic species, towards the nitrogen-fixing associations. A second equally definitive study using signatures is that recently conducted on Australian mulga ecosystems (Erskine et al. 1996; Pate et al. 1998). In this case, a range of Acacia spp. (predominantly A. aneura) in the general mulga were found to be essentially non symbiotic, and this was attributed to i n h i b i t i o n of nodule formation and functioning by the high

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levels of nitrate in soil and ground water in the region. In contrast, legumes growing in the heavily leached sand dunes surrounding salt lakes in the signals indicative of region were heavily nodulated and showed buoyant symbiotic activity. To summarise, the quantitative information concerning the nitrogenfixing performances of microorganism:higher plant associations is very incomplete and mostly of a qualitative rather than quantitative nature. Technological and logistical difficulties beset measurement of nitrogen fixation inputs by woody taxa of natural ecosystem and further progress in this direction w i l l continue to be slow u n t i l much greater resources of finance and manpower become available, particularly towards evaluation of autotrophic inputs of N in survival and functioning of pristine and anthropogenically-modified ecosystems.

4. Conservation issues relating to symbiotic associations There is ever increasing concern among a wide-ranging body of conservation-minded biologists about the alarming loss worldwide of natural habitat and associated attrition of flora and fauna. There is every reason for believing that the threats to biodiversity and ecosystem structure should partnerships as to all other associated apply as much to symbiotic plants and microorganisms of a habitat. Dealing in turn with non the higher plant components considered in this chapter one would single out cycads as a particularly threatened group, due to the extraordinary large demand for a number of cycad taxa in the horticultural trade, combined with the failure of many of the highly localised populations of certain species to provide seed for such purposes and the limited success of ex situ conservation programs. However, on the positive side, appropriate cyanophycean partners to cycads are generally prevalent, judging from the very high frequency of coralloid root formation in natural habitats, pot, glasshouse and garden culture. However, the purist might well argue that irreplaceable cyanophycean resources would be lost wherever the natural populations of the host are debilitated to the point of extinction (e.g. in the case of several Encephalartos species in South Africa). Symbioses i n v o l v i n g Frankia and actinorhizal higher plants, rhizobiumtype symbioses of Parasponia and cyanophycean symbiosis with Gunnera provide little or no evidence of associations of this kind being significantly more at risk in an ecosystem than cohabiting taxa. Just as with cycads, high levels of promiscuity in the abilities of potential microbial partners to nodulate taxa within each of the above host groupings would would be suggest that, when cultivated outside its normal range, more or less guaranteed, albeit possibly only after artificial inoculation.

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The overall role of nodulated legumes in complex ecosystems is still poorly understood and many questions remain unanswered (see Sprent 2002). Given the world’s large resource of legumes, many instances of taxa from this group are to be found in listings of rare and endangered taxa, or in the gazetted listings of species requiring further evaluation. Woody shrub and tree taxa appear to be particularly vulnerable in this connection (e.g. Sophora toromiro (Maunder et al. 1999)). However, it is also not possible to conclude that legumes as a whole are more at risk than other taxonomic groupings, nor, indeed, to find cases in which demise of a particular legume can be directly attributed to prior loss of a suitable rhizobial partner. However with the a b i l i t y of rhizobia to interchange being restricted in certain cases to specific cross inoculation groupings, especially among temperate herbaceous legumes, a situation may be pictured in which an endangered taxon with increasingly sparsely distributed individuals might suffer ‘loss’ of its own specific rhizobial associate. It would then be able to regain symbiotic competency only if able to ‘borrow’ compatible rhizobia from other legumes in its habitat. These issues are considered further in a recent article by Parker (2001), where a legume may fail to colonise if mutualistic partner bacteria are scarce or absent. The same constraints are also discussed more broadly by Richardson et al. (2000) in relation to other organisms and their hosts. mutualisms as well as those between Mycorrhizal association may be a potential aid in rehabilitation where mutualistic partner bacteria are scarce (Franco et al. 1997; Sprent 2002). Considering the effects of anthropogenic pollution, one might argue that where run-off or atmospheric fall-out of a nitrogenous nature is prominent, symbiotic associations might be especially at risk, whether directly from the well authenticated adverse effects of added N on formation and functioning of symbiotic organs, or indirectly by increasing competitive stress on the symbiotically-operating taxa in a community by non taxa better nourished by the additional input of N. Finally, one may attempt to forecast possible effects of increased atmospheric and associated global warming on performance of associations compared to that of cohabiting non symbiotic f i x i n g plant taxa. U n f o r t u n a t e l y , judging from the large and often conflicting information regarding plant and ecosystem responses to elevated it would be extremely difficult to generalise on this issue. However, an attractive scenario is that with rising levels, photosynthetic inputs of plants would increase relative to water utilised. This might then lead to host plants of associations becoming less penalised when providing photosynthate specifically to symbiotic activity, under conditions where limitation of water is the overriding factor in species survival and productivity. This and other possible suggestions clearly need to be

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evaluated against the whole range of complex interactions which may condition competitive responses of plants to long-term climate change. Acknowledgements I am extremely grateful to Russell Barrett for his superb assistance in preparation of earlier versions of the manuscript and in the formatting and checking of the final version. I am also indebted to Emeritus Professor Janet Sprent and Dr Jenny Chappill for information regarding the phylogeny of legumes and a current synopsis of records of their nodulation. References Ahern CP, Staff IA (1994) Symbiosis in cycads: The origin and development of coralloid roots in Macrozamia communis (Cycadaceae). American Journal of Botany 81, 1559–1570. Akimov VN, Dobritsa SV, Stupar OS (1990) Grouping of Frankia strains by DNA homology: How many genospecics are in the genus Frankia? In ‘Abstracts of the international symposium on nitrogen fixation with non-legumes.’ (Florence, Italy) Allen MJ (1992) ‘Mycorrhizal functioning. An integrated plant-fungal process.’ (Chapman Hall: New York, London) Allen ON, Allen EK ( 1 9 8 1 ) The Leguminosae. A source book of characteristics, uses and nodulation.’ (The University of Wisconsin Press: Madison) Aziz, T, Sylvia DM (1993) U t i l i s a t i o n of vesicular-arbuscular mycorrhizal fungi in the establishment of nitrogen-fixing trees. In ‘Symbioses in nitrogen-fixing trees.’ (Eds NS Subba Rao and C Rodriques-Barrueco) pp. 167-194. (International Science Publisher: New York) Becking JH (1977) Dinitrogen-fixing associations in higher plants other than legumes. In ‘A treatise on dinitrogen fixation. Section I I . Biology.’ (Eds RWF Hardy and WS Silver) pp. 763. (Wiley: New York) Benson DR, Silvester WB (1993) Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiological Review 57, 293–319. Bergman B, Matyeyev A, Rasmussen U (1996) Chemical signalling in cyanobacterial-plant symbioses. Trends in Plant Science 1. 191–197. Bergman B, Rai AN, Johansson C, Soderback E (1992) Cyanobacterial-plant symbioses. Symbiosis 14 , 61–81. Berry AM (1994) Recent developments in the actinorhizal symbioses. In ‘Symbiotic nitrogen f i x a t i o n . ’ (Eds PH G r a h a m , MJ Sadowsky and CP Vance) pp. 118–215. (Kluwer Academic Publishers: Dordrecht) Bloom RA, M u l l i n BC, Tate RL (1989) DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry). Applied and Environmental Microbiology 55, 2155–2160. Bond G (1963) The root nodules of non-leguminous Angiosperms. In ‘Symbiotic associations.’ (Eds PS N u t m a n and B Moss) pp. 72–91. (Cambridge University Press: Cambridge) Bond G (1967) Fixation of nitrogen by higher plants other than legumes. Annual Review of Plant Physiology 18, 107–126. Bond G (1974) Root nodule symbioses with actinomycete-like organisms. In ‘The biology of nitrogen fixation.’ A Quispel (ed.) pp. 342–378. (Elsevicr/North Holland Publications: Amsterdam)

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Chapter 4 BACTERIAL ASSOCIATIONS WITH PLANTS: BENEFICIAL, NON N-FIXING INTERACTIONS

Berndt Gerhardson Sandra Wright Plant Pathology and Biocontrol Unit, P.O. Box 7035, S-750 07 Uppsala, Sweden.

1. Introduction Like all other organisms on earth, the higher plants are surrounded by smaller, mostly harmless creatures, the microorganisms. For plants and other organisms l i v i n g outdoors, one could say that they are bathing in microbes. The microflora on and around the aerial plant parts mainly consists of bacteria and small fungal spores, and is in many aspects similar to the air-borne flora. However, since the green terrestrial plants that are soil anchored by root systems are also soil organisms, like the earthworms for example, they have to cope with a soil flora and fauna. In comparison to the aerial microflora, the soil microflora usually is much larger, more diverse, and commonly also more aggressive. During evolution, these close contacts between plants and the microorganisms infecting or invariably surrounding them have developed into various dependencies on both sides. These in turn have in many cases led to specific biological interactions, or symbioses, presumably resulting from a long co-evolutionary process, and in other cases to more or less loose, or even chance associations. We now find these dependencies as hostpathogen interactions, which may be biotrophic or necrotrophic, clear symbiotic interactions (e.g. with certain N-fixing microbes, and the mycorrhizal f u n g i ) and as a variety of looser, probably facultative associations. In these interactions, many microorganisms are found in association either with specific plants or with plants generally. They

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commonly induce no typical pathogenic symptoms or morphological changes, even though they may give measurable and significant effects on plant growth and development. The broader and typically non-associated, ecological relations between plants and microorganisms, e.g. that saprophytes are dependent on plants as carbon sources and plants in turn on microbial mineralisation of nutrients, are not treated here. Furthermore, the bacteria that typically infect plants, e.g. plant pathogens and N - f i x i n g , symbiotic bacteria, are treated in other chapters of this book (3 and 9). We here concentrate on the predominantly non-pathogenic, plant-associated bacteria that are non N-fixing. Although they are less well investigated than the typically pathogenic microorganisms, these also show many different kinds of spatial as well as functional associations with plants. We have the phyllosphere flora on leaves, flowers, fruits and shoots, the spermosphere flora on seeds, the rhizosphere flora on and in the roots, and in addition a possibly fairly specific endophytic bacterial flora within the plant tissues (Hallman et al. 1997). All these floras contain their clearly characteristic groups of bacteria, and within all of them there are bacterial strains that interfere with plant growth and development, either directly by interacting w i t h the plants themselves, or indirectly by interacting with other organisms that affect the plant. 2. The plant growth affecting bacteria The scientific awareness of the existence of beneficial, non N-fixing bacterial interactions with plants is relatively recent. While interactions i n v o l v i n g plant pathogenic bacteria received an obvious attention in the dawn of bacteriology (Postgate 1992), and the N-fixing interactions were described at the beginning of the century, the earliest records of other clearly plant beneficial bacterial-plant interactions are from the middle and century (Tveit and Wood 1955; Bowen and Rovira second part of the 1961; Baker and Snyder 1965). An often-quoted early study was the report of growth increase of cereals and carrots obtained after treatment with certain Bacillus and Streptomyces strains (Merriman et al. 1974), and Brown (1974) wrote one of the earlier reviews in this subject. These and other early studies (e.g. Kerr 1972; Howell and Stipanovic 1979; Kloepper et al 1980) were then followed by an increasing number of reports and also reviews (Schroth and Hancock 1981; Suslow 1982; Lynch 1982; Burr and Caesar 1984; Schippers et al. 1987; Cook 1993) until we at present have an imposing amount of literature treating this subject. There are also regular international conferences covering this topic, especially the plant growth promoting rhizobacteria (PGPR) (see http://www.ag.auburn.edu/pgpr/). Since there is no clear or generally accepted delineation of different functional groups of plant growth affecting bacteria, for the purpose of this

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chapter we will use the general term ‘plant beneficial bacteria’, which are divided into two main subgroups: ‘plant growth affecting bacteria’ and ‘disease suppressing bacteria’. In the literature terms like PGPR (Kloepper et al. 1980), deleterious rhizobacteria (DRB) (Suslow and Schroth 1982; Åström 1990), biocontrol agents, pathogen antagonists, disease suppressing agents (Cook and Baker 1983), resistance inducers (Kuc 1995) and others have been used, partly interchangeably. One of the problems in classifying and naming these bacteria, especially those directly affecting plants, is their dependency on external conditions for inducing effects. A bacterial strain that is clearly a plant growth promoter under certain conditions may, thus, have a plant deleterious effect in association with another plant species, or under other environmental conditions (Schroth and Hancock 1981; Åström 1990). The bacterial strains so far reported as affecting plants usually belong to the bacterial species commonly found in the soil and in the rhizosphere. Most described isolates have been identified as pseudomonads and within the Pseudomonas genus, the fluorescing species such as P. fluorescens, P. putida and P. aeruginosa predominate. The genus Bacillus is also well represented, and the species B. subtilis especially seems to contain many active isolates. Other genera of soil or rhizosphere bacteria reported as plant growth-affecting are Serratia and Azospirillum (Glick et al. 1999). On the plant leaves we have, among others, the intriguing associations between plants and the methanol-utilising bacteria, Methylobacterium spp. (Holland and Polacco 1994). However, even though these genera are well represented in literature reports, there could well be doubts as to what extent this mirrors the actual situation in nature. All of the genera mentioned are fairly easy to isolate from environmental samples including plants, on commonly used laboratory media, with free access to oxygen, and after incubation at room temperature. Since the bacteria tested for plant affecting abilities have often been isolated using such standard methods (e.g. Gerhardson et al. 1985; Kloepper et al. 1988a; Glick et al. 1999), many other bacterial species that are not competitive under these conditions probably do not grow, and thus, have not been isolated and tested. A strong selection already at the isolation stage thereby probably occurs. The suspicion that we have hitherto had a very unclear picture of the nature of the bacteria that are able to interact with and affect plant growth and development in nature is further substantiated both directly by microscopy (Foster 1983) and by using DNA-based methods. Analysis of 16S rDNA from environmental samples has given evidence for a much broader bacterial diversity than hitherto appreciated (Ward et al. 1995). Based on such studies, we can conclude that only a very small fraction of the bacteria present in nature so far have been cultured, studied and described.

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Presumably this applies to the plant-associated bacteria equally well as to bacteria from other habitats. Certain plant-associated bacteria may be specialised and have a biotrophic association with their plant hosts, and therefore may contain a higher proportion of bacteria that are difficult or impossible to culture, than do bacteria isolated from other sources. This inability at present to culture and study but a fraction of the bacteria in nature is also aggravated by the findings that many common bacterial strains that normally are easy to isolate and culture, do enter non-culturable stages (Roszak and Colwell 1978; Ward et al. 1995). Although the full implications of these findings in soil and plant bacteriology is still unclear, this ability has been established for certain Pseudomonas strains (Dendurand et al. 1994) and other soil bacteria (Oliver et al. 1995; Bogosian 1998). In light of this, what we presently know and what we have so far seen of beneficial plant-bacterial associations may be just the tip of the iceberg. 3. Plant reactions to non-pathogenic bacterial associations A majority of the data that has been gathered on plant reactions to specific non-pathogenic (non-infecting) bacterial strains derives from inoculation experiments on various crop plants. Typically, the laboratory produced cultures of specific bacterial isolates have been inoculated on seeds, roots or in the soil matrix, and the effects induced have then been recorded as differences in shoot dry weight, after harvesting the treated plants grown in the field, in the greenhouse or in growth chambers (Merriman et al. 1974; Gerhardson et al. 1985; Kloepper et al. 1980; Kloepper et al. 1988a; Glick 1995). Shoot growth increases of 30-50 % or more are not unusual in such experiments (Kloepper et al. 1980; Glick 1995), and observations of earlier and faster germination and of greener leaf colour for example in treated plants than in control plants, are also often reported (Kropp et al. 1996). However, in cases where screenings of randomly chosen bacterial strains isolated from nature, e.g. from plant rhizospheres are carried out, various kinds of plant deleterious effects may be as frequent as plant growth promoting effects (Bolton and Elliott 1989; Åström 1990; Suslow and Schroth 1982), and in these cases, not just weaker shoot growth, but also various plant symptoms like epinasty, dwarfing, leaf spots or stripes, and necrosis of leaf edges may be induced (Gerhardson et al. 1985). The significance of these results, plant beneficial as well as plant deleterious effects, may in certain cases be questioned, and then mainly on the grounds that they represent artificial and non-natural conditions. Another major objection is that the bacteria tested have often been applied in larger amounts than they would occur under natural conditions, and furthermore, that the material applied include not just the bacterial cells, but also the bacterial culture supernatant, which may contain active metabolites.

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The plant effects observed, might thus have been drastically exaggerated because of large bacterial numbers. Such experiments are reasonable from a scientific point of view, but they may tell little about natural conditions. Also, where several millilitres of inoculum per litre of soil are applied, the inoculum may partly act just as a readily available fertiliser supplying extra N and P, or minor elements. However, even though such effects may have been at hand in many cases, and results have to be interpreted carefully, the overall evidence that significant plant growth effects are induced by plant associated non-infecting bacteria is overwhelming. We have, for example, cases where seed inoculation with only tiny amounts of bacteria give dramatic growth effect on the plant (Kloepper et al. 1980), and the plant growth promoting effects also have been amply tested in field experiments (Suslow 1982; Kloepper et al. 1988b, Johnsson et al. 1998). Specificity is often encountered, where a certain active bacterial strain may induce effects in one plant species, but not when tested on several others, giving us evidence that specific biological, or molecular interactions are at hand. Such specificity is probably common in these kinds of interactions (Lemanceau et al. 1995; Chan way et al. 1988a), but it has been most clearly shown for bacteria inducing plant deleterious effects (Åström and Gerhardson 1988), where the experimental recording of effects may be easier than the recording of growth promotion. Interestingly, a clear specificity in such interactions has been shown on a plant species level (Åström 1990), as well as on the cultivar level in wheat (Chanway et al. 1988b; Åström and Gerhardson 1989). A strain that induces deleterious effects in one plant may also induce growth promotion in another species, or when tested on the same species under other conditions (Åström 1990). Specificity on a plant cultivar level, most probably driven by differences in exudates/bacterial nutrient sources, has been shown also for indirectly plantaffecting, pathogen-antagonistic bacteria. One example of this is the experiments performed on isogenic lines of tomato, where the plant genotype directly influenced the degree of suppression of the seed pathogen Pythium torulosum by Bacillus cereus (Smith et al. 1999). It is intriguing that the plant may affect bacterial growth and activity not only by exuding specific metabolizable substances, but also by specific regulating molecules. Most of those studied are various N-acyl homoserine lactone mimic compounds. By producing and exuding such molecules, the plants may to a certain degree govern the activity and composition of their resident microflora (Teplitski et al. 2000). These findings concerning specificity, together with the indications that we probably have a very common natural occurrence of bacteria with plant growth–affecting abilities (Gerhardson et al. 1985; Glick et al. 1999), give rise to a number of questions concerning their role in nature and in plant ecology.

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In the case of plant deleterious effects, the effect-inducing bacteria may be regarded as soil-borne pathogens, and, if so, there are far more root pathogens in the soil than we have ever been aware of. The similarity in plant reaction to inoculation w i t h the typically non-infecting, plant deleterious bacteria, and to weak root pathogens, has also given these microorganisms denotations as exo-pathogens, or non-invading pathogens (Woltz 1978; Timonin 1946; Salt 1979). Regarded as such, they have been suggested as causes of crop rotation effects (Schippers et al. 1987) (such as the crop losses almost invariably experienced in narrow rotations of crop plants) and replant diseases (Sewell 1979). Since many clearly plant deleterious bacterial strains show specificity in relation to plant species, they have even been tested and shown efficacy as weed biological control agents in bioherbicides (Boyetchko 1996; Kennedy 1997). The plant growth promotion effects have in some cases also been attributed to competition between growth deleterious and growth promoting strains in the rhizosphere (Schippers et al. 1987), viz. a biological control of deleterious, plant-associated bacteria (Schippers et al. 1986), but so far, the experimental evidence for such effects is generally lacking. For crop production specialists, the occurrence of effective plant-associated, growth promoting bacteria raises the question of the growth and yield potential of our crop plants. Has it hitherto been seriously underestimated? The plant ecologist could equally question to what extent these plant-bacterial interactions may interfere with plant development, plant reproduction and plant competition in natural systems. 4. The interaction mechanisms and problems of classification The non-pathogenic, plant associated bacteria that convey plant growth promoting ability will most probably be grouped in a number of different classes in the future depending on the manner by which they affect the plant. So far we have too little knowledge to form a reasonable taxonomic framework, but two main classes (as used in this chapter) are immediately obvious: i) those interacting more or less directly with the plant, and ii) those interacting indirectly by suppressing diseases caused by plant pathogens, viz. pathogen antagonists (reviewed by Weller 1988; Handelsman and Stabb 1996; Thomashow and Weller 1996). There are also good candidates for two additional groups, with one containing bacteria that enhance availability of plant nutrients. These could be exemplified by the bacteria that are able to solubilise inorganic or organic phosphorus in the soil. Strong evidence for enhanced growth resulting from such phosphate solubilisation comes from the large number of reports that link phosphate solubilisation by rhizosphere bacteria to growth promotion in a number of different crop plants (Rodríguez and Fraga 1999). The second additional group consists of various “helper

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bacteria”, e.g. bacteria that are beneficial to plant growth through their enhancing effect on other plant beneficial microorganisms, like N-fixing symbionts, or the disease antagonists. These are also treated elsewhere in this book and are only exemplified here by a brief mentioning of the “mycorrhiza helper bacteria” below. Any such coarse classifications naturally meet problems when it comes to details. We have, for example, ample reports of bacteria that are primarily classified as pathogen antagonists, but which also affect the plant by inducing disease resistance (Liu et al. 1995; Leeman et al. 1995; van Loon et al. 1998). That is, even though they may not directly antagonise the pathogen, they are plant disease suppressers. We also have a group of plantassociated bacteria that may be denoted as pathogen synergists (Vancura and Stanek 1976; Huber and McKay-Buis 1993). These increase disease severity, and, thereby, are indirectly plant growth deleterious. They are probably as common as the pathogen antagonists in nature, but will not (being plant deleterious) be treated further here. It is obvious however, from such findings and from microcosm studies (Gerhardson and Clarholm 1985; Schippers et al. 1987) that under natural conditions, the effect on plant growth is, in most cases, exerted by microbial communities, rather than by single bacterial strains, or isolates. Concerted multi-microbial-plant interactions, often also including soil fungi and animals, are probably as important for obtaining plant growth effects as are the direct plant-single bacterial isolate interactions. Isolating single bacterial strains from their environment and studying their mode of action in simplified systems thus may easily lead to erroneous conclusions.

5. Hormonal action and detoxification For the bacterial strains inducing a consistent and significant direct plant growth promoting effect in our measuring systems most commonly applied, we can often deduce a bacterial interference with the plant hormone balance (Young et al. 1991; Loper and Schroth 1986). It is well demonstrated that many plant-associated bacteria, especially rhizosphere bacteria, are able to produce secondary metabolites with phytohormonal activity (Dowling and O’Gara 1994; Glick et al. 1999). Most common is auxin (IAA) production and as much as 80% of rhizosphere bacteria were estimated to produce various auxins (Glick et al. 1999). Cytokinins, gibberellins and ethylene are also established as microbially produced (Frankenberger and Arshad 1995). However, production of these substances by bacteria is not sufficient evidence for inferring bacterial hormonal action on plants. The main evidence that such metabolites are actually involved comes from the numerous studies where increases in seed germination, seedling emergence, growth and yield of various crops is obtained in response to seed

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or root inoculation (Glick et al. 1999), and where the effects are similar to those typically induced by plant growth hormones (Oberhänsli et al. 1991; Selvadurai et al. 1991). Evidence to this end is also added from various other cause-effect studies. Young et al. (1991) screened a collection of plant growth promoting bacteria for production of plant growth hormones and found a correlation between the ability to induce root elongation, or promote germination and emergence, and a significant high production of plant growth regulating substances. Loper and Schroth, (1986) also screened several bacterial isolates for their production of an auxin, indole-3-acetic acid (IAA), and the accumulation of IAA in bacterial supernatants significantly correlated with root elongation of sugar beet. Similarly, Nieto and Frankenberger (1991) demonstrated the production of phytohormones indole acetic acid and cytokinins by Azotobacter species, which are well known to affect plant growth and development. Culture supernatants of Azospirillum spp. often contain both auxins, cytokinins and gibberellins (Steenhoudt and Vanderleyden 2000), but the production and role of these hormones in the rhizosphere has yet to be established. However, the role of microbially produced IAA and the sensitivity of plants to various levels of IAA was demonstrated by the inhibition of canola root growth by IAA overproducing mutants of P. putida (Xie et al. 1996). A picture is now emerging that despite the fact that plants have tuned metabolic systems for regulation of hormone production levels, and can also store excess amounts of the hormones as conjugate for later use (Glick et al. 1999), an excess of a growth regulating substance, or a hormone group, produced by associated bacteria can also result in a plant hormonal response. The plant-associated bacteria here have probably evolved such intimate relationships with their host plants that they may influence plant physiology in different ways, possibly to an extent that the plants partly depend on those partners for their needs of at least certain growth regulating substances (Freyermuth et al. 1996). The ability of the plant to produce the necessary, although usually very small amount, of growth regulating substances for itself may be impeded, especially under less than ideal climatic and environmental conditions (Nieto and Frankenberger 1991), and it will then be more dependent on the exogenous sources. The plant-associated bacteria could here widen the environmental range of a plant species. It is also evident that uptake of excess hormonal substances, such as cytokinins, in the presence of an exogenous supply may be converted into storage forms within the plant, and these can later be transformed into free-base metabolites for active utilisation and control of plant growth (Wareing and Phillips 1981). This conceptual model of m u t u a l b e n e f i t , or symbiosis-like associations, is further strengthened by the fact that specific plant-associated bacteria also can affect the endogenous hormonal pattern of the plant by

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interaction with the synthesis, translocation or regulation of the existing hormonal level (Frankenberger and Arshad 1995). Certain bacteria may thus modify the plant’s own pool of hormones and subsequently stimulate plant growth. A good example here is the findings of Glick et al. (1998) and Jacobson et al. (1994), who demonstrated the ability of beneficial bacteria to lower the production of ethylene in developing seedlings of canola, tomato and lettuce. High amounts of ethylene may impede an optimal development at the seedling stage ( G l i c k et al. 1999). They suggested that the plantassociated bacteria regulate ethylene synthesis in plants by binding to the roots and/or seed coats and hydrolysing the ethylene precursor, 1aminocyclopropane-1-carboxylate (ACC) exuded from the plant seeds or roots through the action of the bacterial-specific enzyme ACC deaminase. This mode of action shows similarities to detoxification processes, which are probably also important mechanisms for directly bacterial-induced plant growth promotion. Both the plant itself and its surrounding microflora, especially the rhizosphere flora, exude substances deleterious to plant growth (Barber and Martin 1976; Bolton and Elliott 1989; Becker et al. 1985) and such substances may also be present in the soil or other growing media (Bruehl 1987). By breaking down or inactivating such deleterious substances, a significant effect on the plant growth may be found (Lynch 1983). Special cases here are the microbes that are able to change the pH, and/or the redox potential in the plant rhizosphere (e.g. Huber and McKayBuis 1993). An intriguing theory is also that the plant growth promoting bacteria are able to antagonise, or eradicate the effect of plant deleterious bacteria, or other “minor pathogens” (Schippers et al. 1987), that according to this theory, are common inhabitants, especially in plant rhizospheres (Elliot and Lynch 1984; Schippers et al. 1987). However, this mechanism can equally well belong under the heading “disease suppressing, plant beneficial bacteria.” 6. Microbial synergists - the example of mycorrhiza helper bacteria Microbial synergists may, as mentioned above be as common in nature as the microbial antagonists that, when antagonising pathogens, are often plant beneficial by suppressing plant diseases. There is then likewise a group of bacteria that enhance the activities of plant beneficial microorganisms, whether these are plant growth affecting bacteria, pathogen antagonists, Nfixing symbionts, or mycorrhizal fungi. However, our knowledge about these microbe-plant-associated microbe interactions that ultimately may become beneficial to the plants is so far very scanty, and only one example is briefly mentioned here: the mycorrhiza helper bacteria. The term “mycorrhiza helper bacteria” refers to certain soil bacteria, mostly Pseudomonas spp., that have shown ability to significantly enhance

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mycorrhizal formations in plants (Garbaye and Bowen 1989). Since certain plants, especially those with ectomycorrhiza, are dependent on mycorrhizal formation for normal growth (Harley and Smith 1983), the helper bacteria could be seen as plant growth promoting. Furthermore, when we consider the bacterial promotion of plant-beneficial mycorrhizal fungi, these bacteria are also plant growth affecting in this respect. However, the mode of action of these bacteria is s t i l l unclear (Garbaye 1994; Frey-Klett et al. 1997) and they may also have an action whereby they enhance the plants receptivity to mycorrhizal infection (Garbaye 1994; Karabaghli et al. 1998). If true, this points to a direct plant growth-affecting ability and, furthermore, to similarities between these bacteria and the pathogen synergists. 7. Disease suppressing, plant beneficial bacteria Bacteria that are plant beneficial by suppressing plant diseases may also be subdivided into two categories, as mentioned above: i) a direct antagonism of the pathogens or ii) indirectly an enhancement of plant disease resistance (Davison 1988). Such microbial-induced plant disease or pest resistance is at present further divided into two types, representing distinct pathways of disease resistance responses. We have either, systemic acquired resistance (SAR) that is induced by pathogens, or an induced systemic resistance (ISR) that is salicylic acid-independent and typically induced by non-pathogenic bacteria (van Loon et al. 1998). The latter group also includes several bacteria that by virtue of their promotion of plant growth, help the plants withstand infection. These direct and indirect modes of disease suppression very probably operate in consort. However, due to the definition of induced resistance: “...operating at a distal plant part” (Kuc 1995), the relative contribution of induced resistance to disease suppression becomes almost impossible to measure and determine at the site of interaction between pathogen, plant and antagonist. Only when the antagonist is applied at a site distal to that of the pathogen, would induced resistance, by definition, be operating. Moreover, disease suppression by beneficial bacteria is usually easier to measure and enumerate than the direct effect on plant growth. Disease suppressing bacteria were originally revealed through their association with naturally ‘disease-suppressive soils’, and by the ability of certain randomly isolated soil and plant bacteria to protect plants against disease after exogenous application to plant parts. Many of these bacteria were also selected as candidates for disease suppression on the basis of their production of anti-microbial metabolites in laboratory media. Commonly reported disease suppressing bacterial species include Agrobacterium radiobacter, Bacillus subtilis, Burkholderia cepacia, Enterobacter agglomerans, E. cloacae, Pseudomonas aureofaciens, P. chlororaphis, P. fluorescens and P. putida (Schippers 1988; Becker and Schwinn 1993;

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Bevivino et al. 1998; Kang et al. 1998; Pierson III 1998), and some strains of P. fluorescens are simultaneously plant growth promoting (Glick et al. 1999). Mechanisms of disease-suppression by bacteria that have direct effects on pathogens include competition for nutrients (and/or space), siderophore production, antibiosis, production of hydrolytic enzymes and other general traits that make them successful colonisers of plant parts and competent survivors in the phyllo-, rhizo- and/or spermo-sphere. However, with a few exceptions, the activities related to nutrient competition has generally been very difficult to demonstrate in vivo. Efficient uptake and utilisation of nutrients is a prerequisite for activity in a highly competitive environment such as the soil. Roots and seeds exude a variety of amino acids and carbohydrates (Rovira 1965; 1969) that are effectively utilised by microbial residents, including many pathogens. A good example is the sporangia of the seedling pathogen Pythium ultimum that germinate in response to the presence of nutrients in form of seed exudates (Nelson and Hsu 1994). In this case, it has been shown that the utilisation of the long chain fatty acid components of cotton seed exudate by the disease suppressing bacterium Enterobacter cloacae prevented the sporangia from germinating (van Dijk and Nelson 1997). Another demonstration of nutrient competition is the observation that disease suppressing strains that are isogenic to the same species as the pathogen and thus share the same nutritional niche as the pathogen are often very successful competitors. Examples include Agrobacterium radiobacter controlling A. tumefaciens (Kerr 1989), a non­ pathogenic strain of Ralstonia solanacearum controlling R. solanacearum (Sunaina et al. 1997), non-pathogenic Clavibacter xyli that control C. xyli, isogenic strains of P. fluorescens out-competing each other in the rhizosphere (Nautiyal 1997), the control of ice nucleation active bacteria by non-ice nucleating isogenic mutants (Lindemann and Suslow 1987) and the control of vascular wilt fusaria (Fusarium oxysporum) by non-pathogenic strains of F. oxysporum (Alabouvette et al. 1993). They are often only effective when provided with an additional competitive advantage, such as higher initial cell numbers (Nautiyal 1997), earlier establishment than the pathogen, or the production of an antibiotic, such as in the example of agrocin 84 (Kerr 1989). Certain antagonists also out-compete pathogens by sequestering the ferric iron available, which expresses itself through the production of siderophores, iron-chelating molecules that facilitate iron uptake for the microorganisms producing them, but may make it unavailable for the pathogens (Teintze et al. 1981, O’Sullivan and O’Gara 1992). A number of such iron-chelating molecules have been studied, e.g. pyoverdin, pyochelin, ferribactin, ferrichrome, ferroxamine B, phytosiderophores and pseudobactin

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(Dowling and O’Gara 1994). Fluorescent siderophores are also what makes fluorescent pseudomonads glow upon exposure to UV light under ironlimiting conditions. The role of siderophores in biological pathogen control depends on a variety of external factors; the nature of the host plant, the pathogen and the soil environment. Thus, in some cases, siderophores have proved to play a role (Buysens et al. 1996), whereas in other cases they have not (Hamdan et al. 1991). Antibiosis, as caused by specific exuded inhibitory molecules, is one of the most extensively studied mechanisms in biological control systems. The presence or absence of antibiotics, like e.g. streptomycin, is relatively easy to demonstrate on laboratory media. The contribution of antibiosis to the pathogen suppressing a b i l i t y of an antagonist is often assessed by constructing mutants that differ only in the augmented presence or total absence of antibiotics, and comparing their disease suppressing efficacy to that of the wild-type strain. In that way, it was established that phenazines (Pierson III et al. 1994, Thomashow and Weller 1988) and 2,4–diacetylphloroglucinol (Keel et al. 1992; Vincent et al. 1991) produced by rhizosphere pseudomonads were involved in suppression of take-all disease in wheat. Pyoluteorin was involved in suppressing Pythium ultimum on cress (Maurhofer et al. 1994), pyrrolnitrin suppressed Pyrenophora tritici­ repentis on wheat (Pfender et al. 1993) and DDR (2,3-deepoxy-2,3didehydro-rhizoxin) produced by Pseudomonas chlororaphis was involved in suppression of Drechslera teres on wheat (Hökeberg 1998). The role of antibiosis is in many cases, like that of siderophore production, dependent on the environmental conditions (Dowling and O’Gara 1994), including the type of plant species colonised (Maurhofer et al. 1994). Some of the antimicrobial compounds produced have activity against fungi, e.g. phenazines (Thomashow et al. 1990) and some against bacteria, e.g. agrocin 84 produced by A. radiobacter (Das et al. 1978), while others have activity against both. Also the production of compounds like of hydrogen cyanide (Voisard et al. 1989) and hydrolytic enzymes such as chitinases, b-1,3glucanases, proteases and lipases have been implicated in disease suppression (Chet and Inbar 1994). Mild pathogens or non-pathogens can, as mentioned above, induce disease resistance in plants (Deacon and Berry 1993). It often acts against both pathogenic viruses, fungi and bacteria (Kloepper et al. 1999), with an effect that is indirect with regard to the interaction of the pathogen and the disease suppressing agent. The resistance response may be induced by individual components of bacteria, such as the O-antigenic side chain of the outer membrane lipopoly-saccharide, siderophores and salicylic acid produced by the bacteria (reviewed by van Loon et al. 1998). This type of resistance has been induced toward several pathogens, for example vascular

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wilt in carnation caused by Fusarium oxysporum (van Peer et al. 1991) and Pseudomonas syringae pv. lachrymans in cucumber (Wei et al. 1996). In the latter case, it also operated under field conditions. Interestingly, the resistance response can be both bacterial strain- and plant species-specific, as noted by van Wees et al. (1997). In cases where directly or indirectly disease suppressing bacteria have been tested or commercialised as biological control agent in biopesticides, their exact modes of action often have been difficult to elucidate (Hökeberg 1998). It is generally very rare for only one of the mechanisms mentioned above to operate alone. Usually, the disease-suppressing bacteria instead utilise a number of biocontrol factors simultaneously, and in order to do so at all it is sometimes crucial that they colonise and establish on the plant part they are protecting (Cook and Baker 1983). Thus, colonisation ability and general ‘rhizosphere, phyllosphere or spermosphere competence’ also may be a prerequisite, in order for the above-mentioned mechanisms to take effect. 8. Disease suppressive soils – involvement of plant-associated bacteria The term “disease suppressive soils” refers to specific soils with characteristics that cause disease levels to remain low even though the pathogen is present and the environmental conditions for disease development is favourable (Alabouvette 1990; Hornby 1983). Usually, when compared to sterile plant growth media, all unsterilised soils will exhibit some disease suppression, especially those with high biological activity, or biomass, but to classify as disease suppressive, disease ratings on plants grown has to constantly remain low, even after pathogen inoculation. Based on its origin, such soil disease suppressiveness is commonly divided into either i) long-standing, natural suppressiveness, or ii) induced suppressiveness (Hornby 1983). Both types are usually specific, which means that in each specific case it is acting on one, or a few related soil­ borne diseases. Natural suppressiveness is often permanent and it has in most cases been found to have a microbiological origin, but its occurrence is probably also dependent on soil physicochemical properties (Höper and Alabouvette 1996). A good example is the early finding of soil disease suppressiveness in banana plantations in Central America, where it was observed that Fusarium wilt of banana developed much more rapidly in certain areas, and soils, than in others (Stotzky and Martin 1963). The induced soil disease suppressiveness may develop after a monoculture of the host plant for several years, or by soil amendments (Hoitink et al. 1996) and it also has a microbiological background (Shipton 1977). Induction of soil disease suppressiveness was often observed when cultivating virgin soils, e.g. in United States and Australia, which commonly involved an initial

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increase in diseases and a decline in crop productivity during the first years, but after a continued c u l t i v a t i o n the productivity returned (Huber and Schneider 1982) (disease decline soils). In experiments where a disease suppressive soil is added to and mixed with a non-suppressive, or conducive soil, the suppressiveness is usually effectively transferred, and often also with low amounts of suppressive soil (Alabouvette et al. 1996), which points to a transfer of specific substances or biological entities. A common approach for assessing the involvement of biotic factors has also been to measure the degree of suppressiveness before and after soil biocidal treatments, such as heat, irradiation with X-rays, or treatment with methyl bromide (Burke 1965; Louvet et al. 1976; Scher and Baker 1980). When such sterilisation treatments result in loss of suppressiveness, some part of the soil microbiota is assumed as the agent(s) directly causing the suppression (Persson 1998). For finding the possible active agents a number of specific organisms have also been isolated from suppressive soils. By reintroducing these into sterilised suppressive soil, or into sterilised or non-treated conducive soil, it has been possible to assess to what extent the disease suppression is re-established (Alabouvette 1986; Scher and Baker 1980). Among the specific organisms isolated from suppressive soils and tested are several Pseudomonas bacteria (Defago et al. 1990; Weller 1988; Alabouvette et al. 1996; Scher and Baker 1982; Raaijmakers and Weller 1998) besides a number of fungal species such as Trichoderma spp. (Simon 1989; Duffy et al. 1996), Pythium oligandrum (Ribeiro and Butler 1995), and non-pathogenic species of F. oxysporum (Alabouvette et al. 1996; Duffy et al. 1996; McQuilken et al. 1990). Antagonistic activities, and/or induced resistance (Alabouvette et al. 1996; Liu et al. 1995) as described in the preceding paragraph, are thought to be the main modes of action, but it is less easy to visualise activities of bacterial-produced antibiotics and siderophores in the soil than on a plant surface, or in the rhizosphere. The competition for nutrients and the restricted availability of these in soil in the absence of plant roots is also thought to induce fungistasis, viz. prevention of germination of dormant fungal spores (Lockwood 1988). The prevention of germination of spores and thereby of infection of the host may also be induced by a generally high microbial activity, obtained by soil amendment by composts, as reported for diseases caused by Pythium and Phytophthora spp. (Hoitink et al. 1996). Various biosurfactants produced by bacteria have also been shown to act especially on such zoosporic pathogens by destroying the membranes of the zoospores (Stanghellini and Miller 1997). We, thus have reasonable evidence of the involvement of the soil microflora and of non-infecting plant beneficial bacteria in this naturally occurring biological disease co n t r o l . However, since the specific

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microorganisms isolated and investigated very seldom give a full effect when reintroduced (Alabouvette et al. 1996), they do not tell us the whole story of the mechanism. The soil physiochemical properties, climate conditions, specific microbial communities, and/or activities, maybe fostered by a very long cropping, or rather vegetation history, probably also have a role. From a plant conservation point of view, the occurrence of plant disease suppressiveness may have two main implications: i) that our ability to grow, or conserve a specific plant may to a great extent depend on the soil and its microbial content, or site chosen, and ii) there may occur specific soil microflora-plant interactions that take a very long time to develop and when and where developed could be well worth conserving because of their uniqueness. 9. Implications for plant conservation and diversity Although they are very common in nature, and often closely associated to plant physiology, plant health and plant growth, the plant beneficial, non-Nfixing bacteria are not considered to be critical for native plant survival and reproduction. This assumption is based on our present knowledge and will possibly be challenged by new findings to come. There may for example, be obligate plant endophytic bacteria in nature that are highly specialised and possibly required for normal plant growth. The plant endophytic bacterial community can, like other plant-associated bacteria, influence root morphology and structure, and it may also have an impact on the bacteria that reside on the outside of the plant (reviewed by Sturz and Novak 2000). However, since the endophytic bacteria hitherto found in many plant species are regarded as usually originating from the bacterial flora residing on the plant surface (Hallman et al. 1997), they have warranted no special treatment in this chapter. It is generally accepted that all plant species are affected to varying degrees by microflora that surround them. Specific strains associated with, or especially beneficial to one, or a number of plant species, may occur only in certain soils or environments and, if so, this probably substantially affects the success of the species concerned, and consequently their distribution. Furthermore, there are probably strong interactions between a plant community and the population and activity the associated, growth beneficial bacteria. By analogy to the conclusions of Newman (1985) working with rhizosphere fungi, a plant community may have a more or less common rhizosphere flora mediating interactions between plant species that are not possible to discern by traditional ecological methods. This would again point to the advantage of community conservation rather than species conservation. The non-invading, plant beneficial bacteria may, in addition, be more or less critical to survival and reproduction of specific plant species

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on marginal or less suitable locations where they, much like mycorrhiza on reclamation land (Brundrett and Cairney, this volume), may extend the range of plant survival. However at present, these assumptions or theories are not substantiated well enough by experimental evidence to be considered as adequate for e x p l a i n i n g broader implications or for developing recommendations for practical plant conservation. In certain practices, we could nonetheless gain substantial advantages from considering beneficial, plant-associated bacteria, such as in the transplanting or growing of specific plants outside their natural habitats, such as in botanical gardens. Inoculations by, or supplying of, an appropriate complement of bacterial flora should be advantageous. A theory that is also clearly supported is the empirically found advantageous practice of enhancing revegetation by using inoculum of native microbes, e.g. by adding a handful of soil from under nearby existing plants at replanting. Straightforward inoculations of cultures of beneficial, direct growth promoting bacteria, as is an increasingly used practice in agriculture and horticulture (Glick et al. 1999), may so far be premature for most wild, noncrop plants from which the experiences of direct beneficial inoculations are comparatively very scarce. Better experimental data is at hand for the indirect growth-promoting, disease suppressing bacteria (Cook 1993; Whipps 1997). However, as the microbiological competence and equipment needed for practical application are usually not freely available, their utilisation in plant conservation is much dependent on commercial preparations. As more of such products become available, these will normally be easier to handle and also environmentally very safe, but in other ways similar to apply and to use as most chemical plant protection products. No specific requirement should here be set in plant conservation. The area where knowledge of plant associated, growth beneficial bacteria presently may have most significant implications for plant conservation and diversity is probably in the appreciation and managing of the natural microflora in the soil or in the growth substrate where plant communities are to be re-established, or where plants are to be conserved. As mentioned above, it could easily be envisioned how disturbed areas, e.g. mined or cleared land, may become defiant of, or suppressive to, beneficial bacterial associations that are critical for full re-establishment. However, the soils or other growth substrates used in replanting are presently more manageable and usually carefully conditioned concerning plant nutrients, texture, pH etc. Managing the soil biology may be similarly critical, and generally high soil microbial activity may not always be optimal. Ideally, suppressiveness to important soil-borne diseases is a preference, and the preceding vegetation, or even the vegetation history important for development of suppression at a site is likewise of importance. Well

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documented examples from crop plants are the apple replant disease problems (Sewell 1979) and the necessity of crop rotations in agriculture (Cook and Veseth 1991) that partly shape our agricultural landscapes. In replanting, there are also, as discussed above, good possibilities for inoculations by specific microbial communities or by selected microorganisms that may have profound influences on the replant results (Hoitink et al. 1996). 10. Implications for microorganism conservation and diversity Where needed for scientific, mainly taxonomic, or for commercial purposes, specific and identified microorganisms are normally conserved as living, or in some cases, as dead and dried samples in culture collections (Allsopp et al. 1995; Gams, this volume), and we know of no natural reserves, or habitats that have been protected because of their microorganisms. Nor has there been the need for conserving specific strains or their habitats for other reasons. The non-infecting microorganisms are cosmopolitan in their occurrence on a specific host, or hosts, and are usually regarded as ubiquitous. They are very easily spread, even at distances, by winds, by migrating birds and animals (including humans) and in light of this we assume that they have few geographic barriers (Andrews 1991). Their occurrence and conservation then is more dependent on availability of suitable habitats. Specific plants growing under specific conditions may be a required niche, and the microbial conservation should in these cases be concurrent with plant conservation. In this connection the assumption that old cultivars of crop plants may have microfloras worth preserving also could have a bearing, but scientific evidence to this end is still scarce. Of importance is here also that we presently have a very inadequate picture of the plant-associated microorganisms, and especially the bacteria. Probably only a small fraction of the non-infecting microorganisms (1 to 10%) can be cultured in synthetic media (Campbell and Greaves 1990) and even less, 1 to 5%, are known and described (Allsopp et al. 1995). The plant-associated, growth beneficial bacteria are no exception. Another difficulty in evaluating the need and possibilities for bacterial conservation and diversity, especially for soil and plant-associated bacteria, is also their fast genetic change and seemingly constant exchange of genes (Reanney et al. 1983; Sonea and Panisset 1983). In light of this, it may be questioned whether effort should be directed towards maintaining their diversity, or if it is at all possible to maintain them. After all, they will evolve much faster than the higher creatures in their own way, depending on their genetic make-up and environment they encounter.

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K.Sivasithamparam, K.W.Dixon & R.L.Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 105–150. © Kluwer Academic Publishers.

Chapter 5 ECTOMYCORRHIZAS IN PLANT COMMUNITIES Mark C. Brundrett CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural Research, Private Bag No 5, Wembley, 6913, Western Australia; Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia (current address) and Science Directorate, Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia (correspondence).

John W.G. Cairney Mycorrhiza Research Group, Centre for Horticulture and Plant Sciences, Parramatta Campus, University of Western Sydney, Locked Bag 1797, Penrith South DC, 1797, New South Wales, Australia.

1. Introduction

1.1. Associations Ectomycorrhizal associations (abbreviated as ECM) are sometimes called ectotrophic associations or sheathing mycorrhizas. They are mutualistic associations between higher fungi and Gymnosperm or Angiosperm plants belonging to the families listed in Table 1. These associations consist of mycorrhizal roots and fungal storage or reproductive structures that are interconnected by soil-borne mycelia (Figure 1). Ectomycorrhizal associations are formed predominantly on the fine root tips of the host. These ECM roots are defined by the presence of a mantle, consisting of interwoven hyphae on the root surface, and a Hartig net, which is a labyrinth of highly branched hyphae between cells of the root epidermis or cortex. These structures are not always both well developed in the same association. These roots and their associated fungal hyphae typically are most abundant in topsoil layers containing humus and are thought to make a substantial contribution to soil biomass and nutrient cycling in many ecosystems (Section 3.1). Detailed descriptions of the structure and development of ECM are available elsewhere (e.g. Kottke and Oberwinkler 1986; Massicotte et al. 1987).

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1.2. Host plants Trees with ECM associations typically are dominant in coniferous forests in boreal or alpine regions, but are also important in some temperate deciduous forest, tropical forest, as well as savannah and mediterranean plant communities (Meyer 1973; Högberg 1986; Brundrett 1991). Plant families reported to have ECM are listed in Table 1. This table excludes

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families with arbutoid or monotropoid ECM associations and those with atypical ECM associations, such as Australian herbaceous plants in the families Goodeniaceae, Asteraceae and Stylidiaceae (Kope and Warcup 1986; Brundrett 1999b). The majority of ECM hosts are trees, or shrubs (Table 1), but associations are formed by a few herbaceous plants, including Kobresia, Polygonum and Cassiope species found in arctic or alpine regions (Kohn and Stasovski 1990; Massicotte et al. 1998). 1.3. Fungi The reproductive structures of ECM fungi include epigeous fungi (mushrooms, puffballs, coral fungi, etc.) and subterranean structures (hypogeous fungi which are called truffles or truffle-like fungi). Most epigeous fungi have a hymenium consisting of gills, pores, teeth, etc. which actively releases spores, but the hypogeous fungi and puffballs have sequestrate fruit bodies which enclose their spores. The majority of ECM fungi are Basidiomycetes, but there also are a number of Ascomycetes and a handful of Zygomycetes (Molina et al. 1992). Most identification guides for larger fungi provide information about their probable host plants from field observations. Lists of known Australian ECM fungal genera and those which associate with Eucalyptus species are provided on the Web (Brundrett and Bougher 1999). Detailed descriptions of the process required to collect, document, store and identify fungal specimens are provided in manuals (e.g. Largent 1986; Brundrett et al. 1996c). However, accurate fungal identification is a time consuming process that requires much expertise. Fungal identification is most difficult in tropical regions and the southern hemisphere, where many species have yet to be described or illustrated in identification guides. In particular, the hypogeous ECM fungi have been neglected in many regions (Bougher and Lebel 2001). Due to these taxonomic difficulties, it is probable that many lists of fungi contain errors. Consequently, it is imperative that voucher specimens of all fungi referred to in publications are lodged with an internationally recognised herbarium, and these specimens contain material of sufficient quality and quantity to allow future taxonomic and DNA-based studies. 2. Fungal biology 2.1. Distribution and diversity Information on the identity and relative abundance of ECM fungi present in a particular habitat is required to understand the relationship between taxonomic and functional diversity (Section 4.1). Approximately 5500 species of ECM fungi have been listed world-wide (Molina et al. 1992). However, this list would significantly underestimate the diversity of these fungi, as the fungal flora in tropical and southern regions is poorly known. For example, it has been estimated that there could be as many as 6500 species of ECM fungi in Australia alone, but only about 700 species from this region have been named so far (Bougher 1995; Brundrett and Bougher 1999). Most estimates of diversity are based on

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surveys of obvious epigeous fruiting bodies, which exclude hypogeous and resupinate fungi.

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Ectomycorrhizal fungi are normally identified by observations of fruiting under their putative hosts, but the relationship between fruit bodies and the activity of mycelia in soils has usually not been established. The recognition of fungi by ECM morphology (colour, texture, structure, size, branching, etc.), has provided a powerful tool for the identification of fungi in individual root tips (e.g. Agerer 1995; Bradbury et al. 1998; Hagerman et al. 1999; Massicotte et al. 1999). Lipid profiles can also be used to identify ECM fungi in soils (Olsson 1999). Methods based on DNA are now often employed to identify ECM roots and provide a much more accurate picture of below-ground fungal activity than observations of fruit bodies (e.g. Dahlberg and Stenlid 1995; Horton and Bruns 1998; Jonsson et al. 1999a,b). A list of molecular sequence data that can help to identify ECM fungi is provided by Bruns et al. (1998). Molecular studies of fungi inhabiting ECM root tips in the field have shown that up to 60% of ECM roots are inhabited by fungi not observed to produce obvious epigeous fruiting bodies (e.g. Gardes and Bruns 1996; Jonsson et al. 1999a,b). These cryptic fungi would likely include those with hypogeous sequestrate (truffle-like) fruiting bodies (Castellano and Bougher 1994), or inconspicuous, epigeous, resupinate fruiting bodies (Erland and Taylor 1999), largely ignored in previous diversity surveys. Fungi such as Cenococcum, which do not produce macroscopic fruit bodies, also appear to be widespread. However, the observed discrepancies between the fungi which form mycorrhizas and those which fruit at a particular site seem to be due to the fact that many fungi reproduce very sporadically. Most genera of ECM fungi are reported to be widely distributed throughout the world, but there are also genera restricted to certain regions. Individual species of ECM fungi are often restricted to particular geographic regions. When considering functional aspects of mycorrhizal associations we should consider isolates of fungi rather than species or genera, because considerable intraspecific physiological variation is known to exist in ECM fungi (Trappe 1977; Brundrett 1991; Cairney 1999). Thus information about the soil and climatic conditions and host species present where fungi occur may be as important as their accurate identification. Future taxonomic studies are likely to reveal much finer differences between taxa of fungi, than are used in current classification schemes, especially for the neglected floras of the tropics and southern hemisphere. Molecular investigations are also revealing that taxa previously regarded as conspecific are complexes of several species. A good example is the cosmopolitan species Pisolithus tinctorius, for which a number of putative species have been identified using restriction fragment polymorphism (RFLP) and sequence analyses of the rDNA internal transcribed spacer (ITS) region (Anderson et al. 1998; Martin et al. 1998; Chambers and Cairney 1999; Sims et al. 1999). These approaches are likely to result in taxonomic revisions of many other genera of ECM fungi and provide us with a greater understanding of correlation between the taxonomy and physiological attributes of fungi (Section 1.3).

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2.2. Lifecycles and inoculum There are a number of distinct stages in the life cycle of a mycorrhizal association (Table 2). These stages often occur at particular times of the year. Mycorrhizal exchange is a short-lived process dependent on root growth (Downes et al. 1992; Cairney and Alexander 1992a,b; Massicotte et al. 1987) – which in turn is regulated by environmental conditions and host plant phenology. Fungal fruiting, and thus the availability of spore inoculum, is also regulated by climatic factors and fungal phenology. It is important to think of ECM associations as dynamic processes as fungi would utilise mycelia and spores to move through soil while competing with other fungi to claim new roots. Thus, the size and shape of individual fungi is constantly changing with time, due to their foraging behaviour (Section 3.1), interactions with other soil organisms (Section 2.3) and environmental factors (Section 3D). These dynamic processes may, in part at least, explain discrepancies between the fungi fruiting above ground and those forming mycorrhizas in a particular location (Section 2.1). Propagules of ECM fungi include individual hyphae, strands (aggregations of hyphae), spores, sclerotia and probably also mycorrhizal roots (Ogawa 1985; Fries 1987; Ba et al. 1991; Miller et al. 1994; Torres and Honrubia 1997). Most ECM fungi do not produce conidia (Hutchinson 1989). Boreal forest soil and leaf litter often contains spores capable of initiating mycorrhizas (Amaranthus and Perry 1987; Parke et al. 1983b). Localised patterns of ECM fungus proliferation depend on the production of hyphae or strands by a particular endophyte (Ogawa 1985; Agerer 1995; Unestam and Sun 1995). Mycelia of some ECM fungi are thought to require attachment to a living host root to initiate new mycorrhizas (Fleming et al. 1984). Ectomycorrhizal short roots live for months (Majdi and Nylund 1996; Rygiewicz et al. 1997) and are often protected by a thick covering of mantle hyphae, suggesting that they may be important fungus survival structures. However, it is not known how long ECM fungi in these roots can survive after detachment from the host. The implications of ECM fungus dispersal and survival are considered in sections 2.3 and 2.4 below. 2.3. Consumption and dispersal Ectomycorrhizal fungus structures are a major structural component of certain soils, and thus are an important food source for many soil organisms (Table 3). Hyphal grazing by soil organisms can reduce mycorrhiza formation and nutrient translocation by hyphae in soils (Hiol et al. 1995; Setälä 1995). Soil organisms which ingest, inhabit, or associate with hyphae or sporophores of ECM fungi include members of most soil trophic levels (Table 3). Dense mats of ECM roots and mycelia in forest soils can have substantially higher populations of microbes and micro-arthropods than other areas (Cromack et al. 1988; Griffiths et al. 1991).

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Dispersal of fungal propagules is required for colonising new habitats, increasing fungal diversity, changing fungal population structure, or introducing new genes to existing fungi. Most localised

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spread is by mycelial growth through the soil, resulting in discrete patches of soil occupied by the hyphal networks of individual fungi, that can be distinguished from others of their species using genetic or DNAbased methods (de la Bastide et al. 1994; Dahlberg and Stenlid 1995). Many fungi forming ECM associations have large fruiting structures (mushrooms) that produce abundant wind-borne spores, but survival and dispersal of these spores may be limited. Certain ECM fungi produce sclerotia which probably are much more resilient than other propagules (Miller et al. 1994). Fungi with hypogeous fruiting bodies are often excavated and consumed by small mammals or marsupials and thus spread to new locations (Table 3). Spores of ECM fungi contained in animal faeces are a viable source of inoculum (Claridge et al. 1992; Cázares and Trappe 1994; Reddell et al. 1997). 2.4. Disturbance Severe disturbance includes situations where vegetation has been lost and topsoil has been removed or mechanically disrupted, or where plants are introduced to new substrates resulting from mining, glaciation, or volcanic activity. Soil disturbance can result in temperature extremes, anaerobic conditions, loss of organic matter, loss of nutrients, structural changes and loss of biological components (Abdul-Kareem and McRae 1984; Danielson 1985). There is a high degree of spatial variability in ECM fungus inoculum in Australian natural habitats (Brundrett and Abbott 1995; Brundrett et al. 1996a), but this variability is even larger in disturbed habitats, where large gaps between patches containing inoculum would prevent many seedlings from encountering these fungi (Brundrett et al. 1996b). Forestry activities can also result in reductions in ECM fungus inoculum due to the absence of host plants and soil degradation (Amaranthus and Perry 1987; Parke et al. 1983a; Harvey et al. 1997; Perry et al. 1987; Visser et al. 1998; Hagerman et al. 1999). After mechanical disturbance of soils, surviving ECM fungal inoculum may be concentrated in localised soil pockets high in organic materials (Christy et al. 1982; Parke et al. 1983b; McAfee and Fortin 1989). Other forms of severe disturbance known to adversely affect ECM fungi include erosion and fires (Table 3). Mycorrhizal inoculum is often limited in recently disturbed habitats (Danielson 1985; Malajczuk et al. 1994; Brundrett et al. 1996b; Reddell et al. 1999). Propagules expected to survive in disturbed soils are thought to include mycorrhizal root fragments, hyphae within organic matter, segments of rhizomorphs, sclerotia and perhaps spores and root pieces (Ba et al. 1991; Brundrett 1991). The networks of fungal hyphae which are the main propagules of ECM fungi would be highly susceptible to disturbance and other more resilient propagules would decline in the absence of host roots (Figure 2). Reductions in fungal diversity could result because fungi have a limited capacity to adapt to major changes in environmental conditions (Table 4). Fungus specificity (Section 3.3) may also prevent mycorrhiza formation if surviving fungi are not compatible with new host plants (e.g. f u n g i from Eucalyptus may not form

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mycorrhizas if Acacia or Melaleuca are dominant after disturbance). The time required for fungi to grow from residual inoculum or re-colonise sites by dispersal, w i l l determine the rate of recovery of fungal diversity.

Spore dispersal by the wind and mycophagous animals (Table 3) are considered important in the colonisation of new habitats by ECM fungi (Cázares and Trappe 1994; Johnson 1995; Brundrett et al. 1996b). The effectiveness of these natural vectors will depend on the proximity of disturbed sites to habitats containing suitable fungi (and their associated animals) as well as the phenology of fruiting of fungi. It is not known if these fungi are more or less readily dispersed than their host plants (Figure 2). Unfortunately, there is insufficient information about the biology of mycorrhizal fungi to make robust predictions about the capacity of particular strains of fungi to survive disturbance, or to adapt to changes in soil conditions following disturbance. Fungal communities in disturbed habitats are considered further in Section 4.1.

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3. Mycorrhizal plants 3.1. Benefits to plants Ectomycorrhizal associations are assumed to have key roles in nutrient cycling processes in ecosystems where their hosts are dominant. This assumption is based on the large biomass of their fruit bodies which appear at certain times (Fogel and Hunt 1979; Vogt et al. 1982) and the pervasiveness of mycelium assumed to belong to ECM fungi in soils. Further evidence is provided by measurements of substantial carbon transfer from host plants to ECM fungi (Rygiewicz and Andersen 1994; Markkola et al. 1995; Setälä et al. 1999) and between interconnected plants (Simard et al. 1997). The mycelia of ECM fungi also are a major structural component of soils (Table 5). Glasshouse experiments have provided many demonstrations of substantial benefits from inoculation with ECM fungi, due to growth responses resulting from enhanced

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nutrient uptake, improved disease resistance, etc. (Table 5). Unfortunately, there have been few attempts to measure these parameters in natural ecosystems and it is dangerous to extrapolate results from highly simplified experimental systems to the real world.

The “foraging behaviour” of ECM fungus mycelia results in proliferation within nutrient rich patches of soil, lowering nutrient concentrations in these zones (Bending and Read 1995). Different parts of the mycelial systems of ECM fungi exhibit different physiological capabilities and structural properties (Unestam and Sun 1995; Cairney and Burke 1996). Only a fraction of the hyphae of an ECM fungus is considered to be capable of nutrient uptake at any one time and this proportion decreases with association age (Taylor and Peterson 1998). Mycelial systems produced by ECM fungi in soils are considered to play a key role in nutrient cycling in many ecosystems and function as the primary soil-plant interface for their hosts, which include many important

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forest trees. Unfortunately, there have been relatively few attempts to study ECM fungus systems in-situ. Some knowledge has come from observations of large wide) patches dominated by the mycelium of some ECM fungi, where soil physical and chemical properties are altered (Griffiths et al. 1994, 1996; Unestam and Sun 1995). It is considered that changes to soil properties (higher ion concentrations, oxalate accumulation, etc.) in these “hyphal mats” result in increased nutrient availability due to accelerated weathering of soil minerals (Griffiths et al. 1994; Paris et al. 1995). Hyphae of ECM fungi have also been implicated in the weathering of rock fragments in soils (Jongmans et al. 1997; Landeweert et al. 2001). The role of ECM fungi in nutrient cycling depends on their ability to acquire the mineral nutrients required by host plants from inorganic and organic sources in soils. It is generally assumed that ECM fungi have a greater capacity to acquire organic forms of nutrients than AM fungi (Marschner 1995; Smith and Read 1997). Ectomycorrhizal roots normally are more abundant in topsoil layers containing humus, than in underlying layers of mineral soil (Meyer 1973; Harvey et al. 1978; 1997). Evidence for the utilisation of organic materials by ECM fungi has been provided by production of enzymes capable of breaking down organic N sources in experimental systems (Table 5) and studies of ECM plants and f u n g i (Michelsen et al. 1998). Some ECM fungi specialise in the breakdown of organic compounds in animal wastes (Sagara 1995; Yamanaka 1999). Observations of substrate utilisation and isotopic composition of fungi have shown that ECM fungi generally have a much lower capacity to degrade complex substrates such as cellulose, lignin or phenolics than saprophytic fungi (Dighton et al. 1987; Bending and Read 1997; Kohzu et al. 1999). However, measurements of content of fruit bodies by Gebauer and Taylor (1999) suggested that some ECM fungi primarily utilised organic N sources from humus, while others depend on inorganic N from the soil. Organic N sources have been shown to be important to plants with ECM in arctic tundra, Australian eucalypt forests and boreal forests (Kielland 1994; Turnbull et al. 1995). The impact of different nutrient sources on competition between plants is considered in Section 4.2. In situ studies of litter decomposition have shown that hyphae considered to belong to ECM fungi were only present in the latter stages of this complex process and most of the work is done by other types of soil microbes and animals (Ponge 1991). The presence of ECM f u n g i may increase, or reduce the rate of breakdown of soil organic matter (Gadgil and Gadgil 1975; Dighton et al. 1987). Even if some ECM fungi have a role in organic matter breakdown, this would be less important than their role in coupling plants into the soil food web. Lindahl et al. (1999) studied interactions between mycelia of several ECM fungi and a wood rotting fungus (Hypholoma) in a microcosm experiment. They observed antagonistic interaction by several ECM fungi on Hypholoma, resulting in substantial transfer of to the mycorrhizal fungus. We still have much to learn about how ECM fungi acquire nutrients directly from

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organic sources or indirectly from other soil organisms responsible for nutrient cycling and how these processes are affected by environmental factors. Ectomycorrhizal fungi are the final step in the soil nutrient cycling process over a large portion of the earth’s surface, a role that is essential for ecosystem sustainability.

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3.2. Mycorrhizal dependency Different categories of mycorrhizal dependency have been defined for plants with AM associations (Chapter 4, Section 3.2), but it is generally assumed that all plants with ECM are obligately mycorrhizal (unable to survive to reproductive maturity without fungi). Experiments demonstrating substantial growth responses to ECM fungus inoculation are too numerous to list here and involve many of the plant genera listed in Table 1. However, these experiments normally use relatively infertile substrates that are initially devoid of ECM fungi and favourable environmental conditions. Demonstrations of growth responses due to mycorrhizal inoculation in the field have been less common, probably because mycorrhizal fungi are already present, soils may be more fertile and environmental conditions are not always suitable for fungal activity (Castellano 1994; Jackson et al. 1995; Brundrett 2000). Practical uses of ECM fungi are considered in section 5. There normally is a strong positive correlation between a plant’s dependency on mycorrhizas and the degree of mycorrhizal formation in its roots (Janos 1980; Brundrett 1991). Reports of ECM host plants in natural habitats without these structures on most of their fine root tips are rare. However, trees growing in flooded soils (e.g. Salix, Populus and Melaleuca) can have low levels of ECM, relative to trees of the same species in drier soils (Lodge 1989; Khan and Belik 1995). Trees with ECM even occur naturally in extremely fertile soils, such as Pisonia on coral islands where nesting birds provide massive fertiliser inputs (Ashford and Allaway 1985). In this case mycorrhizas appear to be involved in acquiring transiently-available nitrogenous products of uric acid degradation prior to leaching from the coral cay soils (Sharples and Cairney 1997). Perhaps the best evidence that hosts have an obligate requirement for mycorrhizas come from plantation forestry, where the failure of trees such as pines to establish without mycorrhizal inoculum has been noted when they are first planted in exotic locations (Trappe 1977; Smith and Read 1997). Further evidence comes from the nature of the root systems of host trees such as conifers with thick, slow-growing roots without long root hairs that contrast starkly with the fine root systems of plants that are able to grow well without mycorrhizas (Brundrett 1991; Marschner 1995). 3.3. Specificity Three different categories of host-fungus specificity were defined by Molina et al. (1992), who provide lists of fungi considered to belong in each category. Narrow host range fungi are only known to associate with one genus of host plants, intermediate host range fungi associate with different species of hosts w i t h i n a single plant family or group such as the Gymnosperms and broad host range fungi form mycorrhizas with plants from unrelated families. Most knowledge about the host specificity of fungi is based on observations of fruit bodies under trees, so it should be assumed that lists of fungal associates for plant taxa contain some errors.

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For example, the fungus Boletinellus merulioides and the tree Fraxinus appear in lists as ECM associates, but this tree only has AM in its roots and this fungus forms a mutualistic relationship with a subterranean aphid (Brundrett and Kendrick 1987). Further evidence of specificity is provided by glasshouse and sterile culture synthesis experiments which confirm that fungi can form normal looking mycorrhizas with some host plants but not others (e.g. Malajczuk et al. 1982; Burgess et al. 1993). However, some host-fungus combinations which form mycorrhizas in soil are incapable of doing so in sterile environments and vice versa (Malajczuk et al. 1982; Ba et al. 1994). Baiting experiments, where different ECM fungi were detected in the same soil using different host plants, have also demonstrated fungus specificity (Jones et al. 1997; Massicotte et al. 1999). Lists of ECM host-fungus associations are based on observations of fungal fruiting under certain plants backed up by the knowledge gained from synthesis experiments. Consequently, we must expect such lists to contain some errors. Although some ECM fungi, including Cenococcum geophilum (LoBuglio 1999) and Hebeloma cristuliniforme (Marmeisse et al. 1999) have been reported to form ECM with a diverse array of host genera belonging to different plant families, many have a much narrower host range (Molina et al. 1992). These narrow range fungi commonly show specificity at the host genus level with, for example, ca. 250 taxa thought to form ECM only with Douglas fir (Pseudotsuga) in North America (Molina et al. 1992) and a much larger number are likely to be restricted to Eucalyptus in Australia (Bougher 1995). We must be mindful, however, that host ranges of the majority of ECM fungi have been inferred from observations of co-occurrence of sporocarps and hosts in the field (Molina et al. 1992). In many cases it is impossible to separate effects of geographical distribution from true host specificity. Some reports of fungi with a single host may result from our limited knowledge of fungal distribution patterns in many regions. Intermediate host range fungi appear to be most common (Molina et al. 1992: Horton and Bruns 1998). This notwithstanding, individual trees in the field are normally colonised by both narrow and broad host range mycobionts and this may have important influences on competitive interactions between tree species (Section 4.2). The number of species of fungi in the broad host range category may well decrease with future taxonomic studies, as widely distributed fungi are found to comprise a number of similar looking species. A good example of this is the species Pisolithus tinctorius – reported to be one of the most common ECM fungi in the world, with a wide range of reported hosts and habitats. However, recent molecular, and biochemical evidence suggests that this taxon consists of a number of species with more restricted host and habitat preferences (Section 2 . 1 ) . Perhaps the best evidence that most ECM fungi are relatively specific comes from the low diversity of fungi which occur in plantations of pines and eucalypts grown in exotic locations (Dunstan et al. 1998; Brundrett and Bougher 1999). In many countries only a few genera have been

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reported to occur with these hosts, but there are many species found under indigenous tree species. The number of species of fungi fruiting in Australian Eucalyptus plantations increases with time in Australia, but remains low, even after many years, in most exotic locations (Lu et al. 1999). Newton and Haigh (1998) found that diversity of ECM fungi associated with particular hosts was positively correlated with the area of the UK occupied by these hosts plants. However, exotic trees introduced from other continents had a lower diversity of ECM fungi than was suggested by the importance of these trees in landscapes. Ecological implications of specificity of host fungal relationships are considered in section 5.2.

3.4. Pollution and climate Numerous reports attest to altered levels of ECM inoculum and/or diversity at field sites affected by anthropogenic pollution from industrial or urban sources (e.g. Danielson and Pruden 1989; Tosh et al. 1993; Kieliszewksa-Roikicka et al. 1997). Because some of these studies were conducted without quantification of soil pollutant status, and since a complex range of pollutants is generally present at such sites, it is difficult to infer any cause and effect relating to ECM fungal diversity and pollutants from these data. A clearer picture has arisen from simple glasshouse- and field-based experiments that have considered the effects of pollutants on ECM associations either singly or in combination (Cairney and Meharg 1999). These studies provide strong evidence that most forms of pollution can result in decreased ECM infection and altered below-ground community structure, although such effects may vary with the level and duration of exposure to the pollutant(s). Nitrogen deposition can lead to a decrease in percentage total root colonisation by ECM f u n g i (e.g. Tétreault et al. 1978; Taylor and Alexander 1989; Haug et al. 1992; Taylor et al. 2000), although such decreases may be rather short-lived, disappearing within a few years of soil treatment (Arnebrant and Söderström 1992; Kårén and Nylund 1997; Nilsen et al. 1998). Nitrogen fertilisation can also shorten the lifespan of mycorrhizal roots (Majdi and Nylund 1996). Nitrogen addition can also profoundly affect the below-ground structure of ECM fungal communities. Taylor and Read (1996) reported a clear pattern of decreased ECM morphotype richness on Picea spp. hosts was associated with increased nitrogen deposition across Europe. Moreover, they observed a change from those ECM fungi that can readily utilise organic nitrogen in favour of those which rely largely or solely upon inorganic nitrogen sources, at sites where nitrogen deposition was greatest. Smaller-scale studies also support marked shifts in ECM fungal community structure in response to nitrogen deposition, with decreases in the relative frequency of particular ECM fungi noted following nitrogen fertilisation (Taylor and Alexander 1989; Kårén and Nylund; 1997). The form of nitrogen in soil may further differentially influence below-ground ECM fungal community structure (Arnebrant and Söderström 1992). Significantly, the data of Arnebrant and

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Söderström (1992) were collected 13 years following fertilisation and indicate that nitrogen-mediated changes to ECM fungal communities may have more long-term ecological relevance than those observed for overall ECM colonisation. Arnebrant (1994) has shown that intraspecific differences exist in the sensitivity of ECM extramatrical mycelial systems to nitrogen additions, suggesting that growth of some fungi through soil can be profoundly affected by nitrogen inputs, while others appear relatively insensitive. Differences of this nature are likely to strongly influence the relative competitiveness of ECM fungi (Arnebrant 1996) and may underpin nitrogen pollution-related shifts in structure of belowground communities. The influence of acid deposition on forest trees and their associated ECM fungi has received considerable attention. Some field and glasshouse investigations indicate that acid deposition may significantly decrease percentage ECM infection (e.g. Danielson and Visser 1989; Esher et al. 1992; Stroo et al. 1988), while others report no obvious correlation between the two (e.g. Nowotny et al. 1998; Adams and O’Neill 1991). Frequently-cited reports of changes in ECM morphotype assemblages resulting from acidification (e.g. Gronbach and Agerer 1986; Roth and Fahey 1998), however, provide convincing evidence that soil acidification effects below-ground ECM fungus communities. Notably several studies recorded a decline in ECM fungal taxa that produce extensive mycelial systems in soil (Dighton and Skeffington 1987; Markkola et al. 1995). Toxic metal pollution may similarly reduce ECM infection and alter below-ground community structure (e.g. Chappelka et al. 1991), as may a range of organic chemical pollutants (e.g. Nicolotti and Egli 1998). Interactive effects between pollutants may further influence the structure of ECM fungal communities (see Cairney and Meharg 1999). In contrast to other forms of pollution, elevated atmospheric concentrations can increase percentage ECM infection of coniferous and hardwood hosts (e.g. Norby et al. 1987; Godbold et al. 1997). This effect may be short-lived under some conditions (e.g. Runion et al. 1997; Walker and McLaughlin 1997; Walker et al. 1999b) and there may be strong interactive effects with edaphic conditions such as soil nutrient and/or moisture status and atmospheric temperature (Conroy et al. 1990; Delucia et al. 1997; Tingey et al. 1997). Recent work reveals profound effects of enrichment on ECM fungal communities, with some taxa being positively influenced at the expense of others. Specifically, these studies suggest shifts in community structure in favour of taxa that produce extensive extramatrical mycelial systems in soil (Godbold and Berntson 1997; Godbold et al. 1997; Rey and Jarvis 1997). Single ECM fungi are also known to produce more substantial mycelial systems under elevated concentrations (Ineichen et al. 1995), the implication being that some ECM fungi may be carbon-limited at ambient concentrations and that increased carbon availability favours taxa that produce more substantial mycelia. Ectomycorrhizal fungi are a major pathway for carbon flow into soils and the magnitude of this pathway can

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be increased by elevated at least in some cases (Runion et al. 1997; Walker et al. 1999b). The distribution and mycorrhizal efficacy of fungi forming ECM associations is influenced by climatic and edaphic factors (Slankis 1974; Smith and Read 1997). These fungi are generally considered to be acidophilic (preferring a low soil pH) inhabitants of litter layers near the soil surface (A horizon), but some “early stage fungi” (Section 4.1) prefer mineral soils which may be calcareous. Tyler (1992) found that for macrofungi in a European forest (dominated by the ECM tree Fagus), the relative importance of ECM fungi increased (and saprobes decreased) in more-acidic soils. In this study, the distribution of many fungi was correlated with edaphic factors, such as soil organic matter and metal ion content. Isolates of ECM fungi show considerable inter- and intraspecific variations in responses to the factors listed in Table 4. Isolates of ECM fungi from polluted soils often have higher tolerance to metal ions under experimental conditions, than isolates of the same species from normal soils and thus may help their hosts to survive in these conditions (Hartley et al. 1997; Meharg and Cairney 2000a). However, results obtained from in vitro experiments are often poorly correlated with responses to similar factors in soils (Cline et al. 1987; Coleman et al. 1989; Hung and Trappe 1983; Hartley et al. 1997). It has been suggested that variations in tolerance to edaphic factors may restrict geographic ranges of fungi, influence the outcome of fungal competition, or responses to factors such as drought (Parke et al. 1983a; Last et al. 1984; McAfee and Fortin 1986). It is apparent that a wide range of variation in tolerance to edaphic and climatic factors (such as temperature extremes, drought, soil toxicity etc.) often occurs, both between and within species of mycorrhizal fungi and that this variation likely represents adaptation to specific site conditions (Trappe 1977; Trappe and Molina 1986). 4. Natural ecosystems 4.1. Fungal communities Forest plant communities that host ECM fungi are often relatively species-poor, however fungal species richness within these forests is characteristically high (Malloch et al. 1980; Allen et al. 1995). Goodman and Trofymow (1998); for example, estimated that up to 100 ECM root morphotypes (considered to representing either species or genera) were present in four 0.36 ha plots of an old-growth Canadian Douglas fir stand. Similar estimates have been derived from ITS-RFLP and /or morphotype data for stands of a range of other forest types dominated by coniferous, deciduous, eucalypt, or dipterocarp trees (Pritsch et al. 1997; Gehring et al. 1998; Goodman and Trofymow 1998; Ingleby et al. 1998; Glen et al. 1999; Jonsson et al. 1999a). It must be stressed that there are also some ECM forest habitats in which fungal diversity is low. These include, Alnus rubra stands in North America which host only a handful of ECM fungal taxa (Miller et al. 1991), and Pisonia grandis on coral cays in the western Indian and eastern Pacific oceans – which may

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be associated with a single ECM taxon across its entire geographical range (Ashford and Allaway 1985; Chambers et al. 1998). The diversity of ECM fungi in recently-disturbed habitats is typically much lower than in undisturbed sites and there are some species which are characteristically found in disturbed sites (Danielson 1985; Mason et al. 1987; Jumpponen et al. 1999). Reductions in fungal diversity from disturbance likely result because many fungi are eliminated, do not have resistant propagules, or have a limited capacity to adapt to large changes to their environment (Section 2.4). Fungal succession occurs under maturing stands of trees as a few pioneering fungi are gradually replaced by increasing numbers of fungi which typically fruit in older habitats (Gardner and Malajczuk 1988; Termorshuizen 1991; Richter and Brun 1993; Keizer and Arnolds 1994; Lu et al. 1999). Visser (1995) examined species richness in regenerating Pinus banksiana stands following fire. For the first six years, roots were colonised by relatively few fungi, but diversity increased markedly in 41 year-old and 65 year-old stands, the latter having a broadly similar community to a 112 year-old stand. Lu et al. (1999) observed that the diversity of ECM fungi fruiting in Eucalyptus globulus plantations in Western Australia steadily increased by approximately two species per year (Figure 3). In another study of ECM fungal successions on previously cultivated land, ECM species richness increased until canopy closure and then declined (Dighton and Mason 1985). Fungal diversity generally increases until late in succession, when the number of species present may decline when fungi with more specialised host or substrate preferences predominate (Bills et al. 1986; Last et al. 1984).

Observations of fungal succession in aging tree stands have resulted in the designation of two groups of fungi which occupy separate ends of this continuum. Fungi which typically associate with young trees in disturbed habitats or plantations have been termed early-stage fungi, while those that associate with old trees are termed late-stage fungi (Dighton and Mason 1985). Most studies of tree monocultures in disturbed habitats have supported this concept (e.g. Chu-Chou and Grace

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1982; Gardner and Malajczuk 1988; Cripps and Miller 1993; Visser 1995; Lu et al. 1999). However, there are cases where succession in young forests does not start with early stage fungi (Newton 1992; Keizer and Arnolds 1994; Helm et al. 1996; Bradbury et al. 1998). Early stage ECM fungi generally are easier to introduce into disturbed sites than late stage fungi (Danielson 1985; Lu et al. 1999). Typical examples of early stage fungi that are often observed in young plantations include members of the genera Pisolithus, Scleroderma and Laccaria. The factors underlying ECM fungal species richness in forest habitats have not been elucidated but, spatial and/or temporal resource partitioning, along with patterns of disturbance and competition may all play a role (Bruns 1995). A number of soil factors, including host plant age and physiological status, soil microbes, litter accumulation, fungal competition and inoculum availability could drive mycorrhizal fungus succession (Danielson 1985; Keizer and Arnolds 1994; Bruns 1995; Smith and Read 1997; Lu et al. 1999). Early stage ECM fungi typically grow in disturbed mineral soils with low organic matter with young host plants, while late stage fungi occur in the litter layer of mature forest soils (Mason et al. 1987; Gardner and Malajczuk 1988; de Vries et al. 1995; Lu et al. 1999). Physiological differences between early and late stage fungi are also apparent in aseptic culture experiments (e.g. Gibson and Deacon 1990). While we do not fully understand the factors influencing changes in ECM species richness with stand age, it is likely that changing soil properties and/or altered patterns of carbon allocation associated with tree maturation play a significant role. Spatial variability in soil properties and fungal population structure are also likely to be important in forests with high ECM fungal diversity. For example, of the 2000 or so fungi which associate with Douglas fir throughout its range, only about 10% of these will be found at any one location (Helm et al. 1996). Some of this high beta diversity may be due, in part, to spatial variations in soil properties which influence the competitiveness of individual fungi. However, it is likely that local variations in site histories and fungal dispersal events were responsible for initiating this spatial variability, which is maintained by processes we do not yet understand. Although generally species-rich, below-ground communities of ECM fungi are characteristically dominated by a small number of common taxa (e.g. Gehring et al. 1998; Horton and Bruns 1998; Jonsson et al. 1999b). These locally-abundant taxa, which inhabit a large proportion of available roots and presumably explore a proportionately large soil volume, may be functionally dominant (Horton and Bruns 1998), but, this remains to be demonstrated. Indeed, while ECM fungal diversity is widely regarded as important in ecosystem functioning and forest sustainability (e.g. Jones et al. 1997; Pritsch et al. 1997), the extent of functional diversity within the taxonomically diverse ECM fungal communities is currently unknown. It is likely that many ECM fungal taxa fulfil broadly similar ecological roles and, as such, that a high degree of functional redundancy exists (Allen et al. 1995). Attempts have been

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made to group taxa based on their abilities to utilise various substrates as sources of nutrients such as amino acids, which some ECM fungi can utilise but others cannot (Abuzinudah and Read 1986; Gebauer and Taylor 1999). It is known that substantial inter- and intra-specific variations occur between ECM fungi in their responses to environmental conditions (Table 4 and see Cairney 1999), growth and survival strategies (Section 2.2), etc. Our relatively poor understanding of ECM functioning in natural ecosystems, severely hampers our ability to predict the extent to which disturbance, pollution and other stresses will influence functioning of ECM-dominated plant communities. 4.2. Plant communities Mycorrhizal associations could influence plant community structure, by affecting the richness or evenness of populations of coexisting plants, or by changing the competitive ability of species. Table 7 in Chapter 4, shows 4 categories of intraspecific and interspecific interactions involving plants with different mycorrhizal requirements and association types. Facultatively mycorrhizal plants are not considered here, as plants with ECM generally are h i g h l y dependent on these associations (Section 3.2). Interactions between non-mycorrhizal plants are considered in Chapter 4. The impact of ECM associations on competitive interactions between plants would differ if competitors have ( 1 ) the same type of association, or (2) different types of associations. These cases are considered separately below. 4.2.1. Interactions between plants with ECM When growing together, plants with the same type of mycorrhizas are likely to be more equal competitors than plants with different types of mycorrhizas (Newman 1988). Plants with ECM presumably compete with other ECM plants for the same pools of soil resources (forms of nutrients), but plants with other nutrient uptake strategies (e.g. AM, ericoid mycorrhizas, non-mycorrhizal roots) may access different forms of nutrients. Competition between ECM hosts is more complex than between plants with AM (Chapter 4, Section 4.2), because ECM fungi vary more widely in their capabilities (e.g. to access organic nutrients) than AM fungi. Also, many ECM fungi are specific to certain hosts (Section 3.3), while AM fungi generally are non-specific. When different species of ECM plants grow together, the nature of competition will differ if they share the same (broad host range) fungi with a common pool of mycelia, or have separate host-specific ECM fungi that compete for soil resources. Finlay (1989) and Perry et al. (1989b) found that fungi which associate with two competing hosts, allow both to grow well together, but more specific fungi stimulated the growth of one host to the detriment of the other plant. The presence of hostspecific fungi in these experiments shifted the balance of nutrient competition in favour of their hosts. However, these experiments were conducted with only a few f u n g i , while plants in nature normally associate with a diverse mixture of broad and narrow host range fungi, so

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the impact of any one fungus on plant competition is likely to be much less dramatic in the field. Nevertheless, we can postulate that the evolution of specific host-fungus interactions may have occurred to provide benefits during nutrient competition and is more likely to occur for dominant trees in forest communities. Broad host range ECM fungi can form mycelial connections between different host taxa in the field, facilitating bi-directional transfer of carbon between them and resulting in a net carbon gain by one host (Simard et al. 1997). Common hyphal networks may assist the establishment of seedlings which share mycorrhizal partners with dominant trees. It has often been suggested that seedlings growing under mature trees of the same species are supported in this way (Newman 1988; Smith and Read 1997). This may explain why Pseudotsuga trees usually become established in patches of the ectendomycorrhizal shrub Arctostaphylos, b u t not under AM plants (Horton et al. 1999). A delta study has shown that most of the carbon received by shared ECM fungi comes from the overstorey trees, which help to support understorey species (Högberg et al. 1999). Preservation of ‘guilds’ of interconnected host plants (Perry et al. 1989a) and their associated fungal partners may help support the sustainability of ECM plant communities. Molina and Trappe (1982) have suggested that p l a n ts such as Alnus rubra and Pseudotsuga menziesii, which often form pure stands during early succession, tend to form specific associations with ECM fungi, while species such as Tsuga heterophylla, which become established in the understorey of other trees, usually have non-specific ECM associates. Thus, the availability of particular strains specifically required by different hosts could be a regulating factor during plant succession in some habitats. Despite the evidence provided above, the ecological importance of carbon and nutrient transfer between interconnected plants in natural ecosystems is not clear. The fact that only a very small proportion of mycorrhizal seedlings survive to become trees, demonstrates that help provided by mycelial interconnections is generally not sufficient to affect the outcomes of c ompetition (Newman 1988; Brundrett 1991). The greatest impact of sharing a common type of mycorrhiza appears to be an increase in the functional similarity of the roots systems of different species, so they are more equal competitors for soil nutrients which limit plant growth (Brundrett 1991). Even if the magnitude of below-ground carbon movements along hyphal interconnections is not sufficient to influence survival and growth of subordinate taxa, these interconnection can still function as a form of Cupertino where plants help each other by supporting a common mycelial network. The most extreme examples of resource transfer between plants with common mycorrhizal fungi are non-photosynthetic plants in the Orchidaceae and Monotropoideae (Ericaceae). Plants such as Monotropa live entirely by tapping into mycorrhizal fungus networks supported by connected autotrophic plants (Björkman 1960; Furman and Trappe 1971). Associations of non-photosynthetic plants appear to be more

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parasitic (from the fungal perspective) than mutualistic. These plants apparently have evolved a high degree of specificity in their associations with ECM fungi (Cullings et al. 1996; Taylor and Bruns 1997, 1999). 4.2.2. Interactions between different types of mycorrhizal associations Vegetation dominated by plants with one mycorrhiza type apparently can be a hostile environment for plants with other associations to grow in. It has been reported that trees with ECM often fail to become established in sites dominated by plants with ericoid mycorrhizas such as shrublands of Gaultheria, Kalmia, Rhododendron or Calluna heathlands (Robinson 1972; Messier 1993; Yamasaki et al. 1998; Walker et al. 1999b). Restricted availability of mineral nutrients and reductions in ECM fungus activity were reported within these habitats. Robinson (1972) suggested that an allelopathic inhibition of ECM fungi by exudates of Calluna contributed to nutrient deficiency problems for tree seedlings in heathlands. Examination of the world-wide distribution of plant communities reveals that most forests are dominated by trees which form ECM or AM associations and forests where both types of trees are equally dominant are rare (Brundrett 1991; Allen et al. 1995; Smith and Read 1997). It has been proposed that ECM-tree dominated plant communities are more likely to occur in soils with high organic matter and a predominance of organic nutrients, while AM-trees are more likely to dominate in mineral soils (Section 3.1). These relationships may explain the predominance of ECM forests in cooler climates. The situation in tropical regions is more complex as there do not appear to be major differences in soil properties between ECM and AM dominated forests in the same regions (Högberg 1986; Högberg and Alexander 1995; Newbery et al. 1997; Moyersoen et al. 1998). In eucalypt-dominated forest in Western Australia, roots of plants with ECM, AM or non-mycorrhizal cluster roots tended to be distributed in different soil patches, so may avoid direct competition for nutrients (Brundrett and Abbott 1995). Changes to soil properties apparently result because host trees produce leaves which are highly resistant to decomposition, resulting in slower nutrient cycling and a predominance of organic nutrient sources which are more accessible to ECM than AM fungi (Girard and Fortin 1985; Allen et al. 1995). It has also been proposed that substances in the leaf litter of ECM plants such as pine trees can inhibit AM fungi (Tobiessen and Werner 1980; Kovacic et al. 1984). Plant communities dominated by ECM plants may have a tendency to be self-perpetuating, by producing a soil environment which is hostile to AM fungi. Some plants with AM have also been reported to adversely affect ECM fungi. Hanson and Dixon (1987) found that ECM fungi could reduce the impact of allelopathy due to fern frond leachates on oak (Quercus rubra) seedlings. The abundance of weeds with AM associations can influence ECM formation by pine trees in plantations (Sylvia and Jarstfer 1997). Colonisation by AM fungi reduces the lifespan of roots of Populus, a tree which predominantly has ECM

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associations (Hooker et al. 1995). Allelopathic interactions between competing plants are considered to be common in plant communities, but the role of mycorrhizal fungi in these interactions has rarely been investigated. A second form of competition between different types of mycorrhizal fungi occurs within the root systems of plants which are hosts to two types of mycorrhizal associations. In Australia, plants with ECM associations usually also have some VAM in their roots. These plants include major species used in plantation forestry belonging to the genera Casuarina, Allocasuarina (Casuarinaceae), Eucalyptus, Melaleuca (Myrtaceae) and Acacia (Mimosaceae) (Brundrett 1999). Plants with dual ECM/VAM associations are less often reported from other parts of the world (Brundrett 1991), but there are exceptions such as Alnus, Populus, Salix and Uapaca (Lodge and Wentworth 1990; Arveby and Granhall 1998; Moyersoen and Fitter 1998). Most gymnosperms with ECM are highly resistant to AM fungi, but their roots may contain vesicles and hyphae if grown with a companion AM plant (Hooker et al. 1995; Smith et al. 1998). A succession from VAM to ECM associations in Eucalyptus and Alnus roots often occurs as trees age in field soils (Gardner and Malajczuk 1988; Bellei et al. 1992; Oliveira et al. 1997; Arveby and Granhall 1998). Ectomycorrhizal fungi have also been observed to gradually displace VAM in Eucalyptus roots in the glasshouse (Lapeyrie and Chilvers 1985; Chen et al. 2000). Plants which can form both ECM and AM associations occur in some ecosystems. For these plants, it seems that AM are most important for their young seedlings, perhaps due to the time required for ECM fungus dispersal and establishment, while ECM usually becomes dominant when they grow older. There are plants, such as some Acacia species, which are highly receptive to ECM and AM associations throughout their lives, but this is rare and most species show a clear preference for a single type of mycorrhiza (Brundrett 1999).

4.3. Animals The roles of animals as consumers and dispersal agents of ECM fungi are considered in section 2.3. Tree-mycorrhizal fungus-dispersing animal interrelationships are important in forests, especially in western North America and Australia (Maser and Maser 1988; Claridge et al. 1992). Animals which disperse ECM fungi facilitate ecosystem recovery after disturbance (e.g. Claridge et al. 1992; Cázares and Trappe 1994; Johnson 1995; Reddell et al. 1997; Cázares et al. 1999). Ectomycorrhizal fungi and their host plants must also be considered during any attempts to manage mycophagous animal species. For example, the occurrence of hypogeous ECM fungi in different vegetation types is thought to limit the distribution of the Northern Bettong (Bettongia tropica) which needs these fungi for food in the dry season (Johnson 1996). The importance of animals as vectors for ECM fungi is suggested by the evolution of truffle-like fruit bodies in most families of ECM fungi in Australian eucalypt forests (Bougher and Lebel 2001). These

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subterranean fruit bodies would only have a selective advantage in habitats where animal dispersal of fungi is more effective than airborne spore dispersal. Marsupials which consume spores are efficient dispersal agents, because they forage near trees over a wide area and have commensal dung beetles which bury their spore-laden faeces (Johnson 1996). Unfortunately, many Australian mycophagous marsupials are now extinct throughout most of their former ranges, and this could effect ecosystem functions, especially the capacity for recovery after disturbance. We must consider secondary symbiotic associations, such as the animal vectors of ECM fungi, as well as the primary tree-fungus associations when we monitor the quality of remnant vegetation, or attempt to restore plant communities. 5. Utilising mycorrhizas This section will focus on plant conservation and ecosystem restoration. However, there also are concerns about the conservation of ECM fungi in regions where their populations are declining due to changes to soil conditions caused by pollution or over-collection by humans (Arnolds 1991; Boujon 1997; Hosford et al. 1997). Ectomycorrhizal fungi exhibit functional diversity and are adapted to local environmental and soil conditions (Table 4). Consequently, it would be important to conserve representatives of all habitats and soils within regions to maintain the functional diversity of mycorrhizal fungi and other organisms. Conservation of particular ECM fungi may also be required for nonphotosynthetic plants in the Ericaceae (Monotropoideae) which are wholly dependent on a type of ECM fungi and have specific fungal partners (Cullings et al. 1996; Kretzer et al. 2000). 5.1. Ecosystem restoration Mycorrhizal technology can be designated as any artificial means of introducing fungi to new habitats or manipulating existing populations of fungi. Most work on the introduction of ECM fungi has been for plantation forestry, or for valuable edible fungi such as the black truffle (Tuber melanosporum). Inoculation technologies have been developed to introduce fungi in the nursery or field, using soil, spores or mycelia produced in sterile culture as inoculum (Brundrett et al. 1996c). Before promoting mycorrhizal technology, it is necessary to evaluate its relative costs and benefits (Brundrett 2000). Costs arise from the resources and time required to acquire appropriate fungi, produce and apply inoculum and implement quality control measures to confirm inoculated fungi are present. Benefits primarily concern short-term increases in survival and/or growth of plants. Some ECM fungi may offer added benefits by influencing the activities of other soil microorganisms, or degradation of persistent organic pollutants in soil (Meharg and Cairney 2000b). Longterm benefits from increased mycorrhizal fungus biomass or functionality are suggested in the literature, but have not been measured in the field (Section 3.1). Potential environmental costs that may arise from introductions of mycorrhizal fungi into different locations should

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also be considered (Section 5.2). When ECM fungi are introduced to a site (usually by planting seedlings inoculated in the nursery), their success depends on their ability to spread through the soil to new roots and the outcome of competition with indigenous fungi (Last et al. 1984; McAfee and Fortin 1986). Hostfungus specificity is also important, as poorly compatible fungal isolates will often fail to establish. Cases where substantial improvements in tree growth due to mycorrhizal inoculation were measured are outnumbered by trials where there was no measurable response (Castellano 1994; Jackson et al. 1995; Brundrett 2000). Nevertheless, there have been cases where ECM fungus inoculation has resulted in significant growth enhancement in the field, especially where trees have been grown in disturbed habitats such as mine sites, or exotic locations with few compatible fungi (Malajczuk et al. 1994; Castellano 1994; Reddell et al. 1999). Responses to mycorrhizal inoculation are also highly dependent on soil conditions, especially soil fertility. For example, Cistus incanus fails to establish in infertile calcareous soils without mycorrhizal fungi, but grows well without fungi if fertilised (Berliner et al. 1986). Mycorrhizal fungi may be an important consideration in rare species recovery programs. They sometimes are included in cultivation attempts as an insurance policy to eliminate nutritional factors as a cause of failure. Inoculation of tissue cultured plants is especially important for subsequent plant survival and growth during critical early stages of establishment in soil (Martins et al. 1996; Reddy and Satyanarayana 1998). 5.2. Potential problems with fungal technology While ECM inoculation programs were required for the successful establishment of plantations of trees such as pines and eucalypts at exotic locations, they have the effect of introducing alien ECM fungal taxa along with their hosts. It is possible that alien fungi may influence local ECM fungal diversity via invasion of native forest systems. The extent to which this occurs will depend upon the ability of the introduced fungi to persist at the exotic sites and the extent to which they are able to form ECM with the native vegetation. There is certainly evidence that some introduced ECM fungi can persist in plantations at exotic locations for many years following introduction (e.g. Martin et al. 1998; Selosse et al. 1999). It has generally been assumed that no environmental costs will arise from introductions of mycorrhizal fungi into different geographic locations. The host specificity of many fungi often prevents fungi from associating with indigenous hosts belonging to other families (Section 3.3). However, introducing fungi that are more, or less effective symbiotic partners than indigenous strains, may impact on plant community structure, by influencing the outcome of competition between plants. We would expect the potential for broad host range fungi to invade indigenous vegetation should be greater than for fungi which associate with few host plants. Fungi associated with Eucalyptus spp. are a useful

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example in this context. The geographical isolation of the Australian flora is considered to have enabled co-evolution of highly specific ECM associations within the genus Eucalyptus, that are poorly compatible with tree species from other continents (Malajczuk et al. 1982, 1990; Bougher 1995). Recent investigations in Kenya, for example, indicate that Pisolithus species introduced during the past 100 years into Eucalyptus and Pinus plantations are only found in association with their respective hosts (Martin et al. 1998). Australian species of Laccaria, Hydnangium and Hysterangium associated with eucalypt plantations at exotic sites also appear to remain confined to that genus (Castellano 1999). None of the ECM fungi introduced to Australia with Pinus are known to have spread into Eucalyptus forests (Molina et al. 1992; Bougher 1994; Dunstan et al. 1998). However, the pine fungus Amanita muscaria is now found under Nothofagus in Tasmania and New Zealand (T. May pers. com., P. Johnson and P. Buchanan pers. com.). Fungi which readily switch to new families of host plants include several Amanita species which have crossed over to eucalypts introduced to North America and Europe (Brundrett and Bougher 1999). These relatively promiscuous fungi apparently can invade indigenous forests as “weeds” that compete with indigenous species. Perhaps we should be concerned about some feral fungi, such as extremely toxic Amanita species, that have the potential to invade native forests and kill indigenous fungus-feeding animals.

6. Conclusions This review attempts to summarise key roles of ECM fungi in natural ecosystems and provide information that should be of value to people who study processes in or help to conserve natural habitats. Plants with mycorrhizas are dominant in most communities (Table 1 in Brundrett and Abbott, this volume). Thus, mycorrhizal fungi typically are the primary soil interface for plants and must be considered in all studies of nutrient cycling or impacts of nutrient supply on plant productivity or diversity. Processes mediated by ECM associations in natural ecosystems are listed in Table 6. There is much scope for future ecological work investigating the processes listed in Table 6. In these studies, the first challenge is to determine what type of mycorrhizal associations the plant(s) being considered have. Information about mycorrhizal associations has been summarised for some locations such as the UK (Harley and Harley 1987) and Australia (Brundrett 1999). Information exists for many other locations, but may be harder to find. Published lists must be expected to contain some errors or misinterpretations and contradictory data exists for some species. Consequently, it will often be necessary for researchers to examine roots of their plant species using microscopic techniques to confirm the presence of mycorrhizal associations (Brundrett et al. 1996c). The second challenge is to determine if mycorrhizal fungi are already present in soils where host plants will be grown. Sampling methodologies exists for detecting inoculum of these fungi (Brundrett et

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al. 1996c). However, we would expect ECM fungi already to be present in most habitats, provided compatible hosts occur nearby and soils have not been substantially altered by disturbance, pollution, etc. A third challenge may be to determine if it is appropriate to introduce fungi to sites and when it is unnecessary or even harmful to do so (Section 5.2). Ectomycorrhizal fungi can be dispersed by wind and animals and may rapidly arrive in new sites. Some host plants may also grow well initially without ECM fungi provided AM fungi are present or soils are relatively fertile.

We hope that we have provided some guidance about where ECM associations need to be considered in scientific studies of ecosystems or individual species. This information would also be relevant to the management of plants, plant communities and mycophagous animals. These fungi have many important roles (Table 5) and the fruit bodies of some species are important as food sources for animals and humans. The importance of ECM associations was first proposed by Frank in 1885. Since then, we have learned how to manipulate these associations in experiments and have amassed a substantial body of information about

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their roles in plant nutrition. However, there is still much to learn about ECM in natural ecosystems, especially concerning: (i) How they acquire soil nutrients from organic and inorganic sources in co-operation/competition with other organisms in soil food webs. (ii) The magnitude of nutrient and carbon transfer between connected individuals of the same and different species and the role of these transfers in ecosystems. The impact of ECM associations on the disease resistance, water (iii) relations, etc. of their hosts. (iv) The influence of changes to soil structure and chemistry caused by ECM fungus hyphae (e.g. hyphal mats, carbon storage, weathering of minerals, etc.) on plant nutrient supplies. (v) The role of taxonomic and functional diversity in these fungi. (i.e. Why there are many species of ECM fungi in some habitats and few in others?)

Perhaps the start of a new millennium is an appropriate time to reconsider where we heading. It is time to stop repeating the same basic experiments demonstrating benefits provided to yet another plant species using highly simplified experimental conditions. Instead we need to shift focus to the roles of ECM in-situ in natural ecosystems, by considering the neglected areas of research listed above. Experiments will be more difficult in complex systems, but results will be much more meaningful. Acknowledgements The authors are grateful to Neale Bougher, Bill Dunstan, Antoni Milewski and a reviewer for comments on the manuscript. References Abdul-Kareem AW, McRae SG (1984) The effects on topsoil of long-term storage in stockpiles. Plain and Soil 76, 357–363. Ahuzinudah RA, Read DJ (1986) The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. I. Utilisation of peptides and proteins by ectomycorrhizal fungi. New Phytologist 103, 481–493. Adams MB, O’Neill EG (1991) Effects of ozone and acidic deposition on carbon allocation and mycorrhizal colonization of Pinus taeda L. seedlings. Forest Science 37, 5–16. Agerer R (1995) Anatomical characteristics of identified ectomycorrhizas: an attempt towards a natural classification. In ‘Mycorrhiza.’ (Eds A Varma and B Hock) pp. 685–734. (Springer Verlag: Berlin) Aggangan NS, Dell B. Malajzuk N (1996) Effects of soil pH on the ectomycorrhizal response of Eucalyptus urophylla seedlings. New Phytologist 134, 539–546 . Aleksandrowicz-Trzcinska M, Grzywacz A (1997) The effect of fungicides used in the protection of forest tree seedlings on the growth of ectomycorrhizal fungi. Acta Mycologica 32, 315–322 . Allen EB, Allen MF, Helm DJ, Trappe JMa, Molin a R, Rincon E (1995) Patterns and regulation of mycorrhizal plant and fungal diversity. Plant and Soil 170, 47–62.

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Chapter 6

ARBUSCULAR MYCORRHIZAS IN PLANT COMMUNITIES Mark C. Brundrett Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia (current address); CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural Research, Private Bag No 5, Wembley 6913, Western Australia (former address); and Science Directorate, Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia (correspondence)

Lynette K. Abbott Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia

1. General introduction Much of our understanding of the role of arbuscular mycorrhizas (AM) comes from experiments with individual plants and fungi using simplified soil conditions, but there have been some attempts to study their functions in natural ecosystems. This review concentrates on the importance of AM associations in plant communities, their role in competition between plants and in plant establishment in disturbed habitats - as these have the greatest relevance to plant conservation. 1.1. Associations Arbuscular mycorrhizas are mutualistic associations between soil fungi and plant roots. These associations are also referred to as vesicular­ arbuscular mycorrhizas and are abbreviated as AM or VAM (the term endomycorrhiza should no longer be used). Arbuscular mycorrhizas consist of hyphae, arbuscules and vesicles within roots, as well as hyphae, spores and other structures in the soil (Figure 1). The partners in these associations include members of the fungus kingdom in the Zygomycete order Glomales and most vascular plants. The host plant usually receives mineral nutrients obtained from the soil by the fungus while, in exchange, the fungus partners obtain photosynthetically derived carbon compounds

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from the plant. Plants may receive other benefits from mycorrhizal fungi, but these are not well understood (see Section 3.1). Mycorrhizas consist of plant roots and mutualistic fungi in their soil environment and these three factors must be considered together when studying these associations.

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1.2. Host plants It has often been stated that most plants in terrestrial ecosystems have mycorrhizal associations, but there has only been one recent attempt to catalogue data supporting this assertion (Brundrett 1991). Information about the mycorrhizal status of plants occurring in major ecosystems and edaphic communities is summarised in table 1. Arbuscular mycorrhizas are the most important type of association in most ecosystems. The only exceptions are ecosystems or zones within ecosystems dominated by trees with ectomycorrhizas and habitats where adverse climatic or soil conditions severely limit plant productivity. However, plants with AM are still also important in most extreme habitats. Summaries of mycorrhizal strategies by plant taxa (Newman and Reddell 1987; Trappe 1987; Peat and Fitter 1993), or within geographic regions (Harley and Harley 1987; Koske et al. 1992; Brundrett 1999) are also available.

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It is common to see statements such as 95% of plants are mycorrhizal in the literature, but this is not accurate. Trappe (1987) provided a more accurate estimate by reviewing data for 6500 angiosperm species whose mycorrhizal status is known from the scientific literature (approx. 3% of angiosperm species). Figure 2 summarises these results. The actual proportion of plants with mycorrhizas is approximately 80% of examined species with about 60% reported to have AM. However, these conclusions may be somewhat biased because more data came from the temperate northern-hemisphere than from other regions. Many gymnosperms and ferns also have AM associations (Brundrett 2001). An understanding of the importance of mycorrhizal associations at the community level requires data on the relative dominance of plants with different types of mycorrhizal associations in natural ecosystems. These data are only available for a few plant communities (Brundrett 1991).

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1.3. Fungi Fungi forming AM associations include over 150 species belonging to the genera Gigaspora and Scutellospora, Glomus, Acaulospora, Entrophospora, Paraglomus and Archaeospora in the Zygomycete order Glomales (Morton and Benny 1990; Redecker et al. 2000b; Morton and Redecker 2001). They are primitive members of the fungus kingdom which are not closely related to any other living group of fungi. Similar looking associations have been found in fossilised rhizomes of early vascular land plants and evidence of their spores extends back to the Ordovician (Stubblefield and Taylor 1988; Pirozynski and Dalpé 1989; Redecker et al. 2000a). Mycorrhizal fungi are thought to live in a particular habitat for thousands of years with little genetic change (Trappe and Molina 1986). The relatively small number of extant AM fungus species and the lack of sexual reproduction in this group of fungi also suggest that the potential for genetic change within these species is limited (Tommerup 1988; Morton 1990). The hyphae and spores of AM fungi are multinucleate and likely also heterokaryotic, so genetic changes may occur through hyphal anastomosis or somatic recombination involving different nuclei (Tommerup 1988; Trappe and Molina 1986; Sanders et al. 1996; Bever and Morton 1999; Lanfranco et al. 1999). Careful taxonomic studies (Morton 1988), the use of isoenzymes and DNA-based methods (Hepper et al. 1988; Sanders et al. 1996; Clapp et al. 1999) and differential responses to soil and environmental conditions (Section 3.3) have demonstrated considerable variation within currently defined taxa of AM fungi.

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2. Biology and ecology of AM fungi 2.1. Distribution, abundance and diversity Knowledge of the biology and ecology of AM fungi is limited by technical difficulties in both identifying and quantifying species present in soils. The methods used to identify glomalean fungi in soils or roots include (i) separation of spores from soil, (ii) isolation into living cultures with a host plant, ( i i i ) recognition of infection patterns in roots and (iv) use of biochemical or DNA-based methods (Brundrett et al. 1996c). Care must be taken in interpreting results obtained using any of these methods in population studies, as the apparent diversity of fungi and their perceived relative dominance will depend on the procedure used (Brundrett et al. 1999a; Douds and Millner 1999). Most of our knowledge about fungal populations comes from looking at spores, because they are relatively easy to separate from soils and used to identify fungal species. However, the other methods discussed below are now considered to provide a more accurate picture of fungal diversity. Surveys of AM spores have found up to 23 species of fungi in one soil sample (Brundrett 1991; Douds and Millner 1999). This represents a fairly high taxonomic diversity of these fungi (considering that only about 150 species have been named) (Bever et al. 2001). Technical difficulties with spore separation from soils contribute to the inaccuracy of surveys, especially if soils are fine textured, fungi sporulate infrequently, spores occur within organic matter, or they are not distributed uniformly. DNA methods have recently been used to identify or quantify AM fungi in soils (Clapp et al. 1995; Helgason et al. 1999). However, data obtained by this means has been limited due to technical difficulties (Sanders et al. 1996; Douds and Millner 1999; Lanfranco et al. 1999). The extraction of lipids and analysis of fatty acid profiles is another promising method for quantifying AM fungi in soils (Graham et al. 1995; Olsson 1999). Glomalean fungi must be grown in association with a living plant to provide material for research purposes, practical applications and taxonomic study. Fungi are usually propagated using “pot cultures” where an inoculated plant is grown in a sandy soil supplied with low levels of phosphorus (Menge 1984; Jarstfer and Sylvia 1993). These fungi can also be grown using aeroponics or root-organ cultures (Jarstfer and Sylvia 1993; Bécard and Piché 1992). Most pot cultures are initiated using spores separated from a field soil, or soil from a field site (Jarstfer and Sylvia 1993; Bever et al. 1996; Brundrett et al. 1996c). Soil-based trap cultures often contain additional species to those found by examining spores extracted from the same soils (An et al. 1990; Stutz and Morton 1996; Watson and Millner 1996; Koske et al. 1997; Brundrett et al. 1999a). However, trap cultures contain a mixture of fungi that changes over time, so must be further purified before use in experiments. Production of living cultures of AM fungi is difficult and time consuming, and consequently, is the main factor limiting research activities and practical applications with these fungi.

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Microscopic examination of cleared and stained roots reveals structures produced by AM fungi (Brundrett et al. 1996c). These structures can be used to identify fungi to determine their relative abundance in natural ecosystems (Abbott 1982; McGee 1989; Thomson et al. 1992; Brundrett and Abbott 1995; Merryweather and Fitter 1998b), or glasshouse experiments (Abbott and Robson 1984a; Jasper et al. 1991; Lopez-Aguillon and Mosse 1987; Pearson et al. 1993; Brundrett et al. 1999b). This method avoids problems with spore-based surveys, because non-sporulating fungi are often important in soils and there are large differences in spore production between species (Brundrett et al. 1999a). This method allows the relative contributions of individual fungi sharing roots with other fungi to be determined. However, working with roots from natural habitats may be difficult if (i) young roots with active associations are hard to obtain due to the seasonality of root growth and long root life-spans, (ii) roots are thick and have abundant secondary metabolites, or ( i i i ) roots of different plant species are hard to separate in mixtures (Brundrett et al. 1996c). 2.2. Lifecycles and inoculum The spread to new roots, long range dispersal and persistence of mycorrhizal fungi in ecosystems is dependent on the formation of propagules which are resistant to soil and environmental conditions. Propagules of AM fungi include asexual spores formed in soil, root fragments containing hyphae and vesicles (storage structures) and soil hyphae (Figure 1). Mycorrhizal fungus propagules are usually concentrated in the topsoil, but can also occur at greater depths (up to 4 m) in arid ecosystems (Virginia et al. 1986; Zajicek et al. 1986). Fungi must be active when root growth activity occurs, since roots have a limited period of receptivity (Brundrett and Kendrick 1990; Hepper 1985; Smith et al. 1992) and efficient colonization of roots is required for an effective association (Abbott and Robson 1984b; Bowen 1987). Soil-borne spores have traditionally been thought to be the most important type of inoculum of AM fungi. However, spore numbers are often poorly related to mycorrhizal formation in soils and fungi which do not produce recognisable spores are important in many soils (Abbott and Robson 1991; Brundrett and Abbott 1995; Stutz and Morton 1996; Merryweather and Fitter 1998a). Living spores of AM fungi in soil may not function as propagules if they are quiescent, dormant, or have been parasitised (Tommerup 1992; Lee and Koske 1994). A link between mycorrhizal colonisation levels and the timing of sporulation has been observed in experiments with some isolates of AM fungi (Table 2). Observations from these experiments suggest that: (i) colonised root length can be a good predictor of sporulation (Douds 1994), (ii) a minimum colonisation level is required for sporulation for some species (Gazey et al. 1992), (iii) mycorrhizal formation may decline after sporulation begins (Pearson and Schweiger 1993) and (iv) soil hyphae may lose viability after sporulation commences (Jasper et al. 1993). It should be noted that these observations apply to particular fungal isolates as others may behave differently (Table 2).

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A pre-existing network of soil hyphae is often considered to be the main source of AM inoculum in ecosystems because spore numbers are not correlated with mycorrhizal formation and colonisation starts immediately after root growth commences (Jasper et al. 1989; McGee 1989; Brundrett and Abbott 1995). Fragments of dead roots present in the soil can also initiate AM (Rives et al. 1980; McGee 1987). Many AM fungi form vesicles within roots which function as storage organs and/or propagules (Biermann and Lindermann 1983) which can be structurally and functionally similar to the spores of AM fungi in soil (e.g. some Glomus species), or may be temporary storage structures that do not persist in old roots (e.g. most Acaulospora species) (Table 2). Old roots and coarse soil organic matter colonised by AM fungi are also thought to contribute to the survival and spread of AM fungi (Warner 1984; Brundrett and Kendrick 1988; Koske and Gemma 1990). Since the most important propagules of AM fungi in soils are generally unknown, it is best to measure the total inoculum potential of these fungi. This value can be estimated by most probable number methods (serial dilutions using sterilised soil), or bioassays which measure the degree of colonisation of a plant grown in that soil (see Abbott and Robson 1991; Brundrett et al. 1996c). A study of AM fungi in tropical soils found there were two major functional categories of these fungi, which either used spores as an important propagule, or rarely produced viable spores (Brundrett et al. 1999a). AM fungus population studies based entirely on spores in soils are likely to be misleading. Major differences in life history characteristics between species of AM fungi are listed in table 2. It should be noted that some pairs of characteristics in table 2 reflect ends of a continuum. Further research is required to determine how these functional characteristics are correlated with the capacity of fungi to grow in soils and provide benefits to plants. 2.3. Predators and dispersal A wide variety of mycophyllous soil microorganisms occupy, or associate with soil hyphae, mycorrhizal roots or spores of AM fungi (Table 3). Spores of AM fungi isolated from soils in natural ecosystems often show signs of predation, which may be responsible for seasonal fluctuations in spore abundance (Ross and Ruttencutter 1977; Lee and Koske 1994). Interactions between AM fungi and soil microbes include (i) occurrence within living hyphae, (ii) necrotrophic associations with old hyphae or spores, or (iii) parasitism of hyphae and spores which may be detrimental to associations (Macdonald and Chandler 1981; Sylvia and Schenck 1983; Paulitz and Menge 1984; Lee and Koske 1994). Fungus-feeding nematodes, springtails and mites feed on AM fungus hyphae (Table 3). Hyphal grazing by soil arthropods reduced the benefits provided by AM in some experiments (Ingham 1988; Rabatin and Stinner 1988), however in others, these animals were found to have little effect on plant yield (Larsen and Jakobsen 1996), preferentially grazing on other types of fungi when given a choice (Klironomos and Kendrick 1996), or were only detrimental at high animal densities (Klironomos and Ursic 1998). It is not surprising that a wide diversity of

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organisms are known to consume, decompose or associate with AM fungi, as these fungi are a major component of the microflora of most soils. However, most mycophyllous organisms seem to have a limited impact on the performance of mycorrhizal fungi in natural habitats.

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The spread of mycorrhizal fungi (Section 2.2) occurs by active processes (hyphal growth through soil) or passive dispersal mechanisms. Dispersal allows the introduction of mycorrhizal fungi to new geographic locations and the transfer of genetic information. Growth of hyphae of AM fungi through soil can slowly spread the association to adjacent plants (e.g. Scheltema et al. 1985). Propagules of AM fungi can be suspended in moving air currents (Tommerup 1982) and wind dispersal has been observed in ecosystems (Warner et al. 1987). AM fungi are probably also transported by water erosion and human activities. Koske and Gemma (1990) observed that rhizome leaf sheaths and old roots containing AM fungi could be transported by wind or water in coastal habitats. The animals which ingest and disperse AM fungi include small mammals, marsupials, grasshoppers, worms, ants, wasps and birds (Table 3). Soil-dwelling arthropods and earthworms are considered to be important vectors of these fungi (Reddell and Spain 1991; Gange 1993; Harinikumar and Bagyaraj 1994). Larger animals that feed on AM fungi can transport viable spores for considerable distances (McGee and Baczocha 1994; Janos et al. 1995). The introduction of AM fungi by animal digging activities was considered important during the establishment of vegetation in areas devastated by the Mt. St. Helens volcanic eruption (Allen et al. 1992). 2.4. Disturbance AM fungi can survive soil freezing (Addy et al. 1997), or wetting and drying cycles (Braunberger et al. 1996). Daft et al. (1987) found that spores were more resistant to topsoil disturbance and storage than root fragments and much more resistant than hyphae. Networks of soil hyphae are an important source of inoculum in natural ecosystems that are highly susceptible to disturbance (Jasper et al. 1989; Evans and Miller 1990; Bellgard 1993). Disturbance is also likely to reduce the effectiveness of root inoculum (Evans and Miller 1988; Rives et al. 1980). Numbers of surviving propagules of AM fungi in soils decline with time in the absence of host plants (see Figure 3 in Chapter 3). Mycorrhizal propagules can be severely influenced by damage to vegetation and soils resulting from natural processes or human intervention. Destructive processes which adversely affect AM fungi include: intense fires, topsoil removal and flooding (Table 4). Mycorrhizal fungi may be absent from soils affected by salinity, aridity, waterlogging, or severe climatic conditions (Brundrett 1991). AM inoculum is i n i t i a l l y absent from very young habitats such as sand dunes (Corkidi and Rincón 1997a), volcanic substrates (Allen et al. 1992; Gemma and Koske 1990), or glacial deposits (Helm et al. 1996). Anthropogenic disturbances which impact on AM fungi include m i n i n g (Section 5.2), forestry (Norani 1996) and agriculture (Alexander et al. 1992). Agricultural practices such as tillage, long fallow periods, soil compaction, or growth of non-mycorrhizal crops may also be detrimental to mycorrhizal associations (Thompson 1987; Wallace 1987; Evans and

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Miller 1988; Abbott and Robson 1991). The role of AM fungi in disturbed habitats is considered in Section 5.2.

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Much research has focussed on the potential for mycorrhizal associations to increase plant productivity in plantation forestry, or during ecosystem recovery after severe disturbance, as well as in agriculture and horticulture. However, it could be argued that we do not know enough about the role of mycorrhizal associations in natural, disturbed, or managed ecosystems to safely evaluate their potential for applied use.

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Benefits provided by AM associations to plants and their interactions with environmental factors are considered here. 3.1. Benefits to plants The main function of AM associations is considered to be mineral nutrient acquisition from soil, but these fungi can also provide many other benefits to individual plants or other organisms (Table 5). Knowledge of the importance in natural ecosystems of many of the benefits listed in table 5 is very limited (Section 4.1). Plants have demands for mineral nutrients determined by their internal requirements and soils have a limited capacity to supply nutrients due to their availability and mobility (Russell 1977; Marschner 1995). Phosphorus is normally considered to be the most important plant-growth limiting factor which can be supplied by mycorrhizal associations, because its restricted mobility in soils causes depletion zones around roots (Bolan 1991; Marschner 1995). Thus hyphae of AM fungi would be primarily responsible for acquiring phosphorus from outside root depletion zones (Marschner and Dell 1994; Smith and Read 1997). These hyphae were considered to utilise the same forms of nutrients as roots, but there now is evidence that they have a greater benefit when phosphorus is present in less-soluble forms (Bolan 1991; Schweiger et al. 1995; Kahiluoto and Vestberg 1998). AM fungus hyphae help plants acquire P from some organic compounds, but not others (Tarafdar and Marschner 1994; Joner et al. 1995). AM fungus hyphae may also help plants acquire some nitrogen and trace elements (Table 5). Hyphae of AM fungi can respond to localised sources of soil nutrients by hyphal proliferation (St John et al. 1983; Warner 1984) and production of fine highly-branched “absorptive” hyphae (Mosse 1959; Bago et al. 1998). Thus, AM fungi may access nutrients which are spatially and chronologically separated from roots. AM fungi may also work synergistically with organisms which decompose organic materials in soils, by providing a link between roots and localised sites where nutrients are made available by saprobic organisms (Table 5). Because some AM fungi clearly increase the uptake of P and hence plant growth, in glasshouse experiments using P-deficient soils, it is generally assumed that the same happens on a larger scale in agriculture, revegetation sites and in natural ecosystems. This assumption underpins most research on AM fungi. However, the P uptake and translocation by hyphae of AM fungi associated with perennial plants is expected to differ from that of annuals such as crop plants (Smith et al. 1994). The great functional diversity of the roots of perennial hosts remains virtually unexplored. Attempts to quantify the benefits provided by AM fungi in natural ecosystems have been complicated by the complexity of these systems. For example, associations may be active only for a short part of the year (Lapointe and Molard 1997) and acquired nutrients may not be utilised immediately by plants (Sanders and Fitter 1992). The distribution of P is

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likely to be patchy in soils of natural ecosystems (Cui and Caldwell 1996) and the distribution of AM fungi in soils can be extremely heterogeneous (Friese and Koske 1991; Brundrett and Abbott 1994; Moyersoen et al. 1998). Thus, AM fungi in natural ecosystems may be important for locating pockets of phosphorus. The lack of information on the role of AM fungi in natural ecosystems is also due to the difficulty of measuring phosphorus uptake and translocation to plants by hyphae. Methodology developed for use in agricultural soils (e.g. Jakobsen 1994) could be used to measure nutrient translocation by AM fungi in soils from natural ecosystems. Some studies have shown that mycorrhizas can provide benefits to plants even when there is little or no short-term growth response. Other benefits that have been reported include greater reproductive success, increased disease resistance, changes to water relations and/or nutrient accumulation (Table 5). It is often difficult to separate indirect effects of mycorrhizas from those caused by improved nutrition in experiments (Brundrett 1991; Smith and Read 1997). Mycorrhizas can also influence the outcome of competition between species (see 4B below). 3.2. Mycorrhizal dependency Observations of plants in natural ecosystems have shown that species generally have (a) consistently high levels of mycorrhizas, (b) intermediate, or variable levels of mycorrhizas, or (c) are consistently not mycorrhizal (Janos 1980; Brundrett 1991). Plants belonging to these categories are often called obligatorily mycorrhizal, facultatively mycorrhizal, or non-mycorrhizal respectively. Obligatorily mycorrhizal plants are defined as those which will not survive to reproductive maturity without being associated with mycorrhizal fungi in their natural habitats, while facultatively mycorrhizal plants benefit from mycorrhizas only in infertile soils and non-mycorrhizal plants have roots that are resistant to colonisation by mycorrhizal fungi (Janos 1980). Dependence on mycorrhizas can be measured by comparing the growth of plants with mycorrhizas in experiments to plants grown without them at a particular soil P level (e.g. Koide et al. 1988; Manjunath and Habte 1991; Hetrick et al. 1992). Mycorrhizal benefits should be examined across a range of soil P levels, by producing nutrient response curves (Abbott and Robson 1991; Schweiger et al. 1995). Growth responses to AM associations are measured by comparing the growth of plants with and without mycorrhizas in a particular soil (Table 6). In practice, it is very difficult to provide adequate controls in mycorrhizal studies because removal of mycorrhizal fungi causes changes to the chemical, biological and physical properties of soil and inoculation with fungi is likely to introduce other organisms (Ames et al. 1987; Hetrick et al. 1992; Baas et al. 1989; Koide and Li 1989). However, the impact of microbial factors or changes to soil fertility due to sterilisation on plant growth has usually been small relative to the impact of mycorrhizal treatments. Some studies attempting to quantify the benefits of AM fungi in plant communities have measured the impact

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of temporary suppression of these fungi by the application of fungicides (e.g. Gange et al. 1993; Newsham et al. 1995; Lapointe and Molard 1997). These studies are complicated by possible non-target effects of

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fungicides, but have shown reduced yields for some plant species when mycorrhizal formation was inhibited. Table 6 lists examples of plants from natural ecosystems whose mycorrhizal dependency has been measured experimentally, by growing plants with and without mycorrhizas at appropriate soil nutrient levels. Plants which are highly dependant on mycorrhizas appear to be important in most habitats, but apparently are less dominant in grasslands and disturbed habitats dominated by herbaceous plants. Non-mycorrhizal plants are generally only prevalent in habitats where plant productivity is severely limited by soil or climatic conditions, such as very dry, wet, saline, cold, or disturbed soils (Table 1). Typically non-mycorrhizal plants include members of the families Amaranthaceae, Caryophyllaceae, Chenopodiaceae, Brassicaceae, Commelinaceae, Cyperaceae, Juncaceae, Proteaceae, Polygonaceae and Scrophulariaceae (Tester et al. 1987; Brundrett 1991; Peat and Fitter 1993; Brundrett 1999). Plants can acquire nutrients both directly through their roots and/or via mycorrhizal associations. Thus, the benefit provided by mycorrhizas will depend on the capacity of roots to directly acquire mineral nutrients from the soil (Janos 1980; Brundrett 1991; Marschner 1995). Plants with facultatively mycorrhizal associations or non-mycorrhizal roots normally have much finer roots and longer root hairs than obligately mycorrhizal species (Bayliss 1975; Manjunath and Habte 1991; Baon et al. 1994; Schweiger et al. 1995). Thus, the roots of non-mycorrhizal plants typically would be much more efficient at direct nutrient uptake than the roots of mycorrhizal species. 3.3. Soil factors Land degradation due to salinity, waterlogging, erosion, etc. are now recognised as serious and growing problem in Australia and other countries. In ecosystem studies and glasshouse experiments soil factors can influence both the diversity of AM fungi and overall levels of mycorrhizal root formation and sporulation (Table 4). Observations in natural ecosystems have shown that AM plants are often less common than non-mycorrhizal species in soils which are waterlogged or saline, but that some mycorrhizal plants are normally present in even the most inhospitable habitats (Table 1). Low soil pH, high aluminium levels or high soil phosphorus levels prevent some AM fungi from providing substantial benefits to the host plants and can influence the distribution of fungi (Table 4). Excessive NaCl levels in soil inhibit mycorrhizal formation and restrict the activity of most mycorrhizal fungi, but some fungi are more tolerant of these conditions than others (Juniper and Abbott 1993). 3.4. Pollution and climate Various forms of pollution can inhibit mycorrhizal formation in experimental systems (Table 4). There is evidence that fungi adapted to high levels of metals help plants to grow in contaminated soils (Diaz et al. 1996; Joner and Leyval 1997). However, the roles of mycorrhizas in soils contaminated by metals are complex, as they increase the uptake of some

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metal ions but not others and their role also varies with the concentration of ions in soil (Heggo et al. 1990; Guo et al. 1996; Joner and Leyval 1997). This suggests that mycorrhizas would increase plant survival in some contaminated soils, but not in others. Forest decline associated with air and/or precipitation borne pollution has become a serious problem in Europe and North America. Mycorrhizal fungi can help plants to survive in soils affected by acidic precipitation (Malcova et al. 1998). Much recent research has focussed on the impact of elevated levels on mycorrhizal associations. can result in increased fungal biomass due to higher root Elevated colonization or increased soil hypha production (Rillig et al. 1998; Sanders et al. 1998). However, direct effects of on fungi can be much smaller than indirect effects mediated by changes in plant growth (Staddon et al. 1999). 4. Natural ecosystems The many roles of mycorrhizal f u n g i in ecosystems include providing food and habitats for other soil organisms and soil-feeding animals (Section 2.3), but only impacts on fungal and plant communities will be considered further here. 4.1. Fungal communities A succession of AM fungi has been observed in pot cultures (Brundrett et al. 1999b) and seasonal variations in fungal activities (measured by root colonisation) also occur in soils (Brundrett and Abbott 1995). Spores of one fungus in the jarrah forest remain dormant for 11 months and this leads to its appearance as a dominant member of the community on a two-year cycle (Jayasundara, pers. comm.). Therefore, for some species, low relative abundance of a fungus can be quickly followed by its dominance in roots. Fluctuations in the relative abundance of f u n g i within roots depend on the relative abundance of infective hyphae of different fungi in the original inoculum. Maximum activity of some fungi may not coincide with the optimum period for plant utilisation of nutrients, as a consequence of AM fungus phenology patterns. Soil disturbance can also alter the relative abundance of AM fungi in roots (Jasper et al. 1 9 9 1 ) . AM fungus community dynamics can also be influenced by competitive interactions between fungi (Pearson et al. 1993). Plants normally have more than one AM fungus simultaneously present in their roots (Abbott and Robson 1978; Abbott and Robson 1981; Merryweather and Fitter 1998a). Indirect evidence shows that different fungi have different roles in field soils (Merryweather and Fitter 1998b). The presence of different host plants influences the diversity of fungi in soils (Schenck and Kinloch 1980; Johnson et al. 1992; Hendrix et al. 1995; Bever et al. 1996). The effect of host species on fungal populations in roots has been called “ecological specificity” (McGonigle and Fitter 1990; van der Heijden et al. 1998). There also can be seasonal variation in colonisation by different fungi (Merryweather and Fitter 1998b) and different host plants can induce sporulation of a fungus at

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different times (Bever et al. 1996). These experiments suggest that mycorrhizal formation within the roots of two plant species by a single fungus may be very different, even where both plants are equally receptive to the fungus. The majority of AM fungi used in experiments have increased plant growth under particular conditions (Table 6). However, they differ in the extent to which they increase the uptake of phosphorus in relation to the amount of carbon that they remove (Pearson and Jakobsen 1993). There are cases where AM fungi reduce the growth of cultivated plants in highly fertile soils (Graham and Eissenstat 1998). The effectiveness of fungal isolates has been attributed to differences between them in the rate and extent of colonisation (Abbott and Robson 1981), but other factors are also important (Sylvia et al. 1993; Dickson et al. 1999). AM fungi differ in their patterns of colonisation of roots (Abbott 1982; Merryweather and Fitter 1998a), characteristics of the plant/fungus interface (Smith et al. 1994) and in the architecture and function of the hyphal networks that they form in soil (Jakobsen et al. 1992; Smith et al. 2000). Further, the efficacy of different fungi is expected to depend on the host plant species (Ravnskov and Jakobsen 1995). Evidence of the physiological diversity of AM fungi has been obtained by comparing responses of different species or isolates to the soil conditions listed in table 4. These comparisons have demonstrated variations between taxa and intraspecific variability within species of AM fungi in their ability to promote plant growth (e.g. Lambert et al. 1980; Stahl et al. 1988). Isolates of AM fungi are often most infective when used in the soil from which they were collected (Molina et al. 1978; Adelman and Morton 1986; Porter et al. 1987; Stahl et al. 1988; Henkel et al. 1989). Unfortunately, AM research has mainly been concerned with plant responses to mycorrhizas with little consideration of differences between specific fungi (Morton 1988; Abbott and Robson 1991; Brundrett 1991). Correlations between the diversity of AM fungi in microcosms and plant productivity (van der Heijen et al. 1998), may be a consequence of the functional diversity of AM fungi. However, in this study plant responses to mixtures of fungi appear to be similar to responses to the most effective species inoculated individually (see also Wardle 1999). Further research, which determines the relative contribution of different species of AM fungi within roots of competing plant species, is required to establish the importance of AM fungus diversity. 4.2. Plant community structure Arbuscular mycorrhizal fungi are a key part of almost all plant communities (Table 1), so they must be considered in all studies of nutrient cycling or impacts of nutrient supply on plant productivity or diversity. Mycorrhizal associations could potentially influence plant community structure by affecting richness or evenness of coexisting plants.

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Table 7 shows 4 categories of intraspecific and interspecific interactions involving plants w i t h different mycorrhizal requirements with the same type of association (facultative and obligately mycorrhizal plants are considered to be equivalent in interactions with non-mycorrhizal plants). Interactions which only involve non-mycorrhizal plants will not be discussed (type IV). Interactions between plants with different types of mycorrhizas are also considered below (type V). The experiments cited below examined the roles of mycorrhizas by withholding fungi from some experimental treatments, or by suppressing AM fungi in soil with fungicides. These studies measured the productivity and diversity of plants relative to treatments with AM fungi. 4.2.1. Competition between mycorrhizal plants Interspecific competition between mycorrhizal plants. The presence of AM fungi can reduce the strength of competition between two species where one has a competitive advantage due to larger size or faster growth (Moora and Zobel 1996). In highly competitive situations small plants may benefit from sharing a common network of mycorrhizal fungus hyphae with larger plants, which may reduce the cost of supporting an association. However, it does not seem likely that mature plants acquire nutrients from competing mycorrhizal plants through these networks (Newman et al. 1992; Fitter et al. 1998; Robinson and Fitter 1999). Most competition experiments have used plants which differ in their mycorrhizal dependency (see II below). Intraspecific competition. Most studies of the impact of withholding AM fungi from competing plants of one species have found greater competition at high plant densities with AM fungi than without (Eissenstat and Newman 1990; Allsopp and Stock 1992; Shumway and Koide 1995; Moora and Zobel 1996; 1998; West 1996). These studies all reported greater size differences between large and small plants when they were mycorrhizal, indicating that mycorrhizas increased variability in the capacity of plants to compete for resources. Mycorrhizal benefits were reduced by high plant densities, since fewer resources were available per plant (Allsopp and Stock 1992; Moora and Zobel 1998).

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4.2.2. Interactions of plants with high and low mycorrhizal dependency Most studies have shown increased competitive ability of obligately mycorrhizal plant species growing with facultatively mycorrhizal species resulting from the presence of AM fungi (Hall 1978; Grime et al. 1987; Gange 1993; Hartnett et al. 1993; Hetrick et al. 1994; van der Heijden 1998). These experiments typically compared a plant with a relatively fine root system (such as a grass) with a herb with coarser roots, that naturally occur together in pastures or meadows. In these cases, the plant with coarser roots would be less efficient at direct nutrient uptake and thus, mycorrhizas would greatly increase its nutrient uptake and competitive ability (Bergelson and Crawley 1987; Brundrett 1991). It is interesting to note that mycorrhizas can help indigenous plants to compete with weeds threatening their establishment (Nelson and Allen 1993; Smith et al. 1998). The presence of facultatively mycorrhizal species can result in increased mycorrhizal inoculum levels in disturbed soils, which may help obligately mycorrhizal species become established later in succession (Allen and Allen 1988). 4.2.3. Competition between mycorrhizal and non-mycorrhizal plants Competition between non-mycorrhizal species and normally mycorrhizal taxa is similar to competition between obligately and facultatively mycorrhizal plants, as described above (Crowell and Boerner 1988; Boerner and Harris 1991; Sanders and Koide 1994). This results because non-mycorrhizal plants typically have similar root systems to facultative species (Brundrett 1991). In many natural ecosystems, non-mycorrhizal plants are out-competed by mycorrhizal species during succession, but the mechanisms involved are not clear (Schmidt and Reeves 1989; Brundrett 1991; Francis and Read 1995). Most examples of mycorrhizal plants out-competing nonmycorrhizal species (with adequate inoculum of suitable AM fungi) probably result because the mycorrhizal species are more efficient at acquiring limiting soil nutrients such as P. However, AM fungi may also have more direct adverse effects on non-host plants. Francis and Read (1995) found that hyphae of AM fungi damaged the roots of nonmycorrhizal plants. Allen et al. (1989) also observed wounding reaction induced by AM fungi, which apparently resulted in reduced growth and survival of the non-host plant. This may have resulted in the suppression of competing non-host plants, by AM fungi when inoculum levels were high in the presence of host plants. There are also cases where non-mycorrhizal plants adversely affect AM fungi. Non-mycorrhizal plants have poorly understood mechanisms which keep most fungi out of their roots (Brundrett 1991). Many nonmycorrhizal plants are considered to accumulate secondary metabolites which may have defensive roles (Kumar and Mahadevan 1984; Brundrett 1991; Schreiner and Koide 1993). Roots of non-host plants in soils can reduce germination of AM fungus spores or mycorrhizal formation in hosts (Schreiner and Koide 1993; Fontenla et al. 1999). The implications of this form of allelopathy in natural ecosystems are worthy of further investigation.

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4.2.4. Plants with different types of mycorrhizas Interrelationships between plants with different mycorrhizal types are complex. For example, trees with ectomycorrhizas (ECM) are dominant in some forests and coexist with AM trees in others, while AM trees are dominant in other similar habitats (Högberg and Piearce 1986; Newbery et al. 1988; Brundrett 1991). Climatic factors seem to be important in determining the outcome of competition between plants with different types of mycorrhizas (e.g. ECM trees dominate boreal and alpine forests, while many forests in tropical regions are dominated by AM trees). It has been suggested that trees with ECM are more likely to be important in soils with low pH, high levels of aluminium or other toxic ions (Högberg and Piearce 1986), or where nutrient cycling occurs slowly because leaf litter is resistant to decomposition (Gebauer and Taylor 1999; Lindahl et al. 1999). However, the relative importance of these factors has not been established. It is usually considered that the outcome of competition between plants with different types of associations will be determined by the relative success of their associated fungi in acquiring different forms of mineral nutrients which l i m i t plant growth. In an Australian forest, direct competition between plants with ECM, AM or non-mycorrhizal roots may be avoided because their roots occurred w i t h i n separate zones within soils and may have used different forms of nutrients (Brundrett and Abbott 1995). 4.2.5. Conclusions Interpretation of results of competition experiments is difficult because mycorrhizal effects on plant performance can be counteracted by changes to the nature of competition (Watkinson and Freckleton 1997). Mycorrhizas can result in higher plant diversity due to increased intraspecific and reduced interspecific competition (Moora and Zobel 1996). Mycorrhizal dependency primarily determines the impact of mycorrhizas on competitive interactions between plants. 5. Utilising mycorrhizas This section will focus on plants because we do not know enough about the distribution and abundance of arbuscular mycorrhizal fungi to know if any are rare. AM fungi exhibit functional diversity (Table 2) and are adapted to local environmental and soil conditions (Table 4). Consequently, it would be important to conserve representatives of all habitats and soils within regions to maintain the functional diversity of AM fungi and other organisms. Substantial reductions in fungal diversity occur when ecosystems are disturbed or converted to agriculture (Schenck and Kinloch 1980; Hetrick and Bloom 1983; Allen et al. 1987; 1998; Brundrett et al. 1996a,b), but it is not known if this loss is permanent, or represents depletion below the level of detection. 5.1. Propagation of rare plants Attempts to inoculate plants with AM fungi are less common than for ectomycorrhizas, because AM fungi are ubiquitous in soils and are

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considered not to have host preferences. Thus, it is believed that any AM fungus has the capacity to form mycorrhizas with any host. Mycorrhizal inoculation technology has been developed for forestry and horticulture, especially when soils have been sterilised to remove pathogens, but has rarely been applied to plants from natural ecosystems (Brundrett et al. 1996c). However, the mycorrhizal literature provides many examples that may be relevant to the propagation of indigenous plants. For example, AM fungi can greatly reduce the mortality of transplanted micropropagated plants (Subhan et al. 1997). Manipulation of mycorrhizal fungi may sometimes be required to rehabilitate habitats or conserve rare species. Many plant species are highly dependent on mycorrhizas (Table 6) and the potting mixes used to propagate them are likely to be devoid of AM fungi. Koske and Gemma (1995) worked with endangered species of Hawaiian plants which were difficult to propagate. They found that inoculation of nurserygrown seedlings and cuttings resulted in substantial increases in plant growth and survival in certain growth media. Barroetavena et al. (1998) also found that AM f u n g i were important to the survival of propagated plants of an endangered Astragalus species. It may be worthwhile to routinely include mycorrhizal inoculation as a precautionary measure in any programs which attempt to propagate rare species, if AM inoculum is commercially available and its expense is insignificant relative to other costs in a species recovery program. However, inoculated fungi may persist after plants are transplanted in field soils and compete with indigenous fungi which are better adapted to local conditions. 5.2. Ecosystem restoration Mycorrhizal inoculation is only likely to be valuable in disturbed habitats with little or no mycorrhizal inoculum (Section 2.4), because AM fungi are relatively non-specific and already occur in most soils (Abbott and Robson 1991; Brundrett 1991). Consequently, the primary focus of most practical research with AM fungi has been the restoration of disturbed habitats, such as mine sites. Research with AM fungi has also focused on sand-dune stabilising grasses in coastal habitats, where AM colonisation establishes slowly (Sylvia and Will 1988; Corkidi and Rincón 1997a,b). These studies have found that out-planted grasses inoculated with AM fungi grow better than uninoculated plants in sand dunes initially devoid of vegetation (Sylvia 1989; Gemma and Koske 1997). Massive destruction of native vegetation is occurring throughout the world primarily because of land use changes by people. Severe disturbance of ecosystems is likely to result in the loss of some mycorrhizal fungi which have adapted to local conditions. During subsequent attempts at ecosystem reconstruction, the impact of this reduction in mycorrhizal fungus genetic resources will depend on (i) how fungal genetic and functional diversity varies between habitats, (ii) how rapidly surviving fungi adapt to changing soil conditions during succession and (iii) how effectively well-adapted isolates are dispersed from any surviving remnant patches of native vegetation. Most studies of mycorrhizal associations in highly disturbed habitats

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such as mine sites have found reduced levels of mycorrhizal propagules (Jasper et al. 1987, 1991; Pfleger et al. 1994; Brundrett et al. 1996b). Topsoil removal and stockpiling during mining have major impacts on AM fungi (Jasper et al. 1987; Rives et al. 1980; Stahl et al. 1988; Bellgard 1993). Soil disturbance may also have an indirect effect through changes to soil properties which reduce the efficacy of surviving fungi (Abdul-Kareem and McRae 1984; Stahl et al. 1988; Waaland and Allen 1987). Disturbance impacts on AM fungi that have been hypothesised include: (i) a reduction in numbers of viable spores, (ii) loss of a hyphal network in the soil, or (iii) the prevention of hyphal growth from root inoculum to new roots (Evans and Miller 1988; Jasper et al. 1989; Rives et al. 1980). The relative importance of these mechanisms in different situations is unknown. 6. Conclusions AM fungal associations must programs because of their roles The processes listed in table 8 mycorrhizal fungi were absent or conditions.

be considered in plant conservation in key ecosystem processes (Table 8). are likely to be adversely affected if not functioning due to unsuitable soil

Despite the scarcity of direct measurements of mycorrhizal benefits outside the glasshouse, there is considerable indirect evidence to support the assumption that AM fungi have a major role in nutrient uptake in natural ecosystems. This evidence includes the facts that most plant support high levels of mycorrhizal colonisation and many plants have root systems that otherwise would be inefficient at nutrient uptake (Section 3.2). Most attempts to quantify the value of AM fungi have

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focused on short-lived plants growing in relatively fertile soils (e.g. “weeds” of meadows and early successional habitats). These annual species are used in most glasshouse and field experiments because treatment effects can be analysed after one growing season. However, evidence from growth experiments (Table 6) and studies of mycorrhizal colonisation levels in natural habitats (Table 1) have shown that annual plants are often less dependent on mycorrhizas than are perennial plants in undisturbed habitats (Table 6). Studies of annual plants are of little value if they do not consider their reproductive success by measuring survival and growth of subsequent generations (e.g. Stanley et al. 1993; Shumway and Koide 1995). Non-nutritional benefits such as antagonism of pathogens and changes to water relations are also suspected to be important, but have rarely been investigated (Section 3.1). As AM fungi are an integral component of soils (except in disturbed habitats, sites with hostile soil conditions, or a severe climate), experiments where mycorrhizas are w i t h h e l d are artificial situations. Prediction of the benefits resulting from inoculating plants with AM fungi during attempts to restore ecosystems, or for rare species recovery programs are hampered by our lack of knowledge about the basic biology of these fungi. We do not know how many species of these fungi occur in most habitats, or if functional differences between isolates are more important than variations in the distribution of taxa. The community dynamics of AM fungi have the potential to alter the overall contribution of mycorrhizas to plant productivity and dominance of plant species, by mechanisms that are not known (e.g. Moora and Zobel 1996; van der Heijden et al. 1998). The life cycles of AM fungi in soils are not well understood (Section 2.2). Little is known about the contribution of AM fungi to nutrient uptake in natural ecosystems. Such voids in our understanding of AM fungi should be of major concern, because these organisms are one of the most important groups of organisms in plant communities. Mycorrhizal fungi are a major conduit of carbon into the soil and a key part of the plant/soil interface. We need to know how this interface functions if we are to understand the impact of changes to soil or climatic conditions on plant communities. References Abbott LK(1982) Comparative anatomy of vesicular-arbuscular mycorrhizas formed on subterranean clover. Australian Journal of Botany 30, 485–499. Abbott LK, Robson AD (1984a) Colonization of the root system of subterranean clover by three species of vesicular-arbuscular mycorrhizal fungi. New Phytologist 96, 275–281. Abbott LK, Robson AD (1984b) The effect of root density, inoculum placement and infectivity of inoculum on the development of vesicular-arbuscular mycorrhizas. New Phytologist 97, 285–299. Abbott LK, Robson AD (1978) Growth of subterranean clover in relation to the formation of endomycorrhizas by introduced and indigenous fungi in a field soil. New Phytologist 81, 575–585. Abbott LK, Robson AD (1981) Infectivity and effectiveness of five endomycorrhizal

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137–146. Olsson PA, (1999) Signature fatty acids provide tools for determination of t h e distribution and interactions of mycorrhizal fungi in soil. FEMS Microbiology and Ecology 29, 303–310. Olsson PA, Baath E, Jakobsen I, Soderstrom B (1996) Soil bacteria respond to presence of roots but not to mycelium of arbuscular mycorrhizal fungi. Soil Biology and Biochemistry 28, 463–470. Pattinson GS, Hammill KA, Sutton BG, McGee PA (1999) Simulated fire reduces the density of arbuscular mycorrhizal fungi at the soil surface. Mycological Research 103, 491–496. Pattinson GS, McGee PA (1997) High densities of arbuscular mycorrhizal fungi maintained during long fallows in soils used to grow cotton except when soil is wetted periodically. New Phytologist 136, 571–580. Paulitz TC, Menge JA (1986) The effects of a mycoparasite on the mycorrhizal fungus, Glomus deserticola. Phytopathology 76, 351–354. Pearson JN, Abbott LK, Jasper DA (1993) Mediation of competition between two colonizing VA mycorrhizal fungi by the host plant. New Phytologist 123, 93–98. Pearson J N , Jakobsen I ( 1 9 9 3 ) Symbiotic exchange of carbon and phosphorus between cucumber and three a r b u s c u l a r m y c o r r h i z a l fungi. New Phytologist 124, 481–488. Pearson J N , Schweiger P (1993) Scutellospora calospora (Nicol. Gerd.) Walker & Sanders associated w i t h subterranean clover: dynamics of colonization, s p o r u l a t i o n and soluble carbohydrates. New Phytologist 124, 2 1 5 – 2 1 9 . Peat HJ, Fitter AH (1993) The d i s t r i b u t i o n of arbuscular mycorrhizas in the British flora. New Phytologist 125, 845–854. Pfleger FL, Stewart EL, Noyd RK (1994) Role of VAM fungi in mine land revegetation. In ‘Mycorrhizae and plant health.’ (Eds FL Pfleger and RG Linderman) pp. 47–81. (American Phytopathological Society: St Paul) Pirozynski KA, Dalpé Y (1989) Geological history of the Glomaceae with particular reference to mycorrhizal symbiosis. Symbiosis 7, 1–35. Porter WM, Robson AD, Abbott LK (1987) Factors controlling the distribution of vesicular-arbuscular mycorrhizal fungi in relation to soil pH. Journal of Applied Ecology 24, 663–672. Powell CL (1975) Rushes and sedges are non-mycotrophic. Plant and Soil 42, 481–484. Rabatin SC, Stinner BR (1985) Arthropods as consumers of vesicular-arbuscular mycorrhizal fungi. Mycologia 77, 320–322. Rabatin SC, Stinner BR, (1988) Indirect effects of interactions between VAM fungi and s o i l - i n h a b i t i n g invertebrates on plant processes. Agriculture, Ecosystems and the Environment 24, 135–146. Raju PS, Clark RB, Ellis JR, Maranville JW (1990) Effects of species of VA-mycorrhizal fungi on growth and mineral uptake of sorghum at different temperatures. Plant and Soil 1 2 1 , 165–170. Ravnskov S, Jakobsen I (1995) F u n c t i o n a l compatibility in arbuscular mycorrhizas measured as h y p h a l P transport to the plant. New Phytologist 129, 611–618. Reddell P, Milnes AR (1992) Mycorrhizas and other specialised nutrient-acquisition strategies: their occurrence in woodland plants from Kakadu and their role in rehabilitation of waste rock dumps at a local uranium mine. Australian Journal of

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K.Sivasithamparam, K.W.Dixon & R.L.Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 195–226. © Kluwer Academic Publishers.

Chapter 7 ORCHID CONSERVATION AND MYCORRHIZAL ASSOCIATIONS Andrew L. Batty1,2 Kingsley W. Dixon1,3 Mark C. Brundrett1,2 K. Sivasithamparam2 1 Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth 6005, Western Australia. (correspondence) 2 Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia. 3 Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

1. Introduction 1.1. Orchids Orchids (members of the plant family Orchidaceae) include terrestrials that typically occur in temperate regions, and epiphytes, which mostly occur in tropical regions where the taxonomic diversity of orchids is highest (Dressler 1993). The flowers of these highly evolved plants are often beautiful and sometimes bizarre with complex pollination mechanisms involving specific insect pollinators (Dressler 1981; Benzing 1982; Jones 1993). The Orchidaceae contains more species than any other flowering plant family, with estimates ranging from 17,500 to 35,000 species (Garay and Sweet 1974; Gentry and Dodson 1987; Mabberley 1990). The diversity of epiphytic orchid species increases along moisture and latitude gradients and with habitat complexity (Gentry and Dodson 1987). Orchids produce large numbers of minute seeds that favour the expression of genetic variability and high dispersal rates across geographical and ecological barriers. Many orchids are now considered to be at risk of extinction as an indirect or direct result of human activities, which include habitat alteration or destruction and extraction of wild plants for trade (Table 1).

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These processes and the phenomenal diversity of orchids have resulted in many threatened orchid species (Hágsater and Dumont 1996). The impacts of alteration or habitat destruction on orchid taxa will depend on its geographical distribution, habitat specificity, and population size (Rabinowitz et al. 1986). Generally it can be assumed that rare species have more specific habitat preferences than common species. Habitat specificity is likely to be a consequence of distribution of mycorrhizal fungi (see below), pollination mechanisms, seed distribution, etc. Rare orchid species are i n t r i n s i c a l l y prone to extinction due to naturally occurring catastrophes (e.g. intense fires, floods, or severe climatic variations). For example, Epidendrum floridense, a Florida epiphytic orchid species, appears to have nearly become extinct as a consequence of severe unseasonal frosts (Hágsater 1993).

This review focuses on the ecological importance of orchid mycorrhizal associations, and readers should consult other works for details of the physiology and morphology of these associations, and other aspects of the biology and ecology of orchids. The body of scientific knowledge to back up much of what is believed about the mycorrhizal biology and ecology of orchids is limited, so many topics cannot be covered in great depth. Review sections concerning the biology of associations are presented, before ecological consequences of these associations and the practical use of mycorrhizas in orchid conservation are discussed. 1.2. Mycorrhizas Mycorrhizas (fungus-roots) are symbiotic associations between specialised soil fungi and plants involved in nutrient transfer (see Brundrett 2002). Plants with vesicular-arbuscular mycorrhizas (VAM) are ubiquitous, while those with ectomycorrhizas (ECM) are important in many ecosystems, several other types of mycorrhizas occur in particular families of plants, and some plants also have roots that remain nonmycorrhizal (Brundrett and Abbott; Brundrett and Cairney, this volume). Orchid mycorrhizas differ from most other types of mycorrhizas, as they occur in stems as well as roots, and fungi may re-colonise older cells (Hadley 1982; Smith and Read 1997). Orchids mycorrhizas are

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morphologically different from other mycorrhizas and involve a phylogenetically distinct group of soil fungi (Rasmussen 1995; Currah et al. 1997). These fungal associates apparently have limited specialisations as mycorrhizal fungi, and show little evidence of co-evolution with their hosts (Brundrett 2002). Consequently, knowledge about the physiology and ecology of other mycorrhizal associations (that are well studied) cannot be safely applied to orchid associations (of which we know relatively little). In 1886, Wahrlich surveyed more than 50 cultivated orchid species and found fungal infections in every one of them. Subsequent surveys have found mycorrhizas to be ubiquitous in terrestrial orchids and also present in many epiphytes (e.g. Hadley and Williamson 1972; Benzing and Friedman 1981; Ramsay et al. 1986; Currah et al. 1997). However, it needs to be remembered that in most cases the roles of these fungi were not examined and the benefits provided to orchids have rarely been measured (see below). There are considerable variations in the distribution of mycorrhizas within roots or stems of orchids. Mycorrhizal colonisation has been reported to be sporadic in most epiphytes, but is generally more widespread consistent in terrestrial orchids (Burgeff 1959; Rasmussen 1995). Different genera of terrestrial orchids can have distinctive colonisation patterns within their roots (Figure 1) or stems (Ramsay et al. 1986). These mycorrhizal infection patterns in the whole plant (i.e. root, collar, stem, rhizome, etc) may be associated with particular fungal types (Ramsay et al. 1986). Colonisation patterns of mycorrhizal fungi within plants are primarily determined by host cell properties, but mycorrhizal morphological features can also be correlated with the presence of certain fungi (Brundrett 2002).

In the early 1900’s investigators first succeeded in germinating orchids in vitro and observed mycorrhizal formation in embryos and

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seedlings (Bernard 1903; Burgeff 1909). Orchid mycorrhizas are characterised by the presence of coils of undifferentiated hyphae in cortical cells of the root, stem or protocorms of orchid species (Masuhara and Katsuya 1992; Smith and Read 1997; Peterson et al. 1998). Early histological researchers recognised two types of orchid mycorrhizas (i) tolypophagy, which occurs in most species, and (ii) ptyophagy, found in myco-heterotrophic species (e.g. Gastrodia) (Rasmussen 1995). Ptyophagy has been interpreted as hyphal lysis through which fungal cell contents are released into plant cells (Burgeff 1959), but this requires confirmation by observations using techniques such as electron microscopy. The collapse of old coils of hyphae also occurs within autotrophic orchid, and is similar to the collapse of older arbuscules in VAM associations, but the significance of these processes to plant nutrition is unknown (Brundrett 2002). Hyphal digestion allows successive waves of peloton formation, digestion and re-infection within the same root cells (Burgeff 1959; Smith and Read 1997) and would thus primarily be a means of increasing the duration of active associations (Brundrett 2002). Otherwise, the fluxes of substances across the host-fungus interface would have to be much higher than occurs in other types of associations because of the extremely small volume of plant tissues of some orchid mycorrhizas (see below). Most mycorrhizal scientists now consider active transport of metabolites across the host-fungus interface of living cells rather than digestion to be the primary mode of nutrient transfer in all types of mycorrhizas (Smith and Smith 1990). However, there is still much to learn about how orchid mycorrhizas function and how associations of autotrophic and myco-heterotrophic plants differ. The early work by Burgeff (1932) and others provides insight into the complexity of orchid associations, and this work should now be continued by modern orchid scientists. 2. Orchid fungi 2.1. Identity and specificity Most of the endophytic fungi known to form orchid mycorrhizas are Basidiomycetes, and many belong to the form-genus Rhizoctonia (Sneh et al. 1991; Currah et al. 1997). Orchid rhizoctonias are distinguished by their appearance in culture (the presence of short inflated segments which resemble spores and the formation of loose aggregates of hyphae regarded as poorly developed sclerotia or resting bodies (Hadley 1982)). Isolates which form sexual stages in culture (a difficult and poorly repeatable procedure) belong to the basidiomycete genera Ceratobasidium, Ceratorhiza, Epulorhiza, Sebacina, Thanatephorus, and Tulasnella (Warcup and Talbot 1967, 1980; Currah et al. 1997). A diverse assemblage of fungi belonging to other taxonomic groups have also been isolated from orchid roots, but may not all be beneficial (Currah et al. 1997). It is not clear if orchid fungi from different regions are more closely related to each other or to other saprophytic or parasitic groups of Rhizoctonia species. The form genus Rhizoctonia contains saprophytes,

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pathogens and orchid symbionts (and probably also mycoparasites), but its unclear how much overlap and crossover there is been members of these groups (Figure 2). If orchid fungi typically have dual roles as, saprophytes or parasites, they are fundamentally different from the highly specialised fungi forming other types of mycorrhizas. Orchid fungi could not co-evolve with their hosts like other mycorrhizal fungi, as they receive few benefits from their associations with plants (Brundrett 2002). It seems most likely that the orchid fungi are a disparate group with many separate origins and that the recruitment of new fungal lineages by orchids probably continues today. Most of the mycorrhizal associates identified form achlorophyllous orchids are different from those of green orchids (see below). The question of host-fungus specificity within the Orchidaceae has been a point of contention for many years. Knudson (1927) believed there were low levels of specificity for tropical epiphytic species. Conversely, Burgeff (1909, 1959), who worked with many terrestrial orchids, thought that there was strong specificity in the association between orchid and fungus. The evidence available today suggests that both these hypotheses can be correct (Table 2).

Harvais and Hadley (1967) isolated 244 Rhizoctonia strains from Dactylorhiza purpurella and other north British orchids. These belonged to 15 main groups, but most groups were confined to a single habitat, with the exception of R. repens which was widespread. Curtis (1939) argued that ecological distribution of the fungi was related to habitat rather than host. This was supported by Harvais and Hadley (1967) who also showed that Dactylorhiza purpurella was symbiotic with most of the

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isolates they tested. However, there are other orchids, such as Goodyera repens that usually associate with a single fungus (Ceratobasidium cornigerum) as an adult (Hadley 1982). This may explain why G. repens has a more restricted distribution than D. purpurella. Many other orchids have been found to have fairly specific fungal associates that vary much more between hosts than between habitats (e.g. Warcup 1981; Ramsay et al. 1987; Currah et al. 1997; Sen et al. 1999). We may conclude that orchids can associate with either a broad or narrow range of fungal isolates (Table 2). This contrasts with the situation in ECM associations where the fungi seem to primarily regulate specificity and can associate with a narrow or broad range of host plants (Chapter 5). We might expect that the breadth of fungal specificity of orchid taxa would be one of the most important factors determining the breadth of their habitat specificity. However, this hypothesis needs to be tested by obtaining further investigation about the change with habitats and between taxa, using DNA-based fungal identification methods to characterise the diversity of orchid fungi.

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Most of what we know about orchid fungus specificity comes from germination tests conducted under sterile conditions (e.g. Warcup 1981; Ramsay et al. 1986). However, these studies show the importance of mycorrhizal fungi to germination, but do not confirm that the same fungi are important to the survival of adult plants in natural environments. Seed of some orchids will not germinate with fungi isolated from adult plants, but there are also many examples where these fungi promote successful germination (Warcup 1981; Alexander and Hadley 1983; Muir 1989). The establishment of healthy seedlings with leaves or a tuber, not merely successful germination should be regarded as the decisive criterion of host-fungus compatibility (Batty et al. 2001a). Orchids may be able to live in symbiosis with one or several different fungi, but the relative importance of co-occurring fungi is unknown. Differences in fungi within orchid seedlings and adult plants may equate to the successional trends that occur in other types of mycorrhizal fungi (Brundrett and Abbott; Brundrett and Cairney; Dixon and Read, this volume). Co-occurring orchid species with different fungi may minimise competition for the same nutrient resource, if different species of fungi access different soil resources. Relationships between plant productivity and the diversity of mycorrhizal fungi have been observed in microcosm experiments with VAM fungi (van der Heijden et al. 1998). It is generally assumed that greater taxonomic diversity of fungi equates to a greater function diversity that would benefit plants, but further research is required to test this hypothesis. In general, the diversity of orchid fungi associating with a particular orchid species appears to be much lower than for other types of mycorrhizal fungi (i.e. most studies have reported one or two fungi per species). The greatest implication of high-host fungus specificity and low fungal diversity to orchids would be to restrict orchids to certain habitats where these specific fungi occur. 2.2. Distribution in substrates Understanding the distribution of orchid mycorrhizal fungi within soil or other substrates is important for attempts to return orchids to the field and in understanding the distribution of orchids. The patchy distribution of orchids may be a result of the presence or absence of the specific mycorrhizal fungi essential for the survival of the orchid. Orchid dispersal may be a function of mycorrhizal distribution, seed dispersal and conditions suitable for the germination of orchid seed and the establishment of orchid plants. Studies into the in situ germination of orchid seed in field sites have shown that good germination can be obtained (Table 3). A seed burial technique devised by Rasmussen and Whigham (1993) allows the distribution of effective orchid endophytes to be assessed in situ in natural habitats. In a similar study in Western Australia, where seeds remained in the soil throughout the growing season demonstrated tuber development from protocorms, confirming that effective fungi were present (Batty et al. 2001a). In this study the successful germination of orchid seeds was found to be higher in close proximity to adult plants of the same species.

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The spatial distribution of orchid fungi within soils is largely unknown, but we might assume that it is similar to the distribution patterns of other types of mycorrhizal fungi and is likely to be correlated with certain soil properties. Organic matter is likely to be an important resource for orchid fungi, and is unevenly distributed in the soil, as are populations of other mycorrhizal fungi which tend to occur in discrete patches (e.g. Brundrett and Abbott 1995). Some species of orchids tend to grow in clusters, while others tend to be widely spaced. This depends to a large extent on a differential tendency to produce vegetative offshoots, but may also be affected by fungus-mediated intraspecific competition. Competition between orchid siblings for available resources supplied by mycelia occurs in vitro (Alexander and Hadley 1983; Rasmussen et al. 1989; Tsutsui and Tomita 1989), and is also likely to occur in soils. This form of intraspecific competition would affect the number of individuals that can be supported by a patch of fungi and affect seedling recruitment near parent plants. Subordinate plants sharing the same fungi in ECM and VAM associations may receive limited direct or indirect support from dominant plants (Brundrett and Abbott; Brundrett and Cairney, this volume). However, this may not occur in orchids, as there is no evidence of energy transfer to the fungus from the plant.

Orchid fungi are more widespread in the soil than their hosts, as they can be isolated from soil in areas where orchids were absent (e.g. Curtis 1939; Harvais and Hadley 1967; Warcup and Talbot 1967). Orchid fungi such as Ceratobasidium cornigerum and Tulasnella calospora appear to have world-wide distribution, but this requires further investigation as substantial genetic heterogeneity has been found in other wide spread fungal taxa (Chapter 5). Orchid fungi in soils are likely to have substantial temporal as well as spatial variations in distribution (Perkins and McGee 1995).

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2.3. Phenology Rasmussen (1995) noted that there is little data on the phenology of the fungi associated with orchids, or the impacts of low winter temperatures or dry summer weather on the activity of external mycelium. In regions of the World where the growing season is delimited by periods of cold or dry conditions other mycorrhizal fungi are active in the growing season, but persist as networks of hyphae and propagules in soils at other times (Brundrett and Abbott 1994; Braunberger et al. 1997). If it is assumed that orchid fungi behave in a similar manner to other mycorrhizal fungi then there may only be a small seasonal window in which soil conditions and fungal activity are conducive for germination of orchid seedlings (see Batty et al. 2000). In moderate climates, fungi could be active all year, but may have peak periods of activity, such as cool wet periods when the most nutrient inputs from dead plant material occur (Rasmussen 1995; Masuhara et al. 1988). Alexander and Alexander (1984) observed that the terrestrial orchids Bletilla striata and Goodyera repens showed signs of fungal infection throughout the year with a peak from December to May. It appears that the main limiting factor for endophyte (fungal) activity is the lack of soil moisture and not low temperatures (see Rasmussen 1995). In mediterranean climates with dry summers, soil microbial activity is maximised during the cool wet winter months of the year while this activity is limited by soil moisture during the dry summer months (Sivasithamparam 1993). Sivasithamparam (1993) indicates that the saprophytic phase of Rhizoctonia solani (AG8) survives the dry summer both in colonised stubble and as mycelia (probably in a network) in the soil. We might assume that orchids with mycorrhizal fungi behave in a similar manner to plants with VAM fungi, which are known to provide reservoirs of fungal inoculum in older roots, as only a fraction of the root system of most perennial plants is replaced each year (Brundrett and Kendrick 1988). Many terrestrial orchids perenniate only as rhizomes or tubers, but their roots or stems probably often follow the same channels through the soil and these remnants of mycorrhizal tissue may be important reservoirs of fungal inoculum, ensuring early infection. Intact networks of fungal mycelium are key reservoirs of fungal inoculum in many natural habitats for other types of mycorrhizas (Brundrett and Abbott 1995). Propagules of other fungi normally form at the end of the growing season then decline due to predation and are activated during the following growing season. We need to determine the form of saprophytic survival (Sivasithamparam 1993) orchid fungi use to persist in soils and where they are localised in the soil to devise methods of identifying sites where appropriate fungi are present for orchid recovery work (see below). For natural recruitment to occur at a site, a supply of seed must arrive at the location at the end of the growing season prior to the recruitment period. Orchid seed bank size depends on the level of seed production for that season and the life-span of seeds in soil. In Western Australian soils, seed survival is limited to a single year (Batty et al. 200la) and these tiny seeds are unlikely to survive for much longer in

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other habitats with a distinct dry season. Orchid seed germination is likely to be cued to periods of high fungal activity in soil and at the start of a growing period which would enable the pro-embryo to differentiate, germinate, and produce a tuber or plantlet of sufficient size to enable survival until the next growing season (Batty et al. 200la). The presence or absence of an appropriate fungus in an active growth phase at the field site ultimately determines whether seed will convert into an orchid seedling and recruit into the adult orchid population. As seasonal trends in saprophytic activity of orchid fungi have rarely been measured, we can only assume that this is limited to periods when root and fungal activity are not limited by drought or temperature extremes, or when soil is not subjected to excessive dryness or water-logging in seasonal climates. 3. Mycorrhizal associations 3.1. Mineral nutrition and mycorrhizal dependency Early workers believed that orchids could only acquire nutrients through their mycorrhizal fungi. Knudson’s (1922) experiments first demonstrated that seedlings did not have an absolute requirement for mycorrhizas, as they could take up nutrients directly in sterile culture. Some investigators have considered the mycorrhizas of adult plants to be insignificant, except in the chlorophyll-deficient species (Hadley and Pegg 1989). However, there have been many demonstrations of the benefits provided to adult orchids by their fungal partners, and it is probable that most terrestrial orchid have an obligate requirement for mycorrhizas when growing in natural habitats. Experiments with radioactive phosphorus have confirmed that orchid fungi can transport phosphorus into roots (Smith 1966, 1967; Alexander et al. 1984; Alexander and Hadley 1985). Orchid plants typically have very coarse roots with limited lateral branching. Extreme examples are provided by several genera of West Australian terrestrial orchids that are almost completely without roots and form associations in a highly confined space in their stem collars (Ramsay et al. 1986). Thus, orchids typically are not efficient for direct absorption of mineral nutrients from soil, in contrast with the fine highly branched roots of plants that can grow without mycorrhizas in natural habitats (Brundrett 1991). Further evidence of the effectiveness of orchid mycorrhizas is provided by the occurrence of many orchid species in habitats with extremely infertile soils with low accessibility of minerals, or with extremely high or low pH, which often have a loose texture and high humus content (Sheviak 1974). The degree of mycorrhizal dependency of epiphytic orchids is less clear, as their protocorms often become photosynthetic at an early stage and the roots of adult plants often have limited and sporadic fungal colonisation (Arditti 1992). Orchids growing as epiphytes on trees obtain adequate mineral nutrients from dust, organic debris and stem-flow along the bark of the host (Arditti 1992). It has recently been suggested that germinating epiphytic orchid seeds obtain water through mycorrhizal fungi (Yoder et al. 2000).

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Orchid mycorrhizas differ from typical ECM or VAM associations, as orchid fungi can provide a source of energy as well as mineral nutrients to their host plants (Rasmussen 1995). These carbon compounds presumably are derived from the breakdown of organic substances in the surrounding substrate. Radioactive carbon has been traced from fungi to seedlings but not from seedlings to fungi. It has generally been assumed that orchids with chlorophyll provide their fungi with energy in exchange for fertiliser, as is the case with most ECM or VAM associations. The plant may supply essential vitamins or amino acids to fungi in some cases (Leake 1994). However, there is no real evidence that fungi receive substantial benefits from any of their associations with orchids. These fungi appear to be much less specialised than other types of mycorrhizal fungi and presumably are highly independent and often grow without any assistance from orchids (Section 3.2). Little is known about the other ecological roles of fungi that associate with green orchids and some of these fungi may have an adverse impact on other plants. For example, epiphytic orchids sometimes appear to have a detrimental effect on trees that harbour them (Ruinen 1953; Johansson 1977), but this could result from a correlation between epiphyte abundance and tree decline due to other factors. There are some cases where Rhizoctonia isolates from orchids have been identified as pathogens in roots of other plants (Warcup 1985a; Zelmer et al. 1996), or parasites of VAM fungi (Williams 1985). However, most orchids have fairly specific associations with Rhizoctonia strains that are not known to be pathogens of other plants (Warcup 1981; Ramsay et al. 1987; Muir 1989; Currah et al. 1997; Sen et al. 1999). There currently is insufficient information to safely say whether autotrophic orchids normally have mutualistic or exploitative associations with soil fungi. We know very little about the other roles of orchid fungi in soils. This knowledge is essential for us to develop an understanding of the biology and ecology of these beautiful and fascinating plants. If orchid fungi are also plant pathogens or mycoparasites, then orchids are epiparisites of other plants in their communities. Green orchids would be epiparasitic to a lesser extent than myco-heterotrophic orchids which are entirely dependent on fungi supported by other plants. Some terrestrial orchids have a higher degree of shade tolerance than other plants (McKendrick 1996). Numerous observations of orchids in natural habitats also support the conclusion that many adult green terrestrial orchids are less dependant on sunlight for energy than other plants, because of their mycorrhizal fungi, but this requires further investigation (Burgeff 1959; Rasmussen 1995; Smith and Read 1997). Many orchids require full sun to grow, and typically occur in grasslands and open woodlands (Case 1990; Rasmussen 1995). 3.2. Myco-heterotrophic orchids Saprophytic (myco-heterotrophic) orchids without chlorophyll are assumed to have fully-exploitative mycorrhizal associations that supply both the energyt and nutrient requirements of the host (Leake 1994).

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Achlorophyllous plants with VAM or ECM associations such as Monotropa (Ericaceae) have established similar exploitative associations with fungi (Leake 1994; Brundrett 2002). Mycorrhizal associations where fungi do not seem to receive any benefits from plants are also called epiparasitic, myco-heterotrophic, cheating, or exploitative associations (Furman and Trappe 1971; Leake 1994; Taylor and Bruns 1999; Brundrett 2002). The nature of mycorrhizal associations of many of these plants has not been investigated and their nutritional dependency on fungi has been assumed whenever other explanations are lacking. Myco-heterotrophy has evolved independently in approximately 20 separate orchid lineages much more often than in any other group of plants (Molvray et al. 2000). Orchids also appear to evolve more rapidly than other plants (Molvray et al. 2000). Myco-heterotrophic associations involve fungi that belong to separate lineages to those forming mycorrhizas with green orchids, including ECM associates of trees, wood decaying fungi and parasites of other plants (Table 4). These associations have a high degree of hostfungus specificity and orchids such as Corallorhiza, Gastrodia and Galeola are only known to associate with a single fungal genus (Table 4). Achlorophyllous myco-heterotrophic orchids species include Rhizanthella gardneri which is fully subterranean (Dixon and Pate 1984), but other achlorophyllous orchids emerge from the ground for flowering and seed dispersal. The survival of achlorophyllous orchid mutants which are normally green shows that mycorrhizal associations can also supply carbon to the plants (Salmia 1988; Rasmussen 1995). Rasmussen, (1995) summarises the reports where chlorophyllous orchid species have remained underground for a number of seasons. Some Australian species, such as Caladenia spp. and Leporella fimbriata, can to carry out seasonal replacement of tubers without producing a shoot above ground (Dixon 1991). This feature has important conservation implications, as orchid populations can remain unnoticed below-ground for a number of seasons. Seedlings of most plants must develop leaves before the nutrient reserves in the seed are exhausted, and remain phototrophic throughout their life. In contrast orchid seedlings have an option of living for extended periods as myco-heterotrophic organisms, opening habitats to orchids that would not otherwise be accessible. The extremely high degree of fungus specificity in myco-heterotrophs confines these plants to habitats where a particular fungus remains viable for long enough for the orchid to reach reproductive maturity. Many of the fungi listed in Table 4 are vigorous saprophytes or parasites that are dominant in large substrates such as tree trunks that take a long time to decompose, thus providing sufficient time for orchid establishment and reproduction. Myco-heterotrophs will be more difficult to conserve than other orchids if their fungi are hard to manipulate. However, some fungi listed in Table 4 can be grown efficiently (as for mushroom production). The identification of associated fungi and a sound understanding of their biology is a fundamental requirement for the conservation of these orchids.

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3.3. Orchid habitat preferences It can be hypothesised that since orchid seedlings have and obligate requirement for mycorrhizas their establishment will be determined by the distribution in substrates of fungi required for germination and growth (Figure 3). Some of the many questions about the role of mycorrhizal fungi in orchid population biology that still need to be answered are listed below: 1. How important is host-fungus specificity and compatibility? 2. How do fungi vary in effectiveness for promoting seed germination and subsequent growth? 3. How critical is the soil status (‘soil saprophytic growth’ sensu Garrett (1970)) of the orchid endophyte for the germination of the orchid seed? 4. Do plants normally contain multiple fungi? If so, are there seasonal trends in the dominance of individual strains? 5. Are there changes to fungus population in soil during the life of plants? 6. Is the composition of fungal populations for an orchid species determined more by habitat factors or historical events? 7. Is knowledge of habitat preferences of orchid fungi required to identify potential orchid habitats?

These questions cannot be answered without knowledge of the distribution and diversity of orchid fungi within soils. Reasons for the success or failure of orchid establishment from seeds in soil have been examined experimentally in only a few cases (Table 3). In cases where seedlings fail to become established, it is difficult to separate factors

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affecting orchids directly from those affecting their mycorrhizal fungi (Table 5). The successful establishment of an orchid to a field site is likely only to be achieved when conditions are favourable for both the orchid and associated fungi (Figure 3). In nature the development of a seedling would require the arrival of viable seed at a point containing compatible fungi. Following germination, subsequent seedling development could only occur in the presence of the associated fungus. Establishment of an adult plant capable of producing seed is most likely subject to similar constraints. However, successful establishment occurs in those situations where factors remain conducive for both the level of receptivity to and dependence of adult terrestrial orchid plants on fungi. 4. Utilising orchid mycorrhizas This section focuses on terrestrial orchids, as epiphytes typically are propagated by asymbiotic means. As discussed below, there is strong evidence for the vital role of orchid fungi in the propagation and growth of terrestrial orchids. This contrasts with epiphytic orchids where mycorrhizal fungi may have less importance after seed germination. 4.1. Seed germination The seeds of terrestrial orchids measure from 0.07 to 0.40 mm across and from 0.11 to 1.97 mm in length, including the testa (Figure 4; Arditti and Ghani 2000). These minute seeds have very little stored nutrient reserves available to support seedling development. These limited reserves and the subterranean germination of many terrestrial species, result in the general belief that mycorrhizal fungi are normally essential for seed germination (Figure 5).

Studies comparing the effectiveness of symbiotic and asymbiotic germination have usually shown that symbiotic germination was more

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rapid and effective than asymbiotic germination (Table 6). In some terrestrial orchids successful germination was only achieved by symbiotic germination (Hadley 1982). Muir (1989) screened a wide range of fungal isolates for their capacity to promote germination and growth of European species of Orchis, Ophrys, Dactylorrhiza and Serapias. He found that rare species were compatible with fewer isolates of fungi than common species in same genus. In some cases, different fungi are responsible for the growth of mature plants than those responsible for germination (Section 2.1).

4.2. Germplasm storage Ex situ conservation includes studies into the long-term storage of orchid seed and mycorrhiza in liquid nitrogen (-196°C) as a back up of genetic stock in the event that critically endangered species become extinct in the wild. This may give us a second chance for some species and in no way is meant to replace conserving the species in their natural habitat. Where symbiotic seed germination methods are to be used to propagate terrestrial orchids it is important that both orchid seed and associated fungi are stored successfully. The plunging of orchid seed into liquid nitrogen has also been found to increase germination percentages for a range of orchid species (Batty et al. 2001b). This may be used to increase germination of seed from endangered orchid species where seed is often in short supply. 4.3. Orchid recovery plans When a species is recognised as critically endangered, the first step in developing a conservation strategy normally is the preparation of a recovery plan. Through surveys of existing populations, risks are identified and a list of required actions is drawn up. Actions usually include research into the species biology, to identify factors essential for its survival. This is especially so for terrestrial orchids due to their complex associations with specific mycorrhiza and highly evolved

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pollination systems. The preferred way to conserve a species is to manage the plants in their natural habitat before they become critically endangered and in need of intense off-site conservation effort to prevent extinction. On-site (in situ) management should be of the highest priority in any conservation program. Orchids are well researched taxonomically, but relatively little is known of their conservation biology and importantly, methods for management and translocation to field sites. Unlike other plants, terrestrial orchids are unique in their highly specialised pollination mechanisms and habitat requirements. They also require advanced

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technology for large-scale propagation. These methods include: (i) Collection of seed and fungal symbionts which are effective for germination, (ii) Development of effective propagation methods for the target species; and ( i i i ) Translocation of orchids to safe sites. There are a number of different protocols available for the isolation of orchid mycorrhizal endophytes. These include the block method and the peloton isolation (Dixon et al. 1989). Depending on the isolation protocol being adopted by the researcher and the fate of the isolate(s) this may affect results. For example, isolation of fungi from surface-sterilised blocks of orchid tissue may be unsuitable, if the researcher was attempting to investigate fungal diversity in orchids. This occurs because slow growing or more sensitive cultures will be over-run by fungi that grow more aggressively on agar. We have also observed problems with effective surface sterilisation that result in isolation failures from some tissues. Single peloton isolation methods are often best, as they should allow any isolates that form mycorrhizas to be obtained (Rasmussen 1995). The isolation technique used to obtain orchid fungi may impact on specificity studies, as some methods may select rapidly growing isolates or contaminants. A survey of the literature suggests that there are several different approaches to working with orchid fungi for conservation (Table 3). In some cases, it is assumed that each orchid has a new or specific fungal partner and isolate new fungi for each orchid to be conserved, which are then tested for host-compatibility by a germination assay. In other cases, it is assumed that host specificity is less important than habitat specificity and therefore a wide range of isolates from the same and different orchids are screened to find the most effective partners. Although both approaches have resulted in successful propagation of terrestrial orchids, there may still be regional differences in fungal biology. This may become evident when field introductions are attempted. Examples where symbiotic seed germination has allowed the successful propagation and reintroduction of orchids are summarised in Table 7. 5. Case studies 5.1. The Western Australian Underground Orchid, Rhizanthella gardneri The majority of achlorophyllous orchids develop an above-ground phase which functions in flowering and seed dispersal (e.g. members of the genera Epipogium, Didymoplexis and Gastrodia). However the Australian genus Rhizanthella remains below ground, or, at least, below the litter layer, even when forming flowers (Figure 6) and seeds. This cryptic behaviour has continued to confound observers since accidental discoveries of the orchids half a century ago. Even at present, the eastern Australian species R. slateri has been seen only once in flower in the wild in the past decade, while its western counterpart, R. gardneri, has only recently been rediscovered (George 1980; Dixon and Pate 1984). Although known in several locations, R. gardneri still requires considerable effort to locate specimens non-destructively in its native habitat, even where it is currently known to be prolific. Early discoveries of this mysterious, fully subterranean plant roused great excitement

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among the local public and scientific fraternity of the day, and, within six months of its discovery, wax models of the plant had been placed on exhibition at scientific meetings and museums in Western Australia.

5.1.1. The myco-heterotrophic nutrition of Rhizanthella gardneri The endophyte of Rhizanthella was originally described by Pittman (1929) as being “Rhizoctonia-like” and recent work by Warcup (1985b) has confirmed that at least two strains of the form genus Rhizoctonia may be involved in symbiosis with the orchid and its host Melaleuca uncinata. The endophyte resembles the basidiomycete genus Thanatephorus, a known symbiont of orchids (Hadley 1982).

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It is widely held that holomycotrophic orchids derive carbon from their fungal partner which breaks down the organic matter of surrounding litter or humus. Alternatively or additionally they may engage in a three-way epiparasitic relationship with an autotrophic tree or shrub species. In the latter case, carbon passes from roots of the woody species to a shared mycorrhizal partner and thence becomes available to the orchid. Relationships predominantly of an epiparasitic kind have been suggested for other achlorophyllous orchids such as: Gastrodia (Table 4), and, outside the Orchidaceae, in achlorophyllous representatives of the subfamily Monotropoideae of the Ericaceae (Pterospora, Sarcodes and Monotropa, Robertson and Robertson 1982). However, with few exceptions (Bjoerkman 1960) definitive tracer studies of nutrient flow from woody hosts to epiparasitic partner are lacking, and since the surrounding soil is rich in organic matter, a significant saprophytic element in nutrition of this orchid is highly likely. As stated previously, R. gardneri inhabits soils extremely deficient in organic matter, implying that a purely or predominantly organic matterdependent nutritional base for the orchid would be extremely doubtful. The recent success of Warcup (1985b) and the authors in cultivating the orchid from a seed in an artificial Melaleuca-Rhizoctonia-Rhizanthella system corroborates this view, at least to the extent of demonstrating a highly effective epiparasitism by the orchid when organic matter is absent or at very low level in the medium. 5.1.2. Seed morphology and structure, germination, and early establishment of symbiosis with a fungal partner Germination of seeds in laboratory culture occurs more readily in light than dark coloured seeds. It is preceded by swelling of the embryo and cracking of the seed coat. The h i l u m corresponds to the point of entry of the endophyte into the seed, and adjacent cells of the embryo eventually show intracellular proliferation of the fungus. By three months after germination, the body of the protocorm is already some 2-3 times the size of the seed. Radiating from it are a number of trichomes, each a possible entry site for fungal hyphae. After five months the elongated shape of the mature tuber is clearly apparent and the growing shoot apex clearly demarcated and flanked by trichome primordia. Seedlings germinated in mineral agar in the presence of the fungus are ready for transplanting into the rooting medium of soil grown Rhizoctonia-inoculated Melaleuca uncinata plants 3-4 months after germination. Our observations on seedling growth through perspex window in the side of the potted host plant show rapid tuber growth over the ensuing 9 months. First flowering was recorded by Warcup (1985b) in 15 month-old plants. Rhizanthella gardneri possesses several characters considered primitive for the Orchidaceae (Dressler 1981). These include spiral arrangement of leaves, a terminal inflorescence, non-resupinate flowers, fleshy fruits, large hard seeds, low seed number, an ‘endosperm-like’ embryo, mealy pollen, and stomata lacking subsidiary cells. On the other hand, by virtue of its totally underground habit and ability to survive

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aphotosynthetically in soils extremely low in organic matter, it may well be considered to represent the u l t i m a t e in specialisation among orchids. 5.1.3. Conservation and management As far as we know, Rhizanthella gardneri is an extremely rare species which unfortunately occurs primarily in areas suitable for cereal production. On these grounds it would appear that all but less than 10% of the habitats where it may have once occurred are now under agriculture. Worse still, applications for release of new lands for agriculture in potential Rhizanthella habitats continue to outstrip survey efforts to locate the orchid. The enormous d i f f i c u l t y in locating Rhizanthella, let alone the problem of effectively monitoring the vigor of existing populations, presents great obstacles in management of the species. Clearly the best policy is to direct conservation and management policies towards maintaining adequate healthy stands of Melaleuca uncinata which cohosts the fungus, in the hope that they will provide large and varied resource of habitats in which the orchid may well still be present, or onto which it might be introduced where the species is deemed to be greatly endangered. Frequent fires, invasion of habitats by weeds and exotic animals, deliberate or accidental human interference of known habitats of the orchid, and the general deterioration of native bush caused by aerial drift or surface water leaching of agricultural fertilisers, are all potent deleterious influences (Dixon and Pate 1984). A further, more subtle cause for concern, is the possibility that the few remaining populations will degenerate through elimination of pollinating agents, native mammals which might distribute seeds, or merely by genetic deterioration of the species due to lack of input by sexually derived genotypes. As shown here, natural seed set in our study locations is extremely poor in present populations, but can be increased many fold by hand pollination. This may indicate that current maintenance of the species is largely by vegetative multiplication through formation of daughter tubers, giving little potential for long-distance spread within a habitat. The methodology exists for establishing the RhizanthellaRhizoctonia-Melaleuca association in glasshouse culture, thereby giving the opportunity for replenishing stands of the orchid in existing habitats or even for establishing the species in areas where it currently does not exist. Considerable thought must be given to the relative merits of in situ and ex situ conservation for this species, but the fact that this type of approach is available for conservation of the species must surely offer considerable reassurance. 5.2. Other Western Australian orchids The southwestern botanical province of WA is one of the world’s main centres of biodiversity, where a long period of geographical isolation and highly infertile soils have resulted in many plant species that occur nowhere else. This region has one of the world’s most diverse terrestrial orchid floras, with over 340 species known. Southwestern WA is a

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“living biological laboratory,” where the endemism and diversity of our native orchid flora provides a unique opportunity to study issues relating to the conservation and management of terrestrial orchid species. In particular, it is possible to contrast closely related species which are common and rare, have very general or highly specific habitat requirements, or have geographically disjunct populations. At present, 34 taxa of southwestern orchids are designated as critically endangered (Brown et al. 1998) and a 34 more are only known from a few locations and require further study (Atkins 1999). Even relatively common species are declining in many urban and rural areas due to habitat loss and land degradation. Increasing urban and industrial development in many plants of the Perth region threatens the existence of many once common plant communities and species. Perth’s native orchids are a unique part of our natural and cultural heritage, but are being severely impacted upon by land use changes and habitat degradation. There are many endemic species of terrestrial orchids in WA that are rare because of highly specific habitat requirements, requirements for specific pollinators and/or ineffective dispersal mechanisms coupled with extensive land clearing for agriculture and housing. Other threats to orchid habitats include, weed invasion, frequent fires, changes to the water table, grazing by feral animals and disturbance by humans. No other capital city in Australia has as many threatened native plants and clearly urgent conservation actions are needed if species extinctions on the doorstep of Perth are to be averted. It is suspected that the low recruitment rates from natural seed dispersal observed in WA probably result from the patchy distribution of mycorrhizal fungi in soils and the scarcity of suitable habitats in the landscape. Information about the biology of fungi that associate with WA terrestrial orchids is urgently required as a cornerstone for conservation work – to identify habitat requirements and understand factors that determine the success of seedling establishment. Where possible seed has been collected from rare orchid species, specific endophytes have been extracted from cortical cells and cultured. Seed and endophyte have been combined to produce seedlings of many species for the first time. New protocols for the transfer of seedlings to natural bush sites have resulted in the successful translocation of the Cinnamon Sun Orchid (Thelymitra manginii) (Figure 7), Dwarf Bee Orchid (Diuris micrantha) and the Swamp Donkey Orchid (Diuris purdiei) (Batty et al. 2001 a). Diuris purdiei seedlings were translocated to two sites by introducing tissue-cultured plants and mycorrhizal fungi. These plants were still alive after one year but a lack of funding prevented further monitoring (ANPC 1997). More recently, laboratory produced seedlings of common and rare orchids (Caladenia arenicola, Pterostylis sanguinea, Thelymitra manginii and Diuris micrantha were returned to field sites in attempts to re-establish orchid plants in natural habitats. Plants that were returned to the field as actively growing seedlings or as dormant tubers showed survival beyond the first season where as those initiated from in situ seed germination failed to survive the first summer. These methods now

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require further testing to see if they would be suitable for many other WA terrestrial orchids and to understand further the role of associated mycorrhiza in establishing orchids to field sites.

6. Conclusions Symbiotic associations are generally considered to be essential throughout the Orchidaceae. However, we have a very limited understanding of the ecology of the orchid, their associated symbionts, or the interactions between them. The majority of current knowledge on orchid fungus and host plant interactions is based on in vitro studies using fungal isolates from mature orchid plants. However, in recent years researchers have demonstrated the ability to unlock some of the mysteries of orchid mycorrhizas under field (in situ) situations. Most of our knowledge of the role of orchid mycorrhizal associations is from studies of terrestrial orchids and relatively little is known about their role in the establishment and growth of epiphytes. This type of information is essential if successful large-scale re­ introductions are to be carried out on some of the large number of endangered terrestrial orchid species occurring throughout the world. This review considers the ecological implications of orchid mycorrhizas, especially their implications for the conservation of threatened orchid species. This review considers orchid conservation on a world scale, but also includes case studies which address regional issues. Orchid mycorrhizas have been studied since the relationship between orchids and endophytes was discovered by Noël Bernard (1909) and much data has been massed, mostly from in vitro studies (for recent reviews see Arditti 1990; Peterson 1998). Recently, research has begun to focus on in situ studies. It has become evident that not all knowledge gained from in vitro studies can be applied to field situations and when

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dealing with critically endangered taxa we need to understand what happens at the ground level, not the Petri dish. Field studies have been difficult in the past until the development of innovative protocols for studying dust seeds in situ (Rasmussen and Whigham 1993). Acknowledgements The financial support of Western Power is gratefully acknowledged. References Alexander C, Alexander IJ (1984) Seasonal changes in populations of the orchid Goodyera repens Br. and in its mycorrhizal development. Transactions of the Botanical Society of Edinburgh 44, 219–227. Alexander C, Alexander IJ, Hadley G (1984) Phosphate uptake By Goodyera repens in relation to mycorrhizal infection. New Phytologist 97, 401–412. Alexander C, Hadley G (1983) Variation in symbiotic activity of Rhizoctonia isolates from Goodyera repens mycorrhizas. Transactions of the British Mycological Society 80, 99–106. Alexander C, Hadley G (1985) Carbon movement between host and mycorrhizal endophyte during the development of the orchid Goodyera repens Br. New Phytologist 101, 657–665. Andersen TF (1996) A comparative taxonomic study of Rhizoctonia sensu lato employing morphological, ultrastructural and molecular methods. Mycological Research 100, 1 1 1 7 – 1 1 2 8 . Anderson AB (1991) Symbiotic and asymbiotic germination and growth of Spiranthes magnicamporum (Orchidaceae). Lindleyana 6, 183–186. ANPC (1997) ‘Guidelines for the translocation of threatened plants in Australia.’ (Australian Network for Plant Conservation: Canberra) Arditti J (1990) Lewis Knudson (1884–1958), his science, his times, his legacy. Lindleyana 5, 1–79. Arditti J (1992) ‘Fundamentals of orchid biology.’ (John Wiley & Sons: New York) Arditti J, Ghani AKA (2000) Tansley review No. 110 - Numerical and physical properties of orchid seeds and their biological implications. New Phytologist 145, 367–421. Atkins KJ (1999) ‘Declared rare and priority flora list.’ (Department of Conservation and Land Management: Perth) Batty AL, Dixon KW, Sivasithamparam K (2000) Soil seed bank dynamics of terrestrial orchids. Lindleyana 15, 227–236. Batty AL, Dixon KW, Brundrett MC, Sivasithamparam K (2001a) Constraints to symbiotic germination of terrestrial orchid seed in a mediterranean bushland. New Phytologist 152, 511–520. Batty AL, Dixon KW, Brundrett MC, Sivasithamparam K(2001b) Long-term storage of mycorrhizal fungi and seed as a tool for the conservation of endangered Western Australian terrestrial orchids. Australian Journal of Botany 49, 619–628. Benzing DH(1982) Mycorrhizal infections of epiphytic orchids in southern Florida. American Orchid Society Bulletin 51, 618–623. Benzing DH, Friedman WE (1981) Mycotrophy: it’s occurrence and possible significance among epiphytic Orchidaceae. Selbyana 5, 243–247.

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Chapter 8 ERICOID MYCORRHIZAS IN PLANT COMMUNITIES

Kingsley W. Dixon Kings Park & Botanic Garden, Botanic Gardens & Parks Authority, West Perth 6005, Western Australia; Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

K. Sivasithamparam Soil Science and Plant Nutrition, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

David J. Read Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK.

1. Introduction The Ericales encompass a large, but monophyletic lineage of taxa, all generally recognised as possessing an anatomically and mycologically unique association with a mycorrhizal partner (Smith and Read 1997; Cairney 2000; Stevens 2001 onwards). The association with ericoid fungi in the Ericaceae (sens. APG 1998) has been the basis of considerable research and debate, issues ranging from the nitrogen (N) and phosphorus (P) impoverishment of soils where these plants grow and the role of the ericoid fungal partner in nutrient acquisition (Read and Kerley 1999), to elegant experiments demonstrating high levels of diversity of the fungal partners of ericads. The relationship between ericoid fungi and the Ericales provides a remarkable link between a cosmopolitan group of plants and mycorrhizal

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fungi based on the unique anatomy of the association where unsuberised epidermal cells are occupied by a typically dematiaceous fungal partner (Read 1983). The description by Harley (1959) of the structure as an ‘ericoid mycorrhiza’ provided reinforcement of the linkage of these fungi with particular anatomical structures (unique to the Ericaceae: Cassiopoideae, Ericoideae, Epacridoideae, Empetreae, Vaccinioideae). The fact that these families represent many heathland groups has been used to contextualise the nutrient uptake benefits of the fungi. For example, the heathlands of the boreal regions with their ombrotrophic status has resulted in many authors concluding that the fungus operates in a environment of prevailing acidity, leaching (among other things), metal ions into solution (Read and Kerley 1999). In low pH environments, this raises issues of phytotoxicity for the plant (Jalal and Read 1983a,b). The fungus is thought to enable a ‘detoxifying’ system to operate (Bradley et al. 1982; Leake and Read 1991) for establishment of plants in otherwise hostile soil conditions (Cairney 2000). The variety and diversity of habitats in which ericads operate is testament to the adaptability and resilience of the ericoid endophyte. From the moor-humus conditions of Calluna heathlands (Read 1983) in the northern hemisphere to the extreme seasonality and nutrient impoverishment of the hotspots of ericad diversity in the fynbos of Southern Africa and the kwongan of south-western Australia is indeed comparatively unique in mycorrhizal systems (Read 1989; Smith and Read 1997). Understanding the relationship between the diversity of ericoid fungi and their attributes and their specificity as a key to the endemism and conservation of Ericales is the subject of this chapter. 2. Anatomical basis to the ericoid state McLennan (1935) illustrated the ericoid mycorrhizal root as comprising hypertrophied epidermal cells, with attendant endophytic hyphal matrix, surrounding an exodermis and a monarch stele. Ericoid mycorrhizal roots or ‘hair roots’ (Beijerinck 1940) are devoid of root hairs and can be produced from all orders of roots. Dixon (pers. obs.) has recorded hair roots being produced directly on the surface of eight year old roots of Leucopogon conostephioides and Conostephium pendulum in kwongan heathland in south west Australia. The hair root apparatus and attendant fungal biomass can occupy up to 80% of the root system of Calluna (Read 1983) and up to 85% in the kwongan ericad Astroloma xerophyllum (Hutton et al. 1994). The percentage contribution of annual hair root production to total root biomass (Bell et al. 1996) and the abundance, seasonality and ecological value of hair root production in the Magellanic tundra of Patagonia (Pisano 1983) and Sphagnum bogs of New Zealand (Wardle 1991) represent systems which

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require further investigation. The production of hair roots is periodic in the seasonally droughted ericads of south western Australia (Figure 1; Hutton et al. 1994). Here, the seasonality of production of hair roots in the winter growth season implies that the ericoid fungus may have more to do with nutrient acquisition than water balance.

The unique morphological and anatomical nature of the hair roots and its fungal associates in the Ericaceae offers a basis for attempting to understand the cosmopolitan success of the Ericales known to participate in ericoid mycorrhizal partnerships. Whereas Straker (1996) details host and ecological specificity in ericoid mycorrhizas and Duckett and Read (1991, 1995) raise the spectre of ericoid fungi co-associating with hepatics, ericoid mycorrhiza in the Ericales is seen as one of the most specific of mycorrhizas. Linking benefit to the plant with the presence of the endophyte within the unique enlarged epidermal cells of the hair root provides a clear mechanism for determination of the extent of the ericoid syndrome in plants. Read and Kerley (1999) go one step further in providing a more rigorous system to

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accurately define not just the ‘occurrence in or on the ericaceous root’. This system, based on the testing of the simplest of Koch’s (1912) postulates provides three steps for estimating the root-fungus association as mycorrhizal: 1. Candidate fungus is isolatable and maintained in pure culture. 2 . The fungus is grown w i t h i t s putative host plant under defined conditions. 3. There needs to be evidence that infection by the fungus leads to enhancement of growth or nutrient uptake. Read and Kerley (1999) indicate that the dark (rarely pale), slow growing, sterile and dematiaceous mycelia commonly isolated from Ericaceae have only rarely been thoroughly evaluated using these principles (Leake and Read 1991). Monreal et al. (1999) described a raft of 34 mycorrhizal root-associated isolates of Gaultheria shallon (Ericaceae) from conifer plantations in Western Canada, yet failed to demonstrate that the fungi isolated from root sections (hair roots were not specifically stated) were truly endophytic. The issue of defining endophytism is addressed by Stone et al. (2000) who follow Petrini ( 1 9 9 1 ) who suggests “Endophytes colonise symptomlessly the living, internal tissues of their host, even though the endophyte may, after an incubation or latency period, cause disease.” This definition covers all microorganisms as well as endophytic vascular plants (Stone et al. 2000). Jansen and Vosátka (2000) screened a wide range of fungi from Rhododendron and found that of 200 strains tested, only 10% exhibited positive effects on the growth of Rhododendron micro-cuttings. In the absence of isolation from within the specialised plant structures from which the ericoid association was originally described, fungi isolated from the surface of roots or dead cells within active regions of roots remain ambiguous in their functional relationship and ecological importance. The presence of the hair root (Figure 2) as a morphological attribute of the ericoid symbiotic system is also unusual compared with many other plant structures involved in symbiotic relationships. What is most surprising is that unlike most other mycorrhizal systems, the hair roots and attendant hypertrophic epidermal cells are formed in ‘anticipation’ of an infection event. Hutton et al. (1994), in a phenological study of hair root production, found that 40% of the hair root system was devoid of endophyte yet retained the anatomical characteristics of the hair root. The hypertrophic collar and stem region of some terrestrial orchids (Ramsay et al. 1986; Dixon 1991; Batty et al 2001a,b) represent other rare instances where plant structures are formed in anticipation of a mycorrhization event. The chemical composition of the cell walls of hair roots differ from that of most other higher pants (Albersheim 1976) in the lack of fucose and polygalacturonic acid residues and the presence of N-acetylglucosamine

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(Straker 1996). While providing endorsement of the unique nature of the hair root, the chemistry of the hair root wall may play a key role in the recognition of ericoid fungal strains. The development of the fibrillar sheath (Gianinazzi-Pearson and Bonfante-Fasolo 1986) by some efficacious ericoid endophytes may be associated with cell wall recognition prior to ‘docking’ of the fungus (Straker 1996).

The hair root provides a simple morphological criterion for the determination of the likelihood of a possible mycorrhizal association. In friable soils, particularly the loose sands of southern Africa and south western Australia, hair roots (Figure 2) are visible in excavated roots as gossamer-like threads, often with fine soil particles adhering to them. Careful clearing and staining either with a general fungal stain or a specific fluorescent dye such as 3,3’-dihexyloxacarbocyamine iodine (Duckett and Read 1991) will help to detect the presence of intracellular hyphae. This may indicate that the ericad is likely to be involved in a mycorrhizal relationship. Thus, for the conservation practitioner, it is possible to quickly determine if an ericoid mycorrhizal association is present or likely to be present. However, to accurately ascribe ecological function, more complex and exhaustive trials are required (Vrålstad et al. 2002). Management of ericad habitats (e.g. nutrient loading, impact of fire or soil disturbance)

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would therefore need to consider the impact of such disturbance on the health and sustainability of the ericoid mycorrhizal system. 3. Potential value of ericoid mycorrhiza as indicators for conservation of Ericaceae species Harley (1959) coined the term ‘ericoid mycorrhiza’ to describe the relationship between the hair root and a fungal endophyte. Ericoid mycorrhiza have been reported to occur in the majority of the genera of Ericoideae (e.g. Calluna, Erica, Gaultheria, Kalmia, Ledum, Phyllodoce, Rhododendron and Vaccinium), Empetreae (e.g. Empetrum) (Read 1996; Read and Kerley 1999) and Epacridoideae (25 genera) (Reed 1987; Hutton et al. 1994; Bell et al. 1994). Cullings (1996) provided a circumscription of the Ericaceae and found that ericoid mycorrhiza-forming taxa are monophyletic, indicating that the Epacridaceae and Empetraceae should be included within Ericaceae sens. lat. Subsequent comprehensive study of the Ericales reinforces this view (Kron 1996; APG 1998; Stevens 2001 onwards). What is clear is that re-evaluation of the phylogenetic relationship of the Ericaceae as proposed by Kron (1996) convincingly shows that the Empetraceae (Cataula) nests well within the Rhododendron/Calluna clade, sharing with those taxa the distinctive characteristics of the ericoid mycorrhizal association (Smith and Read 1997). The analysis provided by Kron (1996) segregates Arbutus as distinctive from the Ericaceae in a basal position (though included as a subfamily of Ericaceae by APG (1998) and Stevens (2001 onwards)) in concurrence with the distinctive arbutoid mycorrhizal system (Smith and Read 1997). With the knowledge of a more natural and rigorous interpretation of the phylogenetic relationships of the Ericaceae, it is of interest to relate the fungal associates with the known evolutionary history of the Ericaceae (see Cairney 2000). The remarkable morphological similarity of the hair root, the relatedness of these three families with a restricted array of distinctive ascomycetous fungi adds further support to the inclusion of the Empetraceae and Epacridaceae within the Ericaceae. Since the Ericaceae encompasses such a broad and cosmopolitan family of some 140 genera and 3,800 species (Mabberley 1997) across Arctic to southern hemisphere mediterranean-type to cool temperate ecosystems, it is tempting to speculate as to the functional attributes and benefits of the mycorrhizal association across this broad range of habitats. 4. Characterisation of ericoid endophytic fungi The identity of mycorrhizal fungi which associate with the Ericaceae has been a source of debate and conjecture (Vrålstad et al. 2002). A variety of

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Ascomycetous and imperfect fungi have been associated with ericoid roots (Smith and Read 1997) although not all of them have been established to be mycorrhizal. Much of the work, especially in the United Kingdom, has been carried out with Hymenoscyphus ericae. Among the fungi imperfecti associates, Oidiodendron species have featured most commonly (Smith and Read 1997). The centre of diversity for extant Ericales is in the southern hemisphere and most likely reflects a Gondwanan origin (Cullings 1996). Ericales-like plants are found in the fossil record from the Cretaceous (Nixon and Crepet 1993). Although no mycorrhizal involvement can be discerned, speculation is that a coincident major radiation of ascomycete fungi (Berbee and Taylor 1993) may have provided the early Ericaceae with access to a fungal symbiont (Cairney 2000). New molecular evidence (Sharples et al. 2000a) points to the similarity of root-associated fungi of Calluna vulgaris from southwest England with endophytes from North America and Australian Ericaceae in contrast to the earlier findings of McLean et al. (1999). Parry et al. (2000) found that there was a level of sero-relatedness in antipodean and boreal Ericaceae endophytes, lending further support to a ‘common origin’ theory for ericoid mycorrhiza. The diversity in the root-inhabiting endophytes of Ericales can be seen to operate at three levels: 1. High diversity within plant (Xiao and Berch 1996; Liu et al. 1998; Monreal et al. 1999; Sharpies et al. 2000a). 2. High diversity within species (Hutton et al. 1994; Chambers et al. 2000; Parry et al. 2000; van Leerdam et al. 2001; Whittaker and Cairney 2001). 3. High diversity within sites (Hutton et al. 1996). The remarkable levels of diversity of endophyte types (either at a cultural, enzymatic or molecular level) recorded within plants (see 1 above) is highlighted in the study of Sharples et al. (2000a) where 14 RFLP-types were assigned to 107 root-associating fungi of Calluna vulgaris. Even within single root segments the level of diversity of root-associating fungi is surprising. For example, Monreal et al. (1999) found four distinctive molecular types of fungi in small sections of root of the common Gaultheria shallon in Canada. In a geographically distant sense, Chambers et al. (2000) found up to four distinct fungi in one root segment of the eastern Australian species Woollsia pungens, with potentially six fungal taxa in four co-located plants. The biological and ecological implications for the wide diversity of fungi that coexist with Ericaceae may be based on an ancestral tolerance as the early ericads radiated from their centre of origin and encountered an

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array of endophytically competent fungi (Straker 1996; Cairney 2000) and/or the development of ‘intense distinctive mutualisms’ (Read 1996). The latter refers to a means whereby more species of Ericaceae may be ‘packed’ into a niche (such as the high species richness of ericads in southern Africa (672 species) and Australia (c. 350 species)) by virtue of participating with a range of ericoid mycorrhizal fungi, which may vary spatially, temporally and taxonomically. The end result remains that a wide and diverse array of ericoid mycorrhiza provide another level of speciation to extract the limited or locked pools of N and P (Gimingham 1972; Pearson and Read 1975) or to cope with organic extremes, metal toxicity (Burt et al. 1986; Turnau et al. 1998) in the soils in which they inhabit, or drought (Hutton et al. 1996). In the highly endemic flora of south-west Western Australia (with 246 species of Ericaceae), Hutton et al. (1996) found that intensive sampling of endophytic fungi from wet (swampy) to dry habitats resulted in a scale in homogeneity of endophytic zymo-types from less to more diverse respectively. Since plant diversity in this region resides mostly on relatively dry sites, Hutton et al. (1996) postulated that it is possible that the same pressures which drive mega-diversity in higher plants in such extreme environments could also ‘exact similarly diverse levels of specialisation within populations of associated soil microorganisms’. It is therefore tempting to consider the co-evolutionary opportunities provided to Ericaceae in these environments through associating with endophytes with competency for particular pedological attributes with a niche (see Cairney 2000 for further discussion of this aspect). 5. Conservation implications of the ericoid mycorrhizal association Benefits of the ericoid association have existed for at least 100 Myr (Cairney 2000), in which time, extant Ericaceae have occupied all continents and a vast array of habitats. The unique attributes of ericoid fungi which enable growth in ecosystems often characterised by extreme nutrient impoverishment relies on the special ability of ericoid fungi to degrade polyphenols and other complex organic materials, attributes which are not available in many other non-mycorrhizal plants (Haselwandter et al. 1990). In addition, the production of proteases, siderophores and other chemical systems from the ericoid fungus provides a remarkable armoury of physiological attributes for the host ericad. The degree to which these attributes act to ensure plant survival is illustrated in the extreme in the study of Sharples et al. (2000b) who demonstrated that the ericoid fungus Hymenoscyphus ericae had the ability to efflux arsenic from its hyphae while providing P to its host, Calluna vulgaris. Similar studies of tolerance to metals by ericoid mycorrhiza (e.g. Bardi et al. 1999; Martino et al.

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2000a,b) serve to illustrate the emerging theme of the possible co­ evolutionary patterns involved with the ericoid mycorrhizal system. Just as the ericoid fungi have the capacity to adapt to extremes, so the fungal associates of Ericaceae appear to be sensitive to habitat alteration particularly i n v o l v i n g nutrient accretion. For example, Heil and Diemont (1983) showed that nitrogen saturation may have been a major contributing factor leading to the replacement of Ericaceae communities by grasslands. Levels of N or indeed P saturation removes the advantages of tight nutrient conservation so typical of the sclerophyllous communities in which many Ericaceae reside and for which the ericoid mycorrhiza provides such benefits (Specht and Rundel 1990; Read 1996). For the biodiverse communities of Southern Africa and south western Australia, nutrient fluxes, particularly associated with fire, may therefore play a crucial role in the conservation of rare or threatened Ericaceae species (Stewart et al. 1993). Equally, the lack of competitive ability in strains of ericoid fungi may lead to displacement by other microorganisms through changes in biotic balances in ecosystems (e.g. Hutton et al. 1997). The impact of habitat changes on the performance of the endophyte and subsequent impacts on the survival of Ericaceae in terms of nutrient uptake, water balance and stress tolerance is virtually unknown. The conservation of Ericaceae and inter alia, ericoid mycorrhizal diversity will initially rely on studies to determine the degree to which both fungus and host can tolerate and/or adapt to e n v i r o n m e n t a l changes. In one of the few studies to investigate the re-establishment of measurable abundance of ericoid mycorrhiza into disturbed sites, Hutton et al. (1997) found that up to 12 years elapsed before root associating endophytes returned to a post-mined site. A caveat on this study was that topsoil was replaced to site and that the m i n i n g operation retained an interface with un-mined native vegetation. How long, if at all, it would take for the return of ericoid fungi to sites where topsoil/natural system interfaces did not exist e.g. revegetated farmland, requires urgent attention. 6. Summary and conclusions The study of the microbial endophytes of Ericaceae may help us to understand the evolution and distribution of the taxa within the Ericales world-wide. It w i l l also indicate whether the fungal associates moved with their plant hosts or whether new associations with resident strains were formed as the plants spread. This information is also likely to tell us whether the genetic diversity of the fungal associates could help to determine the taxonomic relationships within the host order. Much needs to be done on the determination of the role ericoid fungi play in the successful establishment of horticulturally important members of

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the Ericaceae such as species of Rhododendron (see Jansa and Vosátka 2000) and Vaccinium that are difficult to establish in certain environments. The ecological importance of hair roots in certain environments is poorly understood. In the Western Australian Banksia woodlands, for example, their occurrence in the soil profile is often constrained because of excessive competition by the persuasiveness of cluster roots in the Proteaceae (Pate and Watt 2001). Studies by Hutton et al. (1994) for instance showed that the dominant activity of hair roots and endophytes is restricted to the cooler wet months in the highly seasonal mediterranean-type climate of south western Australia. These same mycorrhizas are also u n u s u a l l y sensitive to soil disturbance with long periods elapsing before recolonisation (Hutton et al. 1997). Finally, while horticulturally important Ericaceae are often translocated, little attention is paid to concurrently including the mycorrhizal partner in the translocation or conservation process (especially with rare and endangered taxa) as has been done with members of the Orchidaceae (Batty et al. 200la). The importance of the ericoid association for the long term sustainable management and recovery of rare or threatened Ericaceae remains an important issue for conservation practitioners.

Acknowledgements The authors would l i k e to t h a n k Mark Brundrett for comments on the manuscript.

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Chapter 9 THE DIVERSITY OF PLANT PATHOGENS AND CONSERVATION: BACTERIA AND FUNGI SENSU LATO

D S Ingram St Catharine’s College, Cambridge University, Cambridge, CB2 1RL, UK.

1. Introduction The conservation of plant pathogen diversity is counter-intuitive to any plant pathologist dedicated to the prevention or eradication of plant disease. To the lay public, I suspect, such a notion would seem at best incomprehensible and at worst irresponsible (Ingram 1998a,b, 1999). Yet, although plant pests and diseases cause some 30% of losses of agricultural production world­ wide (Agrios 1997) and have sometimes devastated native species (e.g. Newhook and Podger 1972), plant pathogens are now being recognised as key components of many natural and semi-natural ecosystems and potentially of great benefit to humankind in spheres as diverse as, for example, basic scientific research, biotechnology and novel drug and pesticide production. In the pages that follow, based on Ingram (1999), the nature and significance of plant pathogen diversity and the threats to it will be adumbrated. Then, some approaches to the conservation of plant pathogen diversity will be reviewed, briefly, with due attention being given to the potential risks involved. The discussions will largely be confined to bacteria and fungi, sensu lato. 2. The diversity of plant pathogens It is important to emphasise at the outset that our current knowledge of microbial diversity is woefully inadequate, only about 5% of fungi, 1% of viruses and 0.1% of bacteria having so far been described (Ingram 1999;

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Table 1). Even where species have been named, our knowledge of their life cycles and general biology is frequently very limited (see, for example, Helfer 1993; Parbery 1996; Rodriguez and Redman 1997). The greatest areas of ignorance are the diversity and biology of microorganisms, especially plant pathogens, inhabiting tropical ecosystems (Day 1993) and aquatic ecosystems (Andrews 1976; Canter-Lund and Lund 1995; Smetacek 2001). There is a pressing need for a significantly greater investment in systematics and biological research to rectify such lacunae in the knowledge base (Hawksworth 1991; Anon. 1992; Day 1993; Anon. 1994a; Ingram 1999). That said, the diversity of bacteria and fungi known to be pathogens of plants is immense, and has been classified in a variety of ways: morphologically, ecologically, physiologically, genetically and so on (Hawksworth et al. 1995; Agrios 1997; Holliday 1998).

In recent morphological and molecular classifications, plant pathogens are scattered across two Super-kingdoms, the Prokaryotae and the Eukaryotae, and four Kingdoms, MONERA, PROTOZOA, CHROMISTA and FUNGI (Ingram and Robertson 1999; Table 2). They cause disease in plant groups as diverse as angiosperms, gymnosperms, pteridophytes, mosses, liverworts and algae. Within the MONERA (Prokaryotae), six major genera of Gram negative bacteria (Agrobacterium, Erwinia, Pseudomonas, Ralstonia, Xanthomonas and Xylella) and two major genera of Gram positive forms (Clavihacter and Streptomyces) include plant pathogens. Most species are unicellular, reproducing by binary fission, and many are flagellate. Streptomyces forms a rudimentary branching mycelium of narrow septate filaments. In addition, a number of plant diseases once thought to be of viral origin are now known to be caused by a group of small, prokaryotic organisms known as mycoplasma-like organisms (MLOs). This group includes phytoplasmas and spiroplasmas, which may be regarded as bacteria which lack the ability to form a rigid cell wall. They are pleomorphic and under natural conditions are obligate parasites. Bacteria are generally less important as pathogens of plants in temperate climates, but are of great significance in the warmer regions of the globe, especially the tropics.

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The plant pathogenic members of the group referred to colloquially as the ‘fungi’ (Table 2) are classified in three Kingdoms, PROTOZOA, CHROMISTA and FUNGI (Eukaryotae), with the FUNGI being the largest group. (In the rest of this chapter the word ‘fungi’ will be used in its wider, colloquial sense and FUNGI, printed in upper case, will be used in the strict taxonomic sense to refer to the Kingdom of that name.) The fungi includes the largest group of plant pathogens, although some 92% of the approximately 7730 genera described as fungi are said to be

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entirely saprophytic (Schäfer 1994). This figure probably needs revision, however, for the fungi as a whole are relatively poorly researched (Parbery 1996). Nevertheless, the diversity within the group is considerable, as follows. Plant pathogenic members of the Plasmodiophoromycota, in the Kingdom PROTOZOA, normally grow in l i v i n g host cells as plasmodia, masses of cytoplasm with many nuclei contained within membranes, or as pseudoplasmodia, comprising many smaller masses of cytoplasm, each with a single nucleus. Members of the group reproduce by means of zoospores and may also produce t h i c k walled resting spores. In most cases the positions of meiosis and nuclear fusion in the life cycle is not known with certainty, but the diplophase is probably very short. Pathogenic members of this group are holobiotrophic in their nutrition (see Table 3). The plant pathogenic fungi with cell walls fall into two Kingdoms, the CHROMISTA and the true FUNGI. The CHROMISTA normally grow as branched hyphae containing cellulose as the main structural component of the walls. There are normally no septa, each hypha containing many nuclei, which are usually diploid. Asexual reproduction is usually by means of diploid zoospores produced within a sporangium, although in some phyla zoospores are not produced and the multinucleate sporangium is the main asexual u n i t of dispersal. Sexual reproduction is by means of thick walled, diploid resting spores called oospores, formed following a short haplophase in the unequal gametangia prior to fertilisation and nuclear fusion. The group includes necrotrophs, holobiotrophs and hemibiotrophs (see Table 3). The holobiotrophs and some of the hemibiotrophs form haustoria. Within the Kingdom FUNGI only the Chytridiomycetes produce zoospores. Here the fungal body is a microscopic rounded structure with chitin walls and rhizoids arising from its base. Thick walled resting spores are sometimes formed, often associated with sexual reproduction. The pathogenic members of the group are holobiotrophic, the fungal body being either embedded in the living host cell or attached to it by rhizoids. The three other groups of the FUNGI normally grow as branched hyphae, like the CHROMISTA, but all contain chitinous polymers as major structural components of the walls and are haploid for the greater part of their life cycles. The hyphae of the Zygomycota are normally aseptate and asexual spores, formed in a sporangium, and are non-motile. The sexual spores, the zygospores, are resting spores and are formed by the fusion of two gametangia, followed by nuclear fusion. Meiosis precedes germination. Plant pathogenic members of the group are extreme necrotrophs (Table 3). The hyphae of the Ascomycota are divided into ‘cells’ by septa, but these have a central pore through which nuclei and cytoplasm move quite freely. The naked asexual spores (conidia) are non-motile. There is

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immense diversity in the size, shape, form and manner of formation, deployment and dispersal of conidia. Sexual reproduction involves fusion of haploid nuclei in pairs, immediately followed by meiosis and then mitosis to produce a series of (usually) eight haploid ascospores. The products of each

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fusion/division are contained in a sac, the ascus. In most genera, the asci are protected by a complex fruit body, the ascocarp. The group includes necrotrophs, holobiotrophs and hemibiotrophs. Some of the holobiotrophs form haustoria. The hyphae of the Basidiomycota are septate, and although the septa may possess pores these are usually partially blocked by membranes, restricting the movement of the nuclei. The hyphae are, therefore, effectively divided up into cells. In most species each cell is a dikaryon, containing a complementary pair of haploid nuclei. Clamp connections ensure in some groups that following cell division, each daughter cell contains complementary nuclei. Asexual spores are not produced by all species, but when present, usually take the form of dikaryotic conidia. Sexual reproduction, fusion of complementary nuclei, followed by meiosis to restore the haploid state, occurs in specialised hyphal branches, the basidia. These each form a group of four, occasionally two, haploid uninucleate basidiospores, the product of one fusion/meiotic division. Following dispersal, the basidiospores germinate to form a haploid, monokaryotic mycelium. Hyphal anastomosis restores the dikaryotic state. Within the Basidiomycota, the Basidiomycetes form their basidiospores in structures (toadstools or brackets) composed of differentiated hyphae, which bear the basidia on gills, in pores or on teeth. The parasitic members of this group are mainly necrotrophic (see Table 3) and many are capable of degrading lignin as well as other plant cell wall polymers. There are two other large classes of the Basidiomycota, the Teliomycetes (Rusts) and the Ustomycetes (Smuts and Bunts). These do not form basidiocarps. Instead, teliospores or ustilospores germinate to form hypha-like basidia which bear the basidiospores. Many of the Teliomycetes have complex life cycles, with several spore stages that may alternate between two different hosts. The members of both groups are holobiotrophs (see Table 3), but only the Teliomycetes produce haustoria. The final group of the mycelial FUNGI, the ‘Deuteromycetes’ or mitosporic fungi, has no formal taxonomic status. The term is widely used, however, as a convenient way of referring to fungi that have either lost the ability to reproduce sexually or do so only rarely and cannot therefore be classified in the usual way. Most, but not all, are probably members of the Ascomycota and many are necrotrophic pathogens of plants. The detailed taxonomy of the plant pathogenic bacteria and fungi is dealt with by Hawksworth et al. (1995), Ellis and Ellis (1997), Holliday (1998) and Bradbury (1999), and more general accounts are provided by Agrios (1997) and Ingram and Robertson (1999). Nevertheless, it is clear from this brief survey that the niche of plant pathogenicity has been exploited by many diverse organisms and has evolved many times

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(Pirozynski and Hawks worth 1988). There is, in addition, further significant diversity apparent below the level of the species, as reflected in molecular analyses (Karp et al. 1997a,b, 1998) or as revealed by more conventional analyses of host ranges using genetically defined differential host species, subspecies and cultivars carrying different genes for resistance (Table 4), or of sensitivity to natural or synthetic bacteriocides and fungicides (Allen et al. 1999). Finally, the great taxonomic diversity of the pathogenic bacteria and fungi is reflected in the range of symptoms they cause in infected hosts (Table 5), itself a reflection of the spectrum of ecophysiological (Table 6) and trophic (Table 3) strategies that have resulted from the co-evolution of so many different pathogens with such a diversity of hosts. The diversity of pathogenic bacteria and fungi, especially at the infraspecific level, must be regarded as extremely fluid and subject to rapid changes in both space and time. This is an inevitable consequence of the often rapid generation times and dispersal rates of populations of microorganisms, linked with: high rates of mutation; long haplophases in many groups; the presence of extrachromosomal nucleic acid (plasmids etc), especially among the bacteria; and, in the fungi, complex mating systems including homothallism, secondary homothallism and heterothallism (often with several mating types); and sometimes also, in the fungi, the capacity for hyphal anastomosis with nuclear exchange and parasexual recombination (Fincham 1979; Agrios 1997; Caten 1996; Hartleb et al. 1997; Bradbury 1999). The capacity for interspecific hybridisation is now also known to be significant in pathogen variation (Brasier 2000a). The consequences of such plasticity are well known to plant breeders attempting to produce durable disease-resistant cultivars or to those attempting to produce effective, longlived, bacteriocides and fungicides (Wood and Lenné 1999). This plasticity is of great relevance to the success of plant pathogens in natural ecosystems. 3. The value of plant pathogens The wholesale destruction that is characteristic of plant disease epidemics in genetically uniform crop monocultures does not normally occur in natural ecosystems. This is in part due to the fact that the ecosystems themselves are usually made up of a great diversity of species, with genetically uniform stands being the exception rather than the rule. Moreover, although race specific resistance does occur in natural ecosystems (Heath 1991; Clarke 1997; Burdon 1997), epidemics do not normally follow its breakdown because stable equilibria normally exist between the host plant and pathogen populations (Prell and Day 2001). These equilibria are dependent upon the number of alleles for avirulence and virulence in the pathogen populations and for resistance and susceptibility in the host populations, and on the

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balance of advantage and disadvantage conferred by any new plant and pathogen genotypes. Such stable equilibria ensure the survival of both host and pathogen populations. Occasionally, however, massive epidemics of plant disease do occur in natural ecosystems, causing immense damage over wide areas. Recent dramatic examples include: Jarrah die-back of Eucalyptus and other native species in Australia and New Zealand, caused by Phytophthora cinnamomi (Newhook and Podger 1972); chestnut blight in North America, caused by Cryphonectria (Endothia) parasitica (Agrios 1997); Dutch elm disease in North America and Europe caused by Ophiostoma novo-ulmi (Brasier 2000b); and, more recently, certain forms of oak decline in central Europe caused by species of Phytophthora (Jung et al. 2000). The reasons for such epidemics are not always clear, but may include introduction of alien species and novel genetic forms, as in the case of chestnut blight and Dutch elm disease, or major genetic changes and interspecific hybridisation in the pathogen, as with Dutch elm disease, complicated by changes in environmental conditions, as with Jarrah die-back. In the future, climatic shifts may also be expected to trigger the breakdown of equilibria in host and pathogen p o p u l a t i o n s leading to f u r t h e r epidemics of and in other species. Even though ecosystem recovery may sometimes be possible following such catastrophes (e.g Weste and Kennedy 1997), this is a slow and erratic process and is by no means fully understood. The damage already caused to natural ecosystems by plant pathogens, the threat of further epidemics in the future and recognition that disease control in such situations is rarely if ever possible, must be of great concern to conservationists and must be taken into account in planning future conservation strategies. In this context, the importance of gaining a better understanding of the biology and genetics of plant pathogens in natural ecosystems cannot be over emphasised, but more of that below. But there is another side to the coin. It has already been stated that the concept of conserving p l a n t pathogen d i v e r s i t y is counter intuitive, and knowledge of catastrophic epidemics in natural ecosystems fuels such prejudice. It is important to explore therefore, the positive role of plant pathogens in natural ecosystems and their value in providing scientific, technological and economic benefits to human societies (Table 7).

3.1 The significance of plant pathogens in natural ecosystems Evidence from model ecosystems and natural grasslands has suggested that high biodiversity may lead to greater productivity and stability and, conversely, that reducing diversity lowers productivity (Naeem et al. 1994; Tilman et al. 1996). More recent research suggests, however, that the relationship between diversity, energy availability and productivity in

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natural ecosystems is far more complex than this simplistic statement implies (Naeem et al 2000; Morin 2000; Emmerson et al 2001). With research at such an early stage, it is impossible to assess the significance of plant pathogen diversity in this context. Any visual survey of a natural ecosystem, however, no matter how superficial, will reveal the presence of plant pathogens, often in abundance (Ingram and Robertson 1999). Such observations are confirmed by the many checklists of plant pathogens that have been produced for natural and

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semi-natural habitats, especially in Europe and North America (e.g. Blackwell et al. 1997). The mere presence of pathogens in a plant community, however, no matter how damaging or debilitating to their individual hosts, does not in itself constitute evidence of their contribution to ecosystem dynamics, productivity or stability. They may be nothing more than another level of biological complexity of only limited structural significance (Harper 1990). So, what, if any, is the evidence to the contrary? The significant role of parasitic fungi that establish mycorrhizal associations in natural ecosystems is now well documented (Simard et al. 1997; Helgason et al. 1998; Read 1998; Chapin and Ruess 2001). A recent study of forest diversity in the United States provides dramatic evidence to support the hypothesis that fungi that are pathogens are also of major significance (Packer and Clay 2000). Packer and Clay (2000) examined the problem of why some forests are more heterogeneous than others. In a black cherry forest near Bloomington, Indiana, USA, they observed that black cherry seedlings growing beneath mature black cherry trees died soon after germination, whereas seeds dispersed to some distance from the parent grew on to produce new trees. It could be argued that the cause of seedling mortality was overcrowding beneath the parent trees, but Packer and Clay showed a better correlation between distance from the parent and survival than between overcrowding and mortality. Next, in a series of pot experiments, they found strong evidence that the cause of death of seedlings close to their parent was a species of the soil­ borne pathogen Pythium, which they had previously isolated from dying seedlings. The seedlings of several tree species other than black cherry that were able to establish and grow where black cherry seedlings were killed were apparently resistant to the fungus. Thus the Pythium species seemed to play a vital role in promoting diversity in the cherry forest in a manner similar to that previously attributed to host specific herbivores by Janzen (1970) and Connell (1978). A flaw in this hypothesis is that Pythium spp., being necrotrophs, are usually regarded by plant pathologists as being non-specific pathogens with wide host ranges. However, although there is no doubt that hosts of such necrotrophic pathogens do not normally develop the highly specific genefor-gene resistance-avirulence systems characteristic of relationships involving holobiotrophic and hemibiotrophic pathogens (Agrios 1997; Prell and Day 2001), a measure of broad specificity at the level of the species or family is not at all unusual. Thus a Pythium species could well exhibit the level of specificity ascribed to it by Packer and Clay (2000). The phenomenon described by Packer and Clay (2000) in black cherry forests is very similar to a syndrome long familiar to horticulturists, called

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‘replant disease’ or ‘soil sickness’ (Pollock and Griffiths 1998) in which roses and fruit trees, when dug up, can only be replaced successfully by a young plant of a different species or family. The demise of replants of the same species is usually associated with the occurrence on the roots of lesions of Pythium sylvaticum. van der Putten (2000), in a commentary on Packer and Clay (2000), hypothesises that the existence of soil pathogens may even select for longdistance dispersal traits in their hosts. He cites in support of this hypothesis the work of Wennström (1994) who demonstrated that, in six vegetatively propagated plant species, sensitivity to pathogens correlated positively with the speed and extent of vegetative outgrowth. In further experiments conducted by D’Hertefeldt and van der Putten (1998), the sedge Car ex arenaria exhibited reduced branching and a switch to unidirectional growth of rhizomes when challenged by patches of soil-borne pathogens. The studies of Packer and Clay (2000), and van der Putten (2000), although excellent, are far from complete: the Pythium species pathogenic to black cherry has yet to be identified and its specificity needs to be defined more precisely; the role of the fungus in generating black cherry forest diversity has yet to be established unequivocally; and the interesting hypothesis that the evolution of long-distance dispersal is a response to a pathogen is far from proven. Moreover, although a similar process to that described for black cherry forests may explain species diversity in Douglas fir forests (Holah et al. 1997), the universality of the process is far from clear. It is interesting to speculate, however, that some necrotrophic plant pathogens at least may have a significant widespread role in promoting ecosystem diversity and thus, indirectly, ecosystem productivity and stability. This work of Packer and Clay serves to illustrate in a striking way the point that the role of plant pathogens in natural ecosystems may in the past have been overlooked or underestimated. Nevertheless, some exciting research has been carried out and more is now being done, as was evident from the contributions to the Seventh International Congress of Plant Pathology (Ingram 1999). An essential pre-requisite for such studies is a clear understanding of the distribution and epidemiology of the pathogens concerned. A major contribution to this field has been made by Burdon (1991, 1992, 1993, 1997), who has reviewed his own research, especially with wild populations of flax infected with the rust fungus Melampsora lini, and also the research of others with a great diversity of host and pathogen combinations. As a basis for analysis, Burdon (1993) classified pathogens of wild hosts according to their effect on host survival, fecundity and individual vigour (Table 8).

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The broad conclusion of Burdon (1993) is that the biology of a particular plant pathogen is affected by such a wide variety of factors, including its breeding system, survival strategies, host range and dispersal mechanisms, that the population dynamics and genetics of each pathogen species must be regarded as unique. He recognises, however, the need to be pragmatic and goes on to suggest that, despite the significant differences between pathogens, similarities in the ecological contexts in which they occur impose similar constraints. He lists these constraints as the size, spatial distribution and genetic structure of host populations, coupled with the effective methods of dispersal possessed by most pathogens, and suggests that they reinforce the need to consider pathogen demography and genetics in a metapopulation context. Burdon (1993) also points out that in

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any one deme, population levels rise and fall, often quite sharply, while the genetic structure can change rapidly and very significantly. Such populations rarely exist in isolation, however, and only by following the pattern of change over many demes within a particular area can a complete picture of the total population be determined. The development of methods for the detection of molecular and other markers is now greatly facilitating studies of pathogen epidemiology in natural and semi-natural ecosystems (Lenné et al. 1994; Karp et al. 1997a,b; Wood and Lenné 1999) and is already resulting in a widening and deepening of the knowledge reviewed by Burdon(1993). The complementary question of the interaction between disease and plant competition, especially in i n f l u e n c i n g the structure of plant communities, has recently been analysed in depth by, among several others, Dobson and Crawley (1994) and Alexander and Holt (1998). Dobson and Crawley conclude that there may be a direct influence of pathogens on the structure of plant communities in cases where pathogens reduce the population of adult and seedling plants, as in the extreme case of Jarrah die­ back, caused by Phytophthora cinnamomi in Eucalyptus spp. in Australia (Newhook and Podger 1972) or Dutch elm disease caused by Ophiostoma novo-ulmi on elms in Western Europe (Brasier 1986 and 2000b). The removal of a dominant tree species by a pathogen has in these cases led to forests and woodlands dominated by less competitive species from earlier stages of the succession or has opened the canopy, allowing colonisation by less competitive species. Less dramatic examples, in which the pathogen reduces the competitive ability of a plant in a succession include the studies of van der Putten and colleagues (van der Putten 2000) of the succession of grasses and sedges on foredunes. Dobson and Crawley (1994) conclude that soil-borne diseases may affect both the rate and direction of the succession of plants in specific ecosystems. It is hypothesised that pathogens may both slow down and speed up successions, depending on circumstances. They also cite examples of pathogens that affect the fecundity of their hosts, thereby influencing plant population dynamics, as in the case, for example, of Atkinsonella hypoxylon, which reduces flower production in the grass Danthonia spicata but increases growth rates and survival of infected ramets. Dobson and Crawley (1994) also draw attention to ways in which pathogens may influence seedling recruitment in tropical forests, thereby increasing species diversity, as in the example of the effect of Pythium spp. on the diversity of black cherry forests cited above. Finally, they outline how the effects of plant pathogens on animals exert indirect effects on plant communities through a reduction in grazing pressure. For example, some plant pathogens render their hosts toxic to herbivores, as with certain

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endophytes of grasses (Parbery 1996). These may in turn lead to reductions in grazing pressures, w i t h concomitant effects on the composition or structure of grassland ecosystems. In a comprehensive review, Alexander and Holt (1998) also assess the evidence in support of the hypothesis that plant disease may have a s i g n i f i c a n t effect on the c o m p e t i t i v e i n t e r a c t i o n between plants, with ecological or e v o l u t i o n a r y consequences. They cover some of the same ground as Dobson and Crawley (1994), but also review the more recent research. In addressing the effects of disease on intraspecific competition between host plants, they present a simple model that suggests that a variety of outcomes might be expected. From the evidence available it can be concluded that pathogens may have a large or small effect on intraspecific population dynamics, depending most notably on the density-dependent ability of healthy plants to compensate for loss of diseased individuals, the ability of non-infected leaves or shoots to compensate for infected ones on the same plant and the spatial patterns of infection. Next Alexander and Holt (1998) analyse the effects of disease on the competitive abilities of i n d i v i d u a l genotypes of the same species and thus on the genetic composition of populations. They conclude that such genetic processes feed back on population dynamics, assuming trade-offs between disease resistance and other fitness characters. Finally, they show that the effects of disease on the interspecific interaction between plants may have significant effects on the composition of communities. Host-specific pathogens such as holobiotrophs and hemibiotrophs, for example, may change a competitive hierarchy between a host and a non-host species, while relatively non­ specific, necrotrophic pathogens may i n d u c e indirect competitive interactions between host species. The evidence reviewed by Alexander and Holt (1998) is limited in the following ways: it derives m a i n l y from research with a limited range of pathogens, especially f u n g i ; most of the conclusions were based on laboratory rather than field studies; interactions were studied over relatively few generations; little evidence was available concerning the effects of density-dependent processes in both host and pathogen populations; and pathogen population dynamics were largely ignored. Despite the large number of empirical studies reviewed, Alexander and Holt (1998) are thus unable to conclude with any certainty that pathogens affect plant population dynamics, the genetic composition of host populations or plant community composition. Nevertheless, there is strong circumstantial evidence that they do affect all of these, although significantly more research is required to be certain. Finally, it should be noted that quite apart from any direct effects on plants in natural ecosystems, necrotrophic pathogens, especially those with

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the capacity to degrade cellulose (e.g. Pythium spp.) and lignin (e.g. Heterobasidion annosum) have a key, although as yet unquantified, role in inducing decay and ensuring the cycling of nutrients, especially carbon, in all natural ecosystems (Andrews 1991; Copley 2000; Naeem et al. 2000). 3.2. The economic and scientific value of plant pathogens The positive value of the pathogens of wild relatives of crop plants and in traditional agro-ecosystems is widely recognised. Most of the progenitors of crops have co-evolved with their pathogens in the centres of origin and diversity - the Middle East in the case of cereals, for example (Heath 1987, 1991; Dinoor and Eshed 1990; Frank 1993; Smartt and Simmonds 1995; Wood and Lenné 1999). This has thrown up a great diversity of disease resistance factors in the hosts and these have been a major resource for the farmer and plant breeder in producing new disease-resistant cultivars (Lenné and Wood 1991; Burden 1997; Wood and Lenné 1997, 1999; ten Kate and Laird 1999). Both existing and newly evolving ‘wild’ sources of resistance (and recent evidence suggests that new plant disease resistance factors may arise relatively rapidly and frequently) are likely to be required long into the future for the production of novel disease-resistant crops (Lenné and Wood 1991; Hawtin 1996; Wood and Lenné 1997; Allen et al. 1999; ten Kate and Laird 1999). Other potential economic benefits are numerous. Genetically defined collections of plant pathogens are essential to the process of revealing diversity and in selection for disease resistance in plant breeding. The great range of, for example, cereal cultivars resistant to rust and powdery mildew diseases is testimony to this (Allen et al. 1999). Pathogens may also be important as sources of novel drugs. Ergot alkaloids and other compounds derived from Claviceps purpurea have been widely used in medicine for many years, and ergotamine is currently of great significance in the treatment of migraine. A wide-range of antibiotics has been obtained from both bacteria and fungi, some of them pathogenic on plants (ten Kate and Laird 1999). Other pathogen-produced or pathogen-induced compounds of pharmaceutical significance are likely to be revealed as the search for novel compounds from plants and microorganisms intensifies (ten Kate and Laird 1999). Similarly, plant pathogens or infected hosts may be good sources of novel fungicides, pesticides and herbicides. For example, a protein derived from the necrotrophic pathogen Fusarium oxysporum has recently been shown to have great potential as a herbicide for broad-leaved weeds (Jennings et al. 2000). Moreover, spores of Ascochyta caulina, a pathogen of fat hen (Chenopodium sp.), have been shown to have excellent potential as organic weed-killers for this pernicious weed (W. Seel, University of Aberdeen, UK, pers. comm.). F i n a l l y , plant pathogens may produce or

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cause their hosts to produce chemical molecules which, although not of direct significance to the pharmaceutical or agrochemical industries, may be of indirect importance in producing chemical templates for novel bio-active molecules (ten Kate and Laird 1999). Plant pathogens have also long been of significance to the food and drinks industry. For example, Botrytis cinerea, the widespread cause of grey mould of m o r i b u n d f r u i t s , flowers and leaves, as a pathogen of lateharvested grapes imparts both sweetness and flavour to the resulting dessert wines. Maize infected by the smut fungus Ustilago maydis is considered a delicacy if eaten before the black ustilospores form. The potential for future use of plant pathogens in food production is, however, probably limited. In contrast, the role of plant pathogens as models for scientific research leading to technological innovation cannot be overestimated. Two examples w i l l suffice to make this point. Plants of rice infected by Gibberella fujikuroi, the cause of bakanae or ‘foolish seedling’ disease, are taller, paler green and more spindly than uninfected individuals. Study of the causes of these symptoms, overproduction by the fungus of gibberellic acid, led to the discovery of a major group of p l a n t hormones, the gibberellins. This discovery was not only of great scientific significance but was also of great technological importance, for gibberellins are now widely used in the brewing industry for the synchronisation of barley malting and in fruit production to induce parthenocarpic ripening. Secondly, studies of the way in which Agrobacterium tumefaciens causes crown gall tumours in infected hosts led to pioneering experiments in genetic modification of plants and ultimately to the widespread application of this technology in the production of GM crops. There is little doubt that in the future, scientific studies of plant disease organisms will continue to lead to major scientific and technological advances, either by chance, as in the above two examples, or by design (ten Kate and Laird 1999). Finally, it is important to end this section by making a point that is so obvious that it is all too easily forgotten or overlooked. For as long as there is a need to control the devastating effects of pathogens on plants, there is an equal need to study in depth the ways in which pathogens interact with their hosts and with their environment at every level and the way that epidemics occur and develop, especially in natural and semi-natural ecosystems. This point is particularly well exemplified by the works of Burdon (1997), Clarke (1996), Heath (1987, 1991), Thompson and Burdon (1992), Rausher (2001) and Stuiver and Custers (2001). Only as a result of such research will it be possible to continue to develop effective strategies for the control of diseases into the future. For this reason alone, if for no other, it is essential that the diversity of plant pathogens is conserved in appropriate ways.

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4. Threats to plant pathogen diversity But is plant pathogen diversity threatened? The stark conclusion of the recently published IUCN Red List of Threatened Plants (Walter and Gillett 1998) is that some 34,000 species of plants, representing 12.5% of the world’s flora, face extinction. The list includes wild relatives of almost every major crop and forestry genus or group. Species, of course, are only one component of the totality of plant diversity. The IUCN Red List, however, is an indication that erosion is probably occurring at the genetic, population and ecosystem levels too, although such erosion is not always easy to measure (Groombridge 1992; Day 1993; Watson et al. 1995). The rate of this loss of plant diversity is equally difficult to estimate, but considerable work has been done on rainforests. Evidence suggests that from the rainforests alone 27,000 species of all organisms are being lost per annum (Wilson 1993). This is roughly equivalent to one thousand times the natural rate of extinction of species and is also equivalent to the rate of some of the great extinctions of the geological past. Other estimates of the current rate of extinction of organisms vary widely (e.g. Groombridge 1992; Myers 1993; Myers et al. 2000; Pimm and Raven 2000), but there is general agreement that it is dangerously high. Plant pathogen diversity is, of course, entirely dependent upon the diversity of host plants. More importantly, natural ecosystems and traditional agro-ecosystems are being destroyed at an unprecedented and dangerous rate, including those in the centres of origin and diversity of major crops (e.g. Wilson 1993; Myers et al. 2000), and with them, no doubt, the associated pathogens. Such losses are likely to include both species and, perhaps more significantly, infra-specific diversity for characters such as virulence and avirulence (Allen et al. 1999). However, all this is conjecture and the need for further and better information on the extent and erosion of pathogen diversity in natural ecosystems and traditional agro-ecosystems, and the potential impact of climate change, cannot be over emphasised. Finally, plant pathologists and crop producers are, quite properly, dedicated to the elimination of plant pathogen diversity, for it is a serious threat to production (Agrios 1997). Even though plant pathogens are probably sufficiently plastic, genetically, to adapt to new bacteriocides, fungicides and resistance genes (Fincham et al. 1979) the resulting year on year erosion of pathogen diversity is likely to be significant, although no data are currently available to substantiate this statement (Day 1993). Moreover, the ever widening use of industrial farming and crop production methods is itself likely to be a threat to pathogen diversity in adjacent natural and semi natural ecosystems as well as on agricultural land itself.

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5. Conservation of plant pathogen diversity If plant pathogen diversity has potential value, ecologically, economically and scientifically, and if this diversity is threatened, it is necessary to consider what strategies might be available to ensure its conservation.

5.1. Ex situ conservation One strategy for the conservation of plant pathogenic bacteria and fungi is ex situ in culture collections or spore and gene banks (Hawksworth 1991; Smith and Waller 1992; Sugawara et al. 1993; Anon. 1994b; Suihko 1995; Kirsop 1996; Smith and Ryan 2001). This is a most effective approach, and there are many important collections and banks around the world, their activities in part co-ordinated by the World Federation for Culture Collections (Kirsop and Hawksworth 1994; see also Gams, this volume). Ex situ strategies, however, are not devoid of problems. Firstly, at a time when the emphasis is on short term funding of scientific endeavours and the commercialisation of science is widespread, culture collections and spore and gene banks, which require secure long term funding, must be at risk (e.g. Anon 1994a; Sly 1998). Secondly, plant pathogens are currently poorly represented in most culture collections, which hold no more than 10% of the species so far identified, while infra-specific diversity is largely ignored. In this context, high priority should be given in the future to the conservation of the working collections of individual plant pathologists and plant breeders. These are usually invaluable and are often irreplaceable, yet at present are frequently at risk in financially pressed institutions. T h i r d l y , although methods for the long-term storage and management of microbial cultures, spores and nucleic acids are extensive (Hawksworth 1990; Smith 1997), further development is still required (Day 1993), especially to ensure genetic stability. Fourthly, the ownership of material held in culture collections and spore and gene banks is frequently far from clear (Hawtin 1996; ten Kate and Laird 1999). And finally, since culture collections and banks facilitate the international movement of organisms for study, there is the ever present risk of accidental release of a pathogen far from its place of collection, with potentially devastating consequences. This last point is emphasised by, for example, the widespread destruction of elms in Europe and America f o l l o w i n g the accidental international transport of Ceratocystis species (Brasier 1986). The work of Newcombe et al. (2000), Brasier (2000a,b) and Brasier and Kirk (2001) has further emphasised the risks associated with such events, suggesting that inter-specific hybridisation between related native and introduced species of pathogens may produce a ‘devastating array’ of novel phenotypes. It is recognised, of course, that these ‘escapes’ were not from culture collections or spore banks, but they serve as reminders of the need

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for care. Despite these caveats, culture collections and gene and spore banks probably represent the most effective and certainly the most secure way of conserving plant pathogen diversity. However, being held in suspended animation, isolated from their hosts, plant pathogens conserved ex situ have no opportunity to co-evolve with their hosts and generate yet further diversity.

5.2. In situ conservation The controversial alternative to ex situ conservation is to conserve plant pathogens in situ, together with their hosts (Ingram 1998a, 1998b, 1999). This has the obvious advantage of allowing the evolution of diversity to continue unchecked, and might therefore be regarded as more natural than ex situ conservation. At its simplest, in situ conservation of pathogens merely means taking account of pathogen biology when devising conservation strategies for particular ecosystems (Helfer 1993; Burdon 1998). Because pathogen populations may be unevenly distributed in host populations, especially at the infra-specific level (Burdon 1993, 1998), however, great care must be taken to ensure that host populations chosen for conservation include all known components of pathogen diversity. And herein lies a significant difficulty. It has already been emphasised that knowledge of the general biology and epidemiology of pathogens of wild plants is, with a few notable exceptions, very limited (see Helfer (1993), for example, regarding rusts on rare plants in the UK). Thus if the conservation of plant pathogen diversity in natural ecosystems is to be taken seriously, a major drive to increase knowledge of their biology is urgently required. A danger with this simple in situ approach is that certain pathogens, especially those that are host specific, such as the rusts and smuts, may pose a very real threat to vulnerable populations of an endangered host species (Helfer 1993). In these circumstances, the conservationist is faced with the dilemma of whether to concentrate on conservation of the host at the expense of the pathogen, or vice versa, or whether to attempt to manage host and pathogen populations together, thereby running the risk of losing both. The pragmatic solution, of course, would be to conserve the pathogen in a spore or gene bank and the host both in situ and in a seed bank. A more complex dilemma concerns the proposition that the pathogens of the wild relatives of crop plants might be conserved in situ, in the centres of origin and diversity of those crops. This idea was first proposed for the wild relatives of cereals by Browning (1974), who also suggested the name ‘living gene parks’ for such areas of conservation. Dinoor and Eshed (1990) returned to the idea nearly twenty years later and coined the alternative name ‘genetic reserve’. At first sight such an idea is seductive. The reserves

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could provide a pool of actively evolving host populations, continually challenged by evolving populations of key pathogens. There might therefore be the continued generation and selection of novel genes, thus ensuring an ongoing supply of raw material for plant breeders (Wood and Lenné 1999). The idea merits further consideration (Ingram 1999), but it is important to emphasise certain caveats. Firstly, despite intensive study of the pathogens of major crops, we still need to know far more of the detailed biology, epidemiology and population genetics of populations of plant pathogens and their hosts in agro-ecosystems and in natural ecosystems (see Burdon 1993; Wood and Lenné 1997, 1999) before we shall have sufficient knowledge to manage such reserves effectively. Secondly and more importantly, as Lenné (Hall 1998) and Wood and Lenné (1999) have emphasised, the risks to adjacent crops are very considerable. The centres of origin of most major crops are regions of the world that although rich in biodiversity are, to a large extent, extremely poor in resources (Myers et al. 2000). Thus the presence of a reservoir of pathogen inoculum close to cultivated crops could have serious economic consequences, and the escape of a new, virulent line or strain of a pathogen could create a disaster. The spread of rust from wild to cultivated wheat in India and Pakistan (Joshi 1986) is a salutary reminder of the need for extreme caution. 6. Summary and conclusions The diversity of plant pathogenic bacteria and fungi is immense, at every taxonomic level. To conserve such diversity is, however, counter intuitive to most plant pathologists and conservationists because of the great economic damage caused by plant diseases of crop plants and the dangers pathogens pose to both common and endangered species in natural ecosystems. Nevertheless, it is suggested that plant pathogen diversity may be of positive value as a component of natural ecosystems, although the evidence to support this suggestion is incomplete. Moreover, plant pathogens have been, and will probably continue to be, of significant economic value as sources of novel drugs and agrochemicals. Pathogen diversity is also essential to the plant breeder in selecting for novel disease resistance factors both in wild populations of host plants and in plant breeding trials. And finally, plant pathogens provide model systems for scientific research leading to biotechnological innovations. In view of all this, it is essential that greater weight is given in the future to understanding the basic biology, genetics and epidemiology of pathogens in natural ecosystems than is currently the case. Moreover, the importance of plant pathogen diversity and the threats pathogens impose must be considered in planning conservation strategies. The threats to pathogen diversity are significant, being related to the threats to host plant diversity, intensified by the agricultural tradition of

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destroying plant pathogens whenever possible. Given the desirability of conserving plant pathogen diversity (by no means universally accepted), both ex situ and in situ approaches are of potential value, but pose the everpresent threat of escape, with potentially devastating consequences. Ex situ conservation strategies pose fewer risks than in situ strategies, but are less effective for conserving a broad spectrum of diversity and suffer from the serious disadvantage of not allowing continuing host-pathogen co-evolution. A major d i f f i c u l t y faced by all those concerned with the role of pathogens in natural ecosystems and the conservation of plant pathogen diversity is that with the exception of a small number of pathogens of major crop plants, research on the biology, genetics and epidemiology of plant pathogens has so far been totally inadequate.

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Chapter 10 EX SITU CONSERVATION OF MICROBIAL DIVERSITY

Walter Gams Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD, Utrecht, Netherlands.

1. Introduction No organisms lend themselves better to long-term ex-situ preservation than microorganisms (Kirsop and Kurtzman 1988; Hawksworth and Kirsop 1988; Samson et al. 1996b). Microbial culture collections preserve large numbers of pure cultures, viz. strains of bacteria, fungi and algae, over long periods. The strains are maintained either in an active condition on agar media with regular transfers, or a n a b i o t i c a l l y under conditions that assure as little alteration as possible over decades. Provided no mutation or contamination occurs, a strain retains its identity permanently. The scope of microbial culture collections is not comparable to that of botanical or zoological gardens which are mainly concerned with the conservation of large components of the earth’s biodiversity that elsewhere may be threatened in situ. Preserved microorganisms mainly serve as reference material or standards in comparative research. Because of my personal limitations, most of the examples used in this chapter are drawn from mycology and fungal collections. Collections of l i v i n g cultures are complementary to the indispensable documentation preserved in herbaria and both must be linked as far as possible. Certain forms of sporulation are only seen in nature and rarely in culture; it is crucial to preserve dried specimens of these conjointly with the culture (Agerer et al. 2000). After collecting a fungus in nature, “the availability of cultures also can mean that other than just naming a specimen, you are naming a species in the true sense of the word. Your species concept can now be tested at various levels, and the isolate can also add data to address many other

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exciting questions” (Crous 1999). Mycologists studying fungi in pure culture have a great advantage over those working with fungi in situ only. Factors determining morphology and development can be analysed and conditions for growth optimised, concomitantly yielding basic ecological information. The correlation of different k i n d s of sporulation as components of one fungus (particularly in establishing anamorph–teleomorph connections) is mostly accomplished through study of isolates obtained from sexual spores (mostly ascospores) in culture. In nearly all disciplines in which micro­ organisms are involved, well-preserved pure cultures are indispensable. Thanks to lyophilization and cryo-techniques, culture collections are now in the position to preserve large numbers of axenic (or monoxenic) fungal isolates with a m i n i m u m of alteration. The initial handling of the material is labour-intensive but, once in an inactive state, the material is durable for decades. With older techniques, particularly those requiring serial transfers, degeneration of cultures is a regularly occurring problem. Loss of sporulation, loss of enzyme activities and, particularly, loss of pathogenicity were disadvantages of old collection strains. To circumvent this, special techniques for particular groups of fungi were often successfully used, such as a soil tube method (Schneider 1958) particularly suitable for Fusarium, and preservation of fungal fragments in distilled water (Castellani 1967; Boesewinkel 1976; Ellis 1979). The more permanent techniques have to a large extent displaced these early improvements. Even with modern technologies, some fungal groups, such as mycorrhizal fungi, Oomycetes, and particularly some large-spored fungi, e.g. nematophagous orbiliaceous anamorphs, s t i l l cause problems; the more hypha-like the spores are the more d i f f i c u l t is their preservation (Tan et al. 1994, 1998). With modifications of the cryoprotectant or freezing regime such problems can partly be overcome (Tan 1997; Tan et al. 1994, 1998).

2. Culture collections There are some 480 registered microbial culture collections (Sugawara et al. 1993; Sugawara and Ma 1995). Professional culture collections have a fulltime staff whose primary duty consists of the receipt, verification, preservation, documentation, maintenance and shipping of cultures for s c i e n t i f i c and commercial purposes on a d a i l y basis. Large service collections, who make their catalogues accessible, include the American Type Culture Collection (ATCC), the Belgian Coordinated Collections of Microorganisms in L o u v a i n - l a - N e u v e , Brussels and Gent (BCCM), Centraalbureau voor Schimmelcultures in Utrecht, Netherlands (CBS), the Czech Collection of Microorganisms, Brno (CCM), Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSM), the Institute for Fermentation, Osaka, Japan (IFO), CABI Bioscience,

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Egham, UK (IMI), the Japan Collection of Microorganisms RIKEN, Tokyo (JCM), and the All-Russian Collection of Microorganisms, Pushchino (VKM). 3. Methods for preservation An ample literature describes methods used in culture collections. For the permanent preservation of microbial strains several techniques are available, most of which are summarised by Smith (1988) and Kirsop and Doyle (1991). Lyophilization (freeze-drying) is mainly available for sporulating cultures, while deep-freezing lends itself also for many other fungi. Details of freezing procedures as used in CBS are described by Tan et al. (1994). 4. What is preserved in culture collections? Normally, representative (if possible ex-type and some other) strains of each species should be preserved in one or more culture collections. Also, any subdivision of species down to formae speciales, anastomosis or vegetative compatibility groups and isozyme groups must be represented with reference strains. Principally, it is desirable to maintain strains of all culturable species as pure or at least monoxenic cultures (for obligate mycoparasites) in collections, whether they play beneficial (see below) or deleterious roles to humans, or are neutral. The whole microbial diversity on earth should be documented in this way, though it is still quite incompletely explored, particularly in tropical countries (Hawksworth 199la; Hawksworth and Mound 1991; Subramanian 1992; Hyde 1997). But also in Australia the exploration of the fungal flora is only in an initial phase (Fungi of Australia 1996). The exploration of unusual niches, microhabitats or substrata will undoubtedly yield numerous new species (Subramanian 1992). Deposition of strains of new taxa is in the interest of every taxonomically active mycologist. Culture collections try to acquire strains of all new species appearing in the literature if they are not supplied by the authors spontaneously. Moreover, taxonomists working at culture collections contribute to the enrichment of the collection by their own collecting activities. It is profitable to concentrate search activities in centres of the origin of certain fungal groups, particularly pathogens. To the benefit of scientific work, a free exchange of cultures between countries is highly desirable, with due regard to all necessary safety regulations applying to pathogenic organisms. The countries of origin of strains, particularly when developing countries are involved, should share in revenues obtained by industrial applications as recommended by the Convention on biological diversity, Rio de Janeiro (1992). But this requirement should not prevent free exchange of cultures for research between countries, even though the procedure is not yet sufficiently regulated.

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For bacteria it has been estimated that the approx. 4000 species described represent a m i n o r i t y (estimated to be 0.1%) of the predicted diversity and that the majority may be unculturable with current methods (Stackebrandt 1996; Ward et al. 1995; Hammond 1995). For fungi, less than 100,000 species are now described, which also represents only a small proportion of the total diversity; but estimates of about 5% (Hawksworth 199la; Hyde and Hawksworth 1997) are probably too low. The oldest and most commonly preserved fungal strains in culture collections are those of saprotrophic genera of Mucorales and many ascomycetes and their anamorphs (Penicillium, Aspergillus, Fusarium, Trichoderma, Cladosporium, etc., etc.), isolated from soil, food, dung and various plant substrata. These are the ecological niches that are richest in culturable fungal species. Numerous fungi cannot be cultured, among which are the insectcommensal Laboulbeniomycetes, the Pneumocystidales, many ectomycor­ rhizal fungi, the zygomycete order Glomales comprising arbuscular symbionts of the majority of green land plants, and many biotrophic plantparasites. These fungi defy axenic culture, but many members of the last two groups can be propagated together with a host plant and then their propagules can be stored, though not quite axenically. A special culture collection has been established for the Glomales (Morton et al. 1993). Destructive, necrotrophic plant parasites can usually be cultured readily, though many tend to remain sterile (non-reproductive) in culture. Most biotrophic plant parasites cannot be grown axenically and are not preserved over long periods in culture collections. As an exception, many Ustilaginales are cultured and preserved axenically; but Uredinales, which can grow axenically for limited periods, are not kept in culture collections. Several projects are on-going for the permanent preservation of spore material of non-culturable plant parasites (e.g. Uredinales) encapsulated in alginate beads that allow infection experiments to be carried out later. All human-pathogenic species are preserved in culture collections except for Pneumocystis, Rhinosporidium and Lacazia (de Hoog et al. 1992). The preservation of any new additions to this diversity is highly desirable in order to ascertain their identity (de Hoog and Guého 1985). Among the basidiomycetes, wood-decomposing species in particular are readily cultured and well represented in culture collections. Only a fraction of ectomycorrhizal species can be cultured and they often grow extremely slowly; orchid mycorrhizal partners mostly are more easily grown in vitro. As these fungi normally remain sterile in culture, they are amenable only to deep-freezing, not lyophilization. Zoosporic fungi, which depend on free water for zoospore formation, are difficult to maintain over prolonged periods and at least require frequent

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transfers unless they are deep-frozen. Plant-pathogenic members of the Oomycetes (Chromista, unrelated to true fungi), such as members of Pythium and Phytophthora, grow well on agar media but also require rather frequent transfers. Using cryo-techniques, about 50–70% of the cultures can be successfully preserved. Another ecologically relevant group of aquatic fungi is the Ingoldian fungi (aquatic hyphomycetes), which can be cultured but tend to remain sterile in culture unless they are aerated in water (Bärlocher 1992). Extremophilic organisms can often be cultured if special conditions, imitating those of their natural habitat, are applied. They can be of interest, particularly as producers of enzymes under very low or high temperatures (Horikoshi and Grant 1998).

5. Criteria for selecting material to be conserved Culture collections can generally be divided into service collections that preserve a wide spectrum of taxa (determined according to relevance to society and potential needs of customers), and more or less specialised research collections that, at least for limited periods, preserve larger numbers of strains of particular groups for the sake of research projects. General culture collections must have a wider scope for preserving at least single representatives of all cultured species and, particularly, strains that have been extensively characterised with any advanced method. Staff and space are limiting factors in culture collections and each strain preserved costs money. Equivalent amounts of money are generally not recovered by revenue generated from the sale of limited numbers of cultures - culture collections therefore strongly depend on state support. Still, the diversity of cultures preserved can only increase if culture collections do not charge for preserving offered strains (unless commercial interests are involved). Also the free delivery of cultures on an exchange basis is an important incentive for the enrichment of the culture collection. But culture collections must determine a policy in deciding what strains can be preserved. While it would be ideal that all critically examined isolates were preserved in at least one culture collection, it is essential that a selection of representative isolates is permanently maintained and made accessible, together with voucher material of the fungus as it occurs on the natural substratum. Full documentation of the origin is essential (Agerer et al. 2000). Particularly in the case of genetically variable and poorly defined taxa, only investigation of large numbers of isolates allows an assessment of the population-genetic structure.

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6. The importance of living cultures for taxonomy Living cultures are of prime importance as sources of documentation for determination of the identity of an organism. Bacterial species are typified by living cultures (Lapage et al. 1992). For fungi, cultures are not considered sufficiently stable to serve as nomenclatural types. Only in 1999 was it recognised that permanently preserved, inactive fungal material can also serve as type of a new species. But when part of the permanently preserved freeze-dried or deep-frozen material from ampoules is reactivated, this automatically becomes ‘ex-type’ (Art. 8.4 of the International Code of Botanical Nomenclature, Greuter et al. 2000). The taxonomy of genera like Penicillium, Aspergillus, Fusarium, Verticillium, Acremonium, Phialophora and many others is entirely culturebased. Taxonomy and nomenclature themselves, however, are formally based entirely on immutably preserved (i.e. dried or at least inactive) type material. Ex-type cultures are generally much more relevant as reference material than the corresponding dried holotype specimens. Reference cultures are needed in all kinds of investigations; this may be official ex-type strains or other, well-developed representative strains. To build a stable taxonomy of critical groups a ‘polyphasic approach’ is increasingly adopted: numerous isolates preserved of the same or related taxa from different origin are analysed in detail with many available methods to ascertain species limits. In many groups of plant parasites and saprotrophs which can be grown axenically, cultural work has hitherto been neglected. Culture collections are the most qualified institutions to uphold a high taxonomic standard and to offer identification services. Though databases and other identification software is becoming available at an increasing rate on the Internet and elsewhere, they remain complementary to the human component of the identification process. 7. The use of preserved pure cultures Besides taxonomy, many disciplines benefit greatly from cultured microorganisms, as exemplified here. 7.1. Teaching Many features of microorganisms are observed best in pure cultures. The morphology of many fungi is m a i n l y studied in cultures. The optimal stage of a culture to be used in classroom demonstration can usually be well planned (Emerson 1958). A course on taxonomic mycology, mainly based on culture work, is given annually at CBS (Gams et al. 1998).

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7.2. Genetics Conventional genetics with fungi generally is done with pure cultures. Critical comparison and mating of single-spore isolates of many basidiomycetes contribute to the understanding of species structures, the dynamics of genetic segregation and speciation (Burnett 1983; Petersen 1997; Petersen and Hughes 1999). Comparative analysis of DNA sequences allows the reconstruction of phylogeny. For DNA extraction and molecular work, cultures are the material of choice to provide reproducible results. From a combination of phylogenetic relationships and the geographic origin of related taxa, phylogeographic trends can be inferred (O’Donnell et al. 1998). In some genera, stable, well-delimited species of great evolutionary age can be recognised, in others the process of diversification seems in full progress (de Hoog and McGinnis 1987). This can be seen in plant pathogens such as the causal agents of Dutch elm disease, where new species, in this case Ophiostoma novo-ulmi, develop under the eye of the investigator (Brasier 1991). Population genetics becomes relevant in all kinds of fungal groups. Molecular comparisons among numerous isolates obtained for one taxon allow important conclusions about the genetic structure of this taxon. Thus the development of, e.g. pathogenic populations of a fungus, can be traced. To render the work reproducible, all these disciplines require stable and well-documented collection strains as a basis for comparison. 7.3. Physiology and ecology Nutritional requirements of fungi are mostly determined from growth under in vitro conditions which rely on pure cultures. A wide and complex array of secondary metabolites has also become known from investigations of in vitro cultures. Conditions leading to their production and the roles of other ecological factors are often determined first in vitro. However, the results of such studies require careful consideration and detailed experimentation before relating in vitro requirements to field conditions. 7.4. Biotechnology Production of microbial metabolites has gained tremendous importance in the pharmaceutical industry (e.g. antibiotics, immuno-suppressants). Various microbial fermentations of food and beverages are performed from with pure culture sources (Beuchat 1987; Samson et al. 1996a). A considerable variety of edible macromycete species is cultivated all over the world, for which standardised strains are available (Stamets and Chilton 1993). On the other hand, some fungi are deleterious spoiling agents of foods and are often toxinogenic (Samson et al. 1996a). Microbially produced enzymes find their application in many unexpected areas of human life. The identity of such strains must be fixed by means of stable cultures; moreover, patented

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processes in which certain microorganisms are involved, require a stable deposit of these cultures. The industrial requirements that need to be met by culture collections have been summarised by Gürtler and Sasa (1996). 7.5. Bioremediation Noxious chemicals can be efficiently inactivated by certain microorganisms. As an example, certain white-rot basidiomycetes are able to detoxify polyaromatic hydrocarbons (May et al. 1997; Boyle et al. 1998; Martens and Zadrazil 1998). 7.6. Medically relevant isolates It is important to preserve isolates of organisms found to be causal agents of disease in view of further comparative studies (de Hoog and Guého 1985). Isolates with well-preserved virulence are necessary for in vitro and animal model studies of new drugs, disease mechanisms, natural immunological as well as vaccine responses and immunodiagnostic or molecular reagent preparation. There is also a high demand for standardised quality-control isolates to be used in medical laboratory procedures as well as dedicated training for staff involved in testing strains. Certain beneficial fungi produce known metabolites, mainly antibiotics (see under Biotechnology), while others have various other beneficial actions, e.g. anti-tumor activity (Stametz and Chilton 1993). The importance of preservation of such strains has been emphasised by Subramanian (1992). 7.7. Plant pathology Plant pathological investigations rely on inoculation studies which, among others, are important for determination of pathogenesis, testing of Koch’s postulates, estimation of resistance and susceptibility of host plants and testing for control measures such as biocidal chemicals. Highly qualified tester strains need stable preservation. Then a constant inoculum can be produced at any moment. 7.8. Biological control Bacteria and fungi are gaining importance for controlling insects, nematodes, pathogenic and spoilage fungi, and weeds. Seed coatings which incorporate bacteria or fungi can serve to promote growth and inhibit seedling colonization with pathogens (McQuilken et al. 1990; Callan et al. 1991; Jensen et al. 2000). 7.9. Symbioses Experiments of the physiology of ectomycorrhiza have always been carried out with artificially established 2-partner symbioses. Plants are sometimes

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artificially inoculated with their cultured symbionts in order to improve growth (Jeffries and Dodd 1991). Inoculation of nursery plants with ectomycorrhiza is of great practical importance in reafforestation. The quality of ectomycorrhiza in forest trees can be crucial for reafforestation in problematic areas, e.g. high-alpine regions (Moser 1958). Mycorrhizal syntheses are now practised on industrial scales using cultured mycelium of the most suitable mycorrhizal fungus, for example in alginate encapsulation of mycorrhiza for Eucalyptus plantations in Australia (Thomson et al. 1993; Hardy and Sivasithamparam, this volume); this practice may increase tree growth by a factor of two. In vitro inoculum production from cultures is thus becoming very important for the efficient propagation of mycorrhizal symbionts. Synthesis w i t h arbuscular mycorrhizal fungi can become important in soils with low phosphorus content, particularly if the natural microflora has been reduced by chemical treatments (Jeffries and Dodd 1991; Strullu et al. 1991). Inoculation with symbiotic actinomycetes of the genus Frankia has made it possible to introduce Alnus in a newly reclaimed polder in the Netherlands, where the tree was able to accumulate nitrogen to the benefit of subsequent tree species (van Dijk 1979; Houwers and Akkermans 1981). Orchid symbionts show a wider diversity in Australia than in many other countries and they are becoming important for the artificial propagation of endangered species (Batty et al., this volume). In the case of the slow-growing autotrophic lichen symbioses, culture studies have not resulted in substantial collections of mycobionts. The slow-growing and often sterile fungal partners of lichens are, however, becoming relevant as producers of valuable metabolites (Stocker-Wörgötter 1995; Crittenden et al. 1995).

8. Culture collections and the conservation of biological diversity In any part of the world, the numbers of fungal species are usually higher than those of green plants (Hawksworth 1991a). But the fungal diversity is less localised than that of green plants. Therefore local relationships between the ratio of green plants to fungi, ranging between 1:3 and 1:6 for the UK (Hawksworth 1991a), probably do not hold at a world-wide scale. Of the presently described fungal species (less than 100,000), only about 13,000 are available in culture collections (Sugawara et al. 1993; CBS 2002; Rossman 1997). Much still remains to be explored, particularly in tropical countries (Hyde 1997), and in the diverse range of ecological niches (Hyde and Hawksworth 1997). In all ecosystems, fungi play many decisive roles (Hawksworth 1992; Carroll and Wicklow 1992; Allsopp et al. 1995). They form a major component of the earth’s biodiversity. A rich microbial diversity is an important component of sustainable agriculture (Hawksworth 1991b;

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Hawksworth and Mound 1991; Edwards et al. 1993; Brussaard and FerreraCerrato 1997). In the interest of m a i n t a i n i n g biological diversity and avoiding collateral damage, pathogens and pests that threaten crops, species should be managed to reduce levels below the critical threshold of damage, while m i n i m i s i n g disturbance to other competing organisms and the dynamic biological e q u i l i b r i u m (see Ingram, this volume). In these efforts, integrated control and particularly, biological control, play a crucial role. The application of f u n g i as control agents has been successfully employed against insect pests (Ferron 1978; Clarkson and Charnley 1996), nematodes (Siddiqui and Mahmood 1996), fungi (Vakili 1984; Jeffries and Young 1994; Lynch 1996) and weeds (Pemberton and Hoover 1980; Hasan and Ayres 1990; Evans 1995). Many virulent strains are already present in culture collections, but the spectrum of species with potential benefits in pest control is still to be exhaustively investigated. Red lists of endangered species of fungi have been drawn up for macromycetes in many countries, but no such approach has been undertaken for micromycetes. Nevertheless, there are a few examples. Species of the mycoparasitic genus N y c t a l i s have declined in the Netherlands in recent decades. A microscopic hyperparasite, Pyxidiophora asterophorae (Tul.) Lindau, described as growing on Nyctalis, has not been found in recent decades. Culture collections of such a species therefore play a vital role in the conservation of fungal diversity. There are many cases of single records of a particular fungus where the only known extant material is in a culture collection. Normally, such observations cannot be taken as proof of rarity, but there are exceptions, such as the conspicuous species of the hyphomycete genus Pleurocatena, which through extensive searching of probable habitats are now considered rare and of which two species are now available in culture. Most Red-list macromycetes are ectomycorrhizal fungi (Arnolds 1991, 1992). For example, commercial production of truffles (e.g., Pirazzi et al. 1990; Jeffries and Dodd 1991) is possible from artificial inoculation of associated tree species. The role of microorganisms in the restoration of degraded or altered landscapes is an emerging field of research and a number of recent studies have explored the benefits of introduction of mycorrhizal agents in post-mining restoration (Brundrett et al. 1996; Hutton et al. 1997) and in reintroduction of orchid species (Quay et al. 1995). Microorganisms defy introduction into a complex ecosystem if the biotope does not suit them and if they are not provided with ample nutrient sources. Unless indigenous fungi have survived adverse conditions, somewhere hidden in their biotope or in neighbouring areas, they may require artificial inoculation to return to the site. But many apparently threatened organisms show a remarkably rapid reappearance when environmental changes favour the microorganism. It is

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not yet known to what extent the local destruction of biotopes leads to the extinction of certain fungi, but for macromycetes the possibility exists. Also pollution of biotopes, particularly flowing waters, is a factor that eliminates many aquatic fungi, although certain aquatic hyphomycetes are surprisingly resistant to heavy metals (Krauss et al. 2001). Not only ectomycorrhiza, but also a diverse population of arbuscular mycorrhizal fungi have been shown to promote plant diversity in plots of natural vegetation (van der Heijden et al. 1998). For all the purposes described here, suitable well-preserved strains are needed. Acknowledgements My colleagues Drs D. van der Mei, R. C. Summerbell and J. A. Stalpers kindly contributed suggestions to improve this chapter. References Agerer R, Ammirati J, Blanz P, Courtecuisse R, Desjardin DE, Gams W, Hallenberg N, Halling RE, Hawksworth DL, Horak E, Korf RP, Mueller GM, Oberwinkler F, Rambold G, Summerbell RC, Triebel D, Watling R (2000) Always deposit vouchers. Mycological Research 104, 642–644. Also published in Canadian Journal of Botany 78, 981; Plant Biology 2, 583; Nova Hedwigia 71, 539-543. Allsopp D, Colwell RR, Hawksworth DL (1995) (Eds) ‘Microbial diversity and ecosystem function.’ (CAB International: Wallingford) Arnolds E (1991) Decline of ectomycorrhizal fungi in Europe. Agriculture, Ecosystems and Environment 35, 209–244. Arnolds E (1992) New evidence for changes in the macromycete flora of the Netherlands. Nova Hedwigia 55, 325–351. Batty AL, Dixon KW, Brundrett MC, Sivasithamparam K (2002) Orchid conservation and mycorrhizal associations. In ‘Microorganism in plant conservation and biodiversity.’ (Eds K Sivasithamparam, KW Dixon and RL Barrett) pp. 195–226. (Kluwer Academic Publishers: Dordrecht) Bärlocher F (1992) ‘The ecology of aquatic hyphomycetes.’ (Springer-Verlag: Berlin) Beuchat LR (1987) ‘Food and beverage mycology.’ 2nd edn. (Van Nostrand Reinhold: New York) Boesewinkel HJ (1976) Storage of fungal cultures in water. Transactions of the British Mycological Society 66, 183–185. Boyle D, Wiesner C, Richardson A (1998) Factors affecting the degradation of polyaromatic hydrocarbons in soil by white-rot fungi. Soil Biology and Biochemistry 30, 873–882. Brasier CM (1991) Ophiostoma novo-ulmi sp. nov., causative agent of current Dutch elm disease pandemics. Mycopathologia 115, 151–161. Brundrett MC, Ashwath N, Jasper DA (1996b) Mycorrhizas in the Kakadu region of tropical Australia. II. Propagules of mycorrhizal fungi in disturbed habitats. Plant and Soil 184, 173–184. Brussaard L, Ferrera-Cerrato R (1997) (Eds) ‘Soil ecology in sustainable agricultural systems.’ (CRC Press: Boca Raton) Burnett JH (1983) Speciation in fungi. Transactions of the British Mycological Society 81, 1–14. Calkin NW, Mathre DE, Miller JM ( 1 9 9 1 ) Field performance of sweet corn seed bio-primed and coated with Pseudomonas fluorescens AB254. HortScience 26, 1163–1165.

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Sugawara H, Ma J-C, M i y a z a k i S, Shimura J, Takishima Y (1993) (Eds) ‘World directory of collections of c u l t u r e s of microorganisms.’ (WFCC World Data Centre on Microorganisms: Saitama) Tan CS (1997) Preservation of fungi. Cryptogamie Mycologie 18, 157–163. Tan CS, van Ingen CW, van den Berg C, Stalpers JA (1998) Long-term preservation of fungi. Cryoletters 1998, 15–22. Tan CS, V l u g IJ, Stalpers JA, van I n g e n CW (1994) Microscopical observations on the influence of the cooling rate during freeze-drying of conidia. Mycologia 86, 281–289. Thomson BD, Malajczuk N, Grove TS, Hardy GE St.J (1993) Improving the colonization capacity and effectiveness of ectomycorrhizal fungal cultures by association with a host plant and re-isolation. Mycological Research 97, 839–844. V a k i l i NG (1984) The role of f u n g i c o l o u s f u n g i in biological control of saprophytic and phytopathogenic fungi of corn kernels. Phytopathology 74, 1272. van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T, Wiemken A, Sanders 1R (1998) Mycorrhizal fungal d i v e r s i t y determines plant biodiversity, ecosystem variability and productivity. Nature 396, 69–72. van Dijk C (1979) Endophyte distribution in the soil. In ‘Symbiotic nitrogen fixation in the management of temperate forests.’ (Eds JC Gordon, CT Wheeler, DA Perry) pp. 84–94. (Forest Res. Lab.: Corvallis) Ward N, Rainey FA, Goebel B, Stackebrand E (1995) I d e n t i f y i n g and c u l t u r i n g the ‘unculturables’: A challenge for microbiologists. In ‘Microbial diversity and ecosystem f u n c t i o n . ’ (Eds D Allsopp. RR Colwell and DL Hawksworth) pp. 89–110. (CAB International: Wallingford)

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K. Sivasithamparam, K.W. Dixon & R.L. Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 285–306. © Kluwer Academic Publishers.

Chapter 11 IMPACT OF FUNGAL PATHOGENS IN NATURAL FOREST ECOSYSTEMS: A FOCUS ON EUCALYPTS

Treena Burgess Michael J. Wingfield Forestry and Agriculture Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, RSA.

1. Introduction Fungal pathogens are integral components of healthy natural forest ecosystems, where they play a major role in eliminating weak and unfit trees (Manion 1981; Burdon 1991; Castello et al. 1995). They also affect species occurrence and distribution, especially in the regeneration layer (Castello et al. 1995). Soil-borne pathogens, in particular, are thought to be important in maintaining plant species diversity and distribution (Augspurger and Kelly 1984; Bever et al. 1997; Mills and Bever 1998; Packer and Clay 2000). It is hypothesised that seedlings close to their parents or other conspecific trees are more likely to be killed by host specific soil-borne pathogens than seedlings further away. Over time, this results in a shift in the juvenile population away from the adults. This relationship has been demonstrated between the temperate tree Prunus serotina and the pathogen Pythium (Packer and Clay 2000) and also for seedling damping off of the tropical tree Platypodium elegans caused by a variety of soil-borne pathogens (Augspurger and Kelly 1984). Interestingly, canker fungi also have been shown to impart a similar effect on the distribution of the tropical tree Ocotea whitei (Gilbert et al. 1994). Higher levels of light intensity, such as those experienced in light gaps caused by fallen trees, reduce both pathogen

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activity and the net impact of pathogens (Augspurger and Kelly 1984; Castello et al. 1995). The epidemiology and visual impact of indigenous pathogens in natural forest ecosystems is greatly minimised by genetic diversity. Here, trees and their pathogens have co-evolved and the population structure of the hosts is characterised by genetic and age diversity (Manion 1981; Hansen 1999). Consequently, individuals in a mature tree population will vary in their susceptibility to a particular pathogen. Usually, the tree is not susceptible throughout its life cycle, consequently, some individuals in some age classes may be susceptible, but not the whole population. Large populations of susceptible trees do not develop and therefore, widespread disease epidemics cannot occur. This i m m u n i t y of natural ecosystems to disease epidemics can be overcome in two ways. Firstly, either a natural or human disturbance could result in the growth of an even aged stand of a single species that may then be susceptible to an indigenous pathogen. Secondly, virulent pathogens, to which the trees have no resistance, could be introduced to a natural ecosystem. Introduced pathogens often lead to the elimination of entire species and result in permanent changes in the species composition of an ecosystem. Ineffective quarantine has often led to epidemics in indigenous and exotic tree populations caused by introduced fungal pathogens (Old and Dudzinski 1998; Palm 1999). Examples of mass destruction of indigenous forests due to fungal pathogens i n c l u d e Chestnut Blight caused by Cryphonectria parasitica (Anagnostakis 1987), Dutch Elm Disease caused by Ophiostoma ulmi and O. novo-ulmi in Europe and North America (Brasier 1991; Hubbes 1999) and Cypress Canker in Europe caused by Seiridium cardinale (Graniti 1998). Destructive epidemics caused by fungi in natural woody ecosystems, particularly in the Northern Hemisphere, have been covered extensively elsewhere (Anagnostakis 1987; Brasier 1991; Graniti 1998; Hubbes 1999). In this chapter, we will rather focus on pathogens of eucalypts in their native range in Australia and in exotic plantations throughout the world. We also consider the threat to na tive forests in Australia due to so-called new pathogens that have emerged on exotic plantations elsewhere in the world. Emphasis is placed on the importance of the origin of a pathogen and the structure of the host population.

2. Eucalypts in Australia There are about 700 species of eucalypts, the majority of which are native to Australia (Brooker and Kleinig 1994; Ladiges 1997; Potts and Pederick 2000). A small number of species are native to Papua New Guinea and a few islands in the Indonesian archipelago (Brooker and Kleinig 1994).

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Eucalypts occur naturally from latitude 7°N to 40°S and from sea level to 1,800 m. They have thus adapted to climatic extremes from the desert gums growing under hot dry conditions to snow gums that can tolerate extreme cold (Poynton 1979; Brooker and Kleinig 1994 Ladiges 1997;). In Australia, eucalypts define much of the forest environment and dominate 95% of the forest area. The most impressive examples are the forest eucalypts found in magnificent stands on the eastern seaboard, Tasmania and Western Australia. It is from these large timber trees that species were selected for exotic plantation forestry in the Tropics and the Southern Hemisphere (Poynton 1979; Potts and Pederik 2000). Indigenous pathogens play an integral role in undisturbed eucalypt forests. Fungi capable of causing leaf diseases and stem cankers such Mycosphaerella spp., Endothia gyrosa, Endothiella spp., Botryosphaeria dothidea and Cytospora eucalypticola are commonly found in native eucalypt forests (Davison and Tay 1983; Walker et al. 1985; Old et al. 1986; Barber and Keane 1999; Ivory 1999; Yuan 1999; Old and Davison 2000; Park et al. 2000). There are no reported cases of these fungi causing significant disease in undisturbed forests (Park et al. 2000). However, much of the Australian eucalypt forests has been heavily logged and is now intensively managed as regrowth forests. This disturbance has resulted in localised outbreaks of indigenous pathogens. Between 1995 and 1998, hardwood plantations in Australia almost doubled from 160,000 ha to 290,000 (Love et al. 1999; Turnbull 2000). This increase was mainly due to a trebling in the area under plantations (predominantly E. globulus) in Western Australia from 42,000 to 120,000 ha. A major concern with this rapid expansion is the danger associated with pests and pathogens. There is an increased disease risk in plantations because of reduced genetic variation and site conditions that are often not suited to the species being planted (Potts and Pederick 2000). Although eucalypts are native to Australia, the species used in plantations are often not endemic to the region in which they are planted (e.g. Tasmanian bluegums, E. globulus, in Western Australia). In addition, plantations represent evenaged monocultures and are more susceptible to disease than native forests where epidemics are restricted by the age structure and the diversity of the plant community. To date, the plantations have been relatively free from major diseases. However, considering the experience in other countries, this situation seems likely to change (Old and Dudzinski 1998; Wingfield 1999; Old and Davison 2000; Park et al. 2000). 2.1. Indigenous pathogens in logged forests Armillaria spp. are widely distributed in many forests where they cause little disturbance besides colonising dead stumps and roots (Kile et al. 1991; Kile

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2000). However, Armillaria spp. have been observed as epiphytes of living roots and as primary root and butt pathogens in disturbed forests (Kile et al. 1991). As a primary pathogen, Armillaria spp. cause typical dieback symptoms, crown decline, root rot, basal lesions and ultimately tree death. Sub-lethal infections reduce tree growth (Kile et al. 1991; Shearer 1995). Armillaria spp. spread by aerial dispersal of basidiospores, by rhizomorphs in the soil or by root contacts. Basidiospores seem not to be a common method of dispersal in Australia, likewise the Australian climate is often not conducive to rhizomorph development (Podger et al. 1978). Thus, the most common method of spread is through direct contact between the tree roots (Kile et al. 1991; Shearer 1994). Consequently, tree death tends to be in patches with dead trees at the centre and dying trees at the periphery of patches. As Armillaria spp. are common saprophytes of dead roots and stumps, the removal of this primary source of inoculum can restrict the spread of the disease. Armillaria luteobubalina is an unimportant indigenous pathogen in southern Australian forest communities (Kile et al. 1991). However, this fungus has emerged as a serious primary pathogen in the dry sclerophyll mixed species eucalypt forests, subjected to heavy logging, in central Victoria and Western Australia (Shearer and Tippett 1988; Kile et al. 1991; Shearer 1994). Regeneration of E. diversicolor forests following logging is also impeded by A. luteobubalina because logged stumps provide a source of infection for young seedlings (Pearce et al. 1986) (Figure 1). There is a strong positive relationship between proximity to infected stumps and disease incidence in regenerating E. diversicolor stands. A. luteobubalina is most active in young stands, but mortality is restricted to weaker trees in older stands (Pearce et al. 1986). 2.2. Indigenous pathogens in eucalypt plantations A. luteobubalina has also led to disease outbreaks when native eucalypt forests have been cleared to allow for reafforestation (Kile 2000). In Western Australia, plantations of E. saligna were established after the clearing of native E. marginata forests. Trees close to infected stumps were killed by A. luteobubalina (Shearer 1995). Similarly, death and disease caused by A. luteobubalina in a fast-growing E. regnans plantation in Victoria was initiated from an infected stump at the edge of the plantation (Podger et al. 1978). Endothia gyrosa is a common canker pathogen in eucalypt forests in eastern Australia (Walker et al. 1985; Old et al. 1986; Old et al. 1990; Old and Davison 2000) and its anamorph Endothiella gyrosa is common on cankered trees in Western Australia (Davison and Tay 1983; Shearer 1994). Endothia gyrosa is an opportunistic pathogen that generally causes

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superficial non-lethal cankers, but under experimental conditions it can kill stressed trees (Old et al. 1986; Old et al. 1990; Old and Davison 2000). In Tasmania, E. gyrosa is also found to cause cankers in stressed trees, and in one plantation, infection resulted in severe disease (Waldlow 1999). This outbreak was apparently not due to a more pathogenic strain of E. gyrosa, but more likely an unknown stress event predisposing the trees to infection (Yuan and Mohammed 2000). In Western Australia, low rainfall in a hybrid plantation (E. grandis crossed with E. camaldulensis) led to a severe cankering of stems caused mainly by Endothiella gyrosa (Neumeister-Kemp, pers. comm.).

Botryosphaeria ribis is an opportunistic pathogen and is frequently isolated from stem cankers throughout Australia (Davison and Tay 1983; Shearer et al. 1987; Old et al. 1990; Old and Davison 2000). Death is uncommon, but cankers girdled and eventually killed individuals of Eucalyptus radiata originating from eastern Australia that had been planted

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in a species trial in Western Australia (Shearer et al. 1987). The trees were most likely not suited to the experimental site and were thus predisposed to infection by unfavourable climatic conditions.

Numerous Mycosphaerella spp. and their anamorphs have been described from Australia (Carnegie et al. 1994; Crous 1998; Crous et al. 1998; Barber and Keane 1999; Ivory 1999; Maxwell et al. 2000; Yuan 1999; Park et al. 2000). These fungi form necrotic lesions on leafs and young shoots of eucalypts causing a disease commonly referred to as Mycosphaerella leaf blotch disease (Figure 2). In the most severe cases, they can reduce the photosynthetic capacity of trees to such an extent that death eventually occurs. This does not happen in forest ecosystems. E. globulus, the plantation species of choice in many parts of Australia, is particularly susceptible to Mycosphaerella leaf blotch disease (Crous et al. 1998). E. globulus plantations in Tasmania have experienced severe defoliation of juvenile leaves due to M. cryptica and also to a lesser degree, M. molleriania (Milgate et al. 1997). Recently, the juvenile foliage of E. globulus grown in plantations in Western Australia has been severely affected by M. nubilosa and the adult foliage by M. cryptica (Carnegie et al. 1997, Maxwell et al. 2000). M. cryptica is also widely distributed in

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Queensland where it causes disease on a wide range of eucalypt species (Ivory 1999). Cylindrocladium leaf spot and shoot blight caused by the fungus Calonectria quinquiseptatum has damaged to young eucalypt plantations in tropical areas of Queensland for approximately two decades (Bolland et al. 1985). Lesions cover the foliage and new shoots of susceptible young trees and cause the leaves to be shed and the branches to die-off (Ivory 1999). There are many other indigenous pathogens reported from eucalypt plantations (Old et al. 1986; Kile et al. 1996; Barber and Keane 1999; Yuan et al. 1999; Park et al. 2000), but they are apparently of relatively minor importance. Cytospora eucalypticola is one of the most commonly isolated canker pathogens (Davison and Tay 1983; Shearer et al. 1987; Yuan et al. 1999), but is considered to be less pathogenic than either Botyrosphaeria spp. or Endothiella spp. (Old et al. 1990). Ceratocystis eucalypti has also been isolated from simulated stem wounds in eucalypt regrowth in Victoria, although it is not thought to be a serious pathogen (Kile et al. 1996).

2.3. Introduced pathogens The forests and near-coastal vegetation of south-western Australia support one of the richest regions of floristic biodiversity in the world with over 7,000 native plant species. Eucalyptus marginata (jarrah) is the dominant overstorey tree species in much of this region (Figure 3). The introduced root and collar pathogen Phytophthora cinnamomi has had a major impact on the jarrah forest ecosystem (Shearer and Tippett 1989; Wills 1993; Shearer and Smith 2000). At least 2,000 species in Western Australia are directly susceptible to P. cinnamomi and the growth and germination of many other species would be affected indirectly through the loss of the over­ story and changes in light and moisture conditions. Infection by P. cinnamomi in the jarrah forest causes dieback of species in the overstorey, mid-storey and under-story (Davison and Shearer 1989). This results in changes to the vegetation structure and ultimately species biodiversity. A more open forest with a sedge-dominated understorey emerges and this in turn affects the endemic fauna (Davison and Shearer 1989). This was demonstrated in a detailed study of P. cinnamomi disease centres in Banksia woodlands on the Swan coastal plain, Western Australia (Shearer and Dillon 1996). Infection by P. cinnamomi decreased species number, changed vegetation structure and totally altered the visual and floristic characteristics of the ecosystem. P. cinnamomi infects the roots and collar of trees via motile zoospores and thus, water plays an important role in their movement. The up-slope in the jarrah forest is usually free of P. cinnamomi while the down-slope and the valleys and any area where water collects tend to be infected (Davison

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1994). In the Swan coastal plain, disease is worst where the water table is high (Shearer and Dillon 1996).

The impact of P. cinnamomi on Australian ecosystems is not limited to Western Australia, but causes problems throughout Australia in regions with mediterranean or temperate climates. Comprehensive studies on vegetation changes in forests of Victoria following infestation showed a reduced overstorey and a change in the species composition of the understorey (Weste 1986). P. cinnamomi has also devastated natural ecosystems and irreversibly changed the vegetation structure in Tasmania, the Australian Capital Territory and New South Wales (Davison and Shearer 1989). The extent of the disease associated with P. cinnamomi is so great, this pathogen is considered one of the five major threats to biodiversity in Australia (Commonwealth of Australia 1992).

3. Eucalypts as exotics Eucalypts were introduced from Australia to the rest of the world in the century. They adapted well as exotics and became a part of the landscape in

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many regions of the world. From the beginning, eucalypts were found to be useful for shade, timber and fuel and in stabilising degraded lands. Commercial plantations were established in many countries by the turn of the century, and in the last 40 years there has been a rapid expansion in areas under afforestation, particularly in the tropics and the Southern Hemisphere. In 1990, there were 8 million ha of exotic eucalypt plantations, this increased to over 10 million ha by the year 2000 and is expected to increase to over 20 million ha by the year 2010 (Turnbull 2000). Exotic eucalypt plantations have primarily been established to meet the world’s demand for paper and pulp. Species and provenance trials have been used to select the best p l a n t i n g stock for new regions and subsequently extensive breeding programs have been established world-wide. Hybrid species and local land races have emerged from trial plantings of different species and have become the basis of major planting programs (Cotterill and Brolin 1997). In addition, breeding programs and artificial hybridisation of different species has been actively pursued and vegetative propagation of superior genotypes has led to extensive clonal forestry (Borralho 1997; Cotterill and Brolin 1997). Ultimately, the initial success of eucalypts as exotics has been due to the absence of pests and pathogens naturally associated with them in their regions of origin (Wingfield 1999). Only a small number of diseases were reported in the early days of eucalypt plantation development. However, as the planted areas have increased and more plantations have been established ‘off-site’, disease problems have also increased (Day 1950; Wingfield 1999; Wingfield et al. 2001). Pathogens on exotic eucalypts are predominantly those accidentally introduced to all eucalypt-growing regions of the world along with those trees. The majority of these pathogens infect stems and leaves and have been introduced with the germplasm, either on seed or chaff or as endophytes of vegetative material (Burley 1987). No known root pathogens have been introduced as soil was rarely, if ever, transported with planting stock. Interesting examples of soil fungi introduced world-wide are the beneficial ectomycorrhizal fungi. It was recognised early on that eucalypts, especially from the subgenus Monocalyptus, grew poorly as exotics due to the absence of ectomycorrhizal fungi (Pyror 1956). As a consequence, soil and spores were distributed world wide and now eucalypt-specific ectomycorrhizal fungi such as some species of Pisolithus, Laccaria laccata, Hydnangium carneum and Scleroderma verrucosum are found wherever eucalypts are grown (Dell et al. 2000). These species are common in Australia, but represent only a few of the thousands of ectomycorrhizal fungi of eucalypts (Bougher 1995).

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In many cases, exotic eucalypts are planted in close association with native plant species that are closely related to eucalypts. The destruction of the native forests, coupled with genetically uniform eucalypt plantations, places a very high selection pressure on indigenous pathogens to jump hosts and infect eucalypts. A good example of this is the eucalypt rust, Puccinia psidii, that has moved from native Myrtaceae to eucalypts in South America (Coutinho et al. 1998). However, there are many other examples of new and emerging pathogens of unknown origin. Some pathogens such as Botyrosphaeria dothidea are found in Australia, but is also found world­ wide with many woody plants as hosts. Thus, a population of B. dothidea in one region may include introduced and indigenous individuals. Population studies examining allelic distribution of polymorphic loci will be necessary to determine the origin of such pathogens. Others pathogens such as Cryphonectria cubensis seem to be indigenous to some areas and introduced to others (Hodges et al. 1986; Myburgh et al. 1999).

3.1. Stem pathogens Cryphonectria cubensis causes a serious and destructive stem canker disease on eucalypts in tropical and sub-tropical regions of the world (Figure 4). It was first reported in Cuba (Bruner 1916), but has since been recorded in

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eucalypt growing countries in the tropics and sub-tropics of Asia, Africa and the Americas (Boerboom and Maas 1970; Hodges et al. 1979; Gibson 1981; Sharma et al. 1985; Wingfield et al. 1989). In Australia, C. cubensis has been found only associated with root cankers on Eucalyptus marginata (Davison and Coates 1991). This niche and the mediterranean climate of the region are unusual for the fungus. The impact of the disease was also minimal and the distribution of the pathogen localised. These facts do not support an Australian origin for the fungus. One hypothesis regarding the origin of C. cubensis is that it jumped from cloves (Svzygium aromaticum, Myrtaceae) to eucalypts, possibly in Indonesia, and from there has spread to other eucalypt growing regions of the world (Hodges et al. 1986). This hypothesis is based on the fact that Hodges et al. (1986) showed C. cubensis to be conspecific with the clove pathogen Endothia eugeniae. Studies on the population diversity of C. cubensis in Brazil (van Zyl et al. 1998) as well as in Venezuela and Indonesia (van Heerden et al. 1997) suggest an equal likelihood of origin in South America and South East Asia. Moreover, phylogenetic studies on the fungus by Myburgh et al. (1999), have revealed two distinct clades, one containing isolates of C. cubensis from Asia (including clove isolates and those from Australia) and another containing isolates from South Africa and America. This suggests the pathogen has either crossed over from native plants to eucalypts at least twice or the fungus has been geographically isolated in the two regions for a long time. Although much research is still needed to resolve the origin of C. cubensis, it is an excellent example of a pathogen adapting to a new host. Puccinia psidii provides a classic example of a pathogen that has crossed over to a new host from a known host of known origin. P. psidii causes eucalypt rust, one of the most serious diseases of eucalypts in Brazil and has been found in other South American countries, Central America and Florida (Laundon and Waterson 1965; Marlatt and Kimbrough 1979; Coutinho et al. 1998). P. psidii is a rust pathogen first reported from guava (Psidium guajava), but it has a wide host range among Myrtaceae in South America. To date, this pathogen is unknown in Australia, Africa or Asia. The young leaves and buds of eucalypts under two years of age are the most susceptible to infection (Figure 5). Growth is restricted or terminated, resulting in stunted multi-stemmed trees, with the most susceptible species and individuals being killed. A number of Coniothyrium spp. have been described from eucalypts, most of them causing leaf diseases of little economic importance. In contrast, Coniothyrium zuluense has recently emerged as a serious primary stem pathogen in the sub-tropical regions of South Africa (Wingfield et al. 1997). The disease was first observed in 1988 on a single clone of E.

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grandis in Zululand. Since its discovery, it has become widespread in South Africa and affects many different eucalypt species, clones and hybrids. Subsequently, C. zuluense has been reported in Uruguay, Argentina, Thailand and Mexico where it is also causing significant disease (Wingfield, unpubl.). This disease is typified by lesions on young green stem tissue during the early part of growing season. Small lesions then coalesce to form large necrotic patches and swellings develop and crack (Figure 6). In susceptible clones, the stem cankers coalesce and girdle the tree. Most interestingly, the fungus is associated with two species of the Pantoeae, which are common soft rot causing bacteria. The fungus alone is not as pathogenic as it is in association with the bacteria (van Zyl 1999). C. zuluense probably also represents a component of a newly emerging disease complex and not a pathogen introduced from Australia.

Ceratocystis fimbriata is a well known wilt and canker pathogen of many economically important woody plants (Kile et al. 1996). The fungus requires wounds for the initiation of infection, but until recently was not recognised as a serious pathogen of eucalypts. It has, however, recently been found to cause a serious disease of Eucalyptus clones in the Congo and Brazil (Roux et al. 2000). This appears to represent another newly emerging disease, the origin of which is most likely outside Australia.

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3.2. Leaf pathogens Mycosphaerella spp. and their anamorphs are common pathogens of eucalypts and have become one of the major disease causing agents affecting exotic plantations throughout the world (Crous 1998). Afforestation with E. globulus in South Africa was suspended in the 1930’s due to the severity of infection by M. juvensis (Crous 1998). Mycosphearella leaf blotch disease restricts the growth of infected trees by reducing the photosynthetic area of the leaves (Figure 2). Different species of Mycosphearella infect different species of eucalypts. Many of these (e.g. M. suttoniae) have a broad host range, while others such as M. molleriana, only known on E. globulus, appear to be more specific. Numerous species have been recorded from eucalypts in South Africa and elsewhere (Park and Keane 1984; Carnegie and Keane 1994; Crous 1998; Park et al. 2000). Mycosphaerella spp. found on eucalypts appear to be eucalypt-specific and are thought to be moved about via seed. However, many Mycosphaerella spp. known to infect eucalypts have not been found in Australia, indicating that some species may have emerged on native plants and adapted to eucalypts outside Australia.

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Cylindrocladium spp. include a number of serious pathogens of eucalypts and other woody hosts (Crous and Wingfield 1994). Cylindrocladium spp. have been found wherever eucalypts are grown, but cause the most severe damage in the tropics and sub-tropics. The causal agent of new outbreaks is usually indigenous to the region of the outbreak (Wingfield unpubl.). Unlike Mycosphaerella spp., species of Cylindrocladium found on eucalypts usually have a wide host range (Crous et al. 1991). They cause a range of serious diseases from damping-off of seedlings, seedling and shoot b l i g h t , leaf spot, stem cankers and in some countries, even root diseases. 3.3. Endophytes An intriguing group of eucalypt pathogens are those with an opportunistic habit. In their natural environment, they cause disease only to stressed trees and are considered of minor importance. Exotic trees, however, are exposed to a wide diversity of stresses and these opportunistic pathogens can become very important. Their ubiquitous nature has led researchers to propose and eventually demonstrate that they are endophytes (Bettucci and Saravay 1993; Smith et al. 1996). Endophytic fungi are able colonise healthy plant tissue w i t h o u t causing disease. Many endophytes are seed-borne and are consequently present in the plant tissue from germination, while others enter the plant early in development through lenticels and stomata (Carroll 1988; Stone and Petrini 1997). The most important of these endophytic pathogens on eucalypts is Botryosphaeria rhodina in the tropics and B. dothidea in the sub-tropics and temperate regions. Since first reports in 1989, B. dothidea has become one of the most commonly recognised pathogens of exotic eucalypts in South Africa where it is considered an important and widespread threat to eucalypt production (Smith et al. 1994, 1996). Symptoms of Botryosphaeria infection include twig dieback and stem cankers on the current year’s shoots, terminal leader shoots and main stems associated with the extensive production of kino (Smith et al. 1994). If trees recover, the death of the leader results in a twisted and weak stem susceptible to wind damage (Figure 7). The production of k i n o veins or pockets in older trees renders them unacceptable for solid wood production. Botryosphaeria dieback is always associated with physiological stresses including drought, hot or cold winds, nutritional imbalance, waterlogging, hail wounds, insect damage and damage by other pathogens (Smith et al. 1994, 1996). Both B. dothidea and B. rhodina are cosmopolitan fungi having a wide host range and geographic distribution. As such, they could be introduced or indigenous to a region where they are found. Endothia gyrosa and Colletotrichum gloeosporiodes

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are other opportunistic stress pathogens often associated with Botryosphaeria (van der Westhuizen et al. 1993; Smith et al. 1998). Endophytic pathogens can pose greater threats to plantation forestry than non-endophytic plant pathogens. Most pathogens only cause disease at a certain stage of their host’s life (for example Mycosphaerella spp. on juvenile leaves of E. globulus and E. nitens), while endophytic pathogens can cause disease in response to stress throughout the trees’ life. The dramatic increase in eucalypt plantations world-wide will result in trees being planted outside their natural range. Hence, the risk of stress-induced diseases is also increasing. Clonal forestry also raises additional complications as clones vary in their tolerance to Botryosphaeria (Smith et al. 1996). In the absence of careful screening, a susceptible clone could be severely damaged.

4. Quarantine and emerging diseases Healthy eucalypt forests in Australia are rarely affected by fungal pathogens. Conversely, in disturbed forests and plantations, outbreaks of indigenous pathogens are becoming more common. As the areas of eucalypt plantations in Australia increase, it is likely infections by indigenous pathogens will

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result in more serious disease problems. Of greater concern, however, is the emergence in exotic eucalypt plantations of new diseases not previously encountered in Australia (see Hardy and Sivasithamparam this volume). The introduction of P. cinnamomi into Australia provides one sobering example of the devastating impact a foreign pathogen can have on a natural ecosystem. The accidental introduction of pathogens such as P. psidii, C. zuluense or the virulent South African strains of C. cubensis could have an equally devastating effect on forests in Australia (O'Neill, 2000). In the last 25 years, there have been numerous incursions of exotic forest pests and diseases throughout the southern hemisphere (Eldridge and Simpson 1987; Old and Dudzinski 1998; Wingfield 1999). Introduced pathogens often have no initial impact and are not detected until a sizeable area is affected (Walker 1987; Bright 1998). By this time, the pathogen will have generally spread extensively beyond the diseased area and thus eradication becomes almost impossible (Walker 1987). Currently, global trade agreements and the removal of embargos and tariffs have facilitated the movement of forestry products around the world (Palm 1999). Consequently, the chance of introducing pathogens into new areas has also increased. Some new diseases of eucalypts may already be in Australia, although their presence may not have been detected. Despite this, the only means to prevent introductions of new and damaging pathogens to eucalypts in Australia is by vigilant quarantine at all points of entry. This is perhaps easier for eucalypts than for other timber species, as there is little, if any, importation of eucalypt timber into Australia. A more likely source of infection would be germplasm. Many other countries have been breeding superior quality eucalypt material, especially hybrids and clones. These are very attractive to commercial forestry in Australia and there are plans to bring selected material back i n t o Australia. This material, as seed or vegetatively propagated tissue, should clearly be detained in quarantine until it can be safely released.

5. Conclusions Natural forest ecosystems have many indigenous pathogens associated with them, but genetic and age diversity of the host community prevents disease epidemics. This is the situation in undisturbed eucalypt forests in Australia. Disturbed ecosystems and plantations, however, are more susceptible to outbreaks because of a reduction in both genetic and age diversity and because of increased external stress. Observations and records of eucalypt pathogens and diseases in Australia are increasing. In addition, many new diseases are emerging on exotic eucalypt plantations throughout the world, especially in the tropics and sub-tropics. These emerging diseases pose a threat to native eucalypt forests in Australia. Vigilant and strictly applied

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quarantine measures are necessary to prevent the introduction of potentially devastating pathogens into Australia.

Acknowledgements We are grateful to Bernard Slippers for critically reviewing the manuscript. Financial support was provided by The University of Pretoria and the National Research Foundation (NRF). References Anagnostakis SL (1987) Chestnut blight: the classical problem of an introduced pathogen. Mycologia 79, 23–37. Augspurger CK, Kelly CK (1984) Pathogen mortality of tropical tree seedlings: experimental studies of the effect of dispersal distance, seedling density and light conditions. Oecologia 61, 211–217. Barber PA, Keane PJ (1999) Foliar diseases of Eucalypts globulus (Blue Gum) in plantations in Victoria. In ‘Proceedings of the APPS 12th Biennial Conference, Asia-Pacific plant pathology for the new m i l l e n n i u m , Canberra, 27-30 September.’ pp. 83 (Arawang Communication Group: Canberra) Bettucci L, Saravay M (1993) Endophytic fungi of Eucalyptus globulus: a preliminary study. Mycological Research 97, 679–682. Bever JD, Westover KM, Antonovics J (1997) Incorporating the soil community into plant population dynamics: the utility of the feedback approach. Journal of Ecology 85, 561–573. Boerboom JHA, Maas PWT (1970) Canker of Eucalyptus grandis and E. saligna in Surinam caused by Endothia havavensis. Turialba 20, 94–99. Bolland L, Tierney JW, Tierney BJ (1985) Studies on leaf spot and shoot blight of Eucalyptus caused by Cylindrocladium quinquiseptatum. European Journal of Forestry 15, 385–397. Borralho NMG (1997) Seed orchards or cuttings: which is best? In ‘Proceedings of the IUFRO conference on s i l v i c u l t u r e and improvement of eucalypts.’ Salvador, Brazil, 24–29 August. pp. 330–336 (EMBRAPA: Salvador) Bougher NL (1995) Diversity of ectomycorrhizal fungi associated with eucalypts in Australia. In ‘Mycorrhizas for plantation forestry in Asia.’ (Eds MC Brundrett, B Dell, N Malajczuk and M Gong) pp. 8–16 (ACIAR: Canberra) Brasier CM (1991) Ophiostoma novo-ulmi sp. nov., causative agent of current Dutch elm disease pandemics. Mycopathologia 115, 151–161. Bright C (1998) ‘Life out of bounds: bioinvasion in a borderless world.’ (WW Norton: New York) Brooker MIH, Kleinig DA (1994) ‘Field guide to eucalypts Volume 3 Northern Australia.’ (Inkata Press: Sydney) Bruner SE (1916) A new species of Endothia. Mycologia 8, 239–242. Burdon JJ (1991) Fungal pathogens as selective forces in plant populations and communities. Australian Journal of Ecology 16, 423–432. Burley J (1987) Problems of tree seed certification in developing countries. Commonwealth Forestry Review 66, 151–159. Carnegie AJ, Keane PJ (1994) Further Mycosphaerella species associated with leaf diseases of Eucalyptus. Mycological Research 98, 413–418.

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Old KM, Dudzinski M (1998) Forest pathogen introductions to Australia: experiences, threats and counter measures. Current and potential impacts of pitch canker in radiata pine. In ‘Proceedings IMPACT Monterey workshop, Monterey, CA, USA.’ 30 Nov. to 3 Dec. 1998. (CSIRO Australia: Melbourne) Old KM, Gibbs R, Craig I, Myers BJ, Y ua n ZQ (1990) Effect of drought and defoliation on the susceptibility of eucalyptus to cankers caused by Endothia gyrosa and Botryosphaeria ribis. Australian Journal of Botany 38, 571–581 Old KM, Murray DIL, Kile GA, Simpson J, Malafant KWJ (1986) The pathology of fungi isolated from eucalypt cankers in south-eastern Australia. Australian Forest Research 16, 21–36. O’Neill G (2000) Resistance is useless. The Bulletin, November 28, pp. 44–45. Packer A, Clay, K (2000) Soil pathogens and spatial patterns of seedling mortality in a temperate tree. Nature 404, 278–281. Palm ME (1999) Mycology and world trade: a view from the front line. Mycologia 91, 1–12. Park RF, Keane PJ (1984) Further Mycosphaerella species causing leaf diseases of Eucalyptus. Transactions of the British Mycological Society 89, 93–105. Park RF, Keane PJ, Wingfield MJ, Crous PW (2000) Fungal diseases of eucalypt foliage. In ‘Diseases and pathogens of e u calypts.’ (Eds PJ Keane, GA Kile, FD Podger and BN Brown) pp. 153–240. (CSIRO Publishing: Melbourne) Pearce MH, Malajczuk N, Kile GA (1986) The occurrence and effects of Armillaria luteobubalina in the karri (Eucalyptus diversicolor F. Muell.) forests of Western Australia. Australian Journal of Forest Research 16, 243–259. Podger FD, Kile GA, Watling R, Fryer J (1978) Spread and effects of Armillaria luteobuhalina sp. n o v . in an Australian Eucalyptus regnans plantation. Transactions of the British Mycological Society 71, 77–87. Potts BM, Pederick LA (2000) Morphology, phylogeny, origin, distribution and genetic diversity of the eucalypts. In ‘Diseases and pathogens of eucalypts.’ (Eds PJ Keane, GA Kile, FD Podger and BN Brown) pp. 11–34. (CSIRO Publishing: Melbourne) Poynton, RJ (1979) ‘Tree planting in southern Africa. Vol. 2. The eucalypts.’ (Department of Forestry: Republic of South Africa) Pyror LD (1956) Chlorosis and lack of vigour in seedlings of renantherous species of Eucalyptus caused by lack of mycorrhiza. Proceedings of the Linnaean Society NSW 81, 91–96. Roux J, Wingfield MJ, Bouillet J-P, Wingfield BD, Alfenas AC (2000) A serious new wilt disease of Eucalyptus caused by Ceratocystis fimbriata in central Africa. Forest Pathology: 30, 175–184. Sharma J K , Mohanan C, Florence EJM (1985) The occurrence of Cryphonectria canker of Eucalyptus in Kerala, India. Annals of Botany 106, 265–279. Shearer BL (1994) The major plant pathogens occurring in native ecosystems of south­ western Australia. Journal of the Royal Society of Western Australia 77, 113–122. Shearer BL (1995) Impact and symptoms of Armillaria luteobubalina in rehabilitation plantings of Eucalyptus saligna in forests of Eucalyptus marginala in south-western Australia. Australasian Plant Pathology 24, 77–81. Shearer BL, Dillon M (1996) Impact and disease centre characteristics of Phytophthora cinnamomi infestations of Banksia woodlands on the Swan Coastal Plain, Western Australia. Australian Journal of Botany 44, 79–90. Shearer BL, Tippett JT (1988) Distribution and impact of Armillaria luteobubalina in the Eucalyptus marginata forest of south-western Australia. Australian Journal of Botany 36, 433–445.

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Shearer BL, Tippett JT (1989) ‘Jarrah dieback: The dynamics and management of Phytophthora cinnamomi in the jarrah (Eucalyptus marginata) forests of south-western Australia.’ (Department of Conservation and Land Management: Perth) Shearer BL, Tippett JT, Bartle JR (1987) Botryosphaeria ribis infection associated with death of Eucalyptus radiata in species selection trials. Plant Disease 71, 140–145. Shearer BL, Smith IW (2000) Diseases of eucalypts caused by soilborne species of Phytophthora and Pythium. In ‘Diseases and pathogens of eucalypts.’ (Eds PJ Keane, GA Kile, FD Podger and BN Brown) pp. 259–291. (CSIRO Publishing: Melbourne) Smith H, Kemp GHJ, Wingfield MJ (1994) Canker and die-back of Eucalyptus in South Africa caused by Botryosphaeria dothidea. Plant Pathology 43, 1031–1034. Smith H, Wingfield MJ, Coutinho TA (1998) Eucalyptus die-back in South Africa associated with Colletotrichun gloeosporioides. South African Journal of Botany 64, 226–228. Smith H, Wingfield MJ, Petrini O (1996) Botryosphaeria dothidea endophytic in Eucalyptus grandis and Eucalyptus nitens in South Africa. Forest Ecology and Management 89, 189–195. Stone J, Petrini O (1997) Endophytes of forest trees: a model for fungus-pathogen interactions. In ‘Plant relationships. The Mycota V, Part B.’ (Eds G Carroll and B Tudzynski) pp. 129–140. (Springer-Verlag: Berlin) Turnbull JW (2000) Economic and social importance of eucalypts. In ‘Diseases and pathogens of eucalypts.’ (Eds PJ Keane, GA Kile, FD Podger and BN Brown) pp. 1–10. (CSIRO Publishing: Melbourne) van der Westhuizen IP, Wingfield MJ. Kemp GHJ, Swart WJ (1993) First report of the canker pathogen Endothia gyrosa on Eucalyptus in South Africa. Plant Pathology 42, 661–663. van Heerden SW, Wingfield MJ, Coutinho TA, van Zyl LM, Wright JA (1997) Diversity of Cryphonectria cubensis isolates in Venezuela and Indonesia. In ‘Proceedings of the IUFRO conference on silviculture and improvement of eucalypts, Salvador, Brazil, 24–29 August 1997.’ pp. 142–146 (EMBRAPA: Salvador) van Zyl LM (1999) Factors associated with Coniothyrium canker of Eucalyptus in South Africa. In ‘Microbiology and Biochemistry.’ pp. 193. (University of the Orange Free State: Bloemfontein) van Zyl LM, Wingfield MJ, Alfenas AC, Crous PW (1998) Population diversity amongst isolates of Cryphonectria cubensis. Forest Ecology and Management 112, 41–47. Waldlow TJ (1999) Endothia gyrosa associated with severe stem cankers on plantation grown Eucalyptus nitens in Tasmania, Australia. European Journal of Forest Pathology 29, 199–208. Walker J (1987) Development of contingency plans for use against exotic pests and diseases of trees and timber. 1. Problems with the detection and identification of exotic plant pathogens of forest trees. Australian Forestry 50, 5–15. Walker J, Old KM, Murray DIL (1985) Endothia gyrosa on Eucalyptus in Australia with notes on some other species of Endothia and Cryphonectria. Mycotaxon 23, 353–370. Weste G (1986) Vegetation changes associated with invasion by Phytophthora cinnamomi on defined plots in the Brisbane Ranges, Victoria, 1975-1985. Australian Journal of Botany 29, 261–276. Wills RT (1993) The ecological impact of Phytophthora cinnamomi in the Stirling Range National Park. Australian Journal of Ecology 171, 145–159. Wingfield MJ (1999) Pathogens in exotic plantation forestry. International Forestry Review 1, 163–168. Wingfield MJ, Crous PW, Coutinho TA (1997) A serious canker disease of Eucalyptus in South Africa caused by a new species of Coniothyrium. Mycopathologia 136, 139–145.

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Wingfield MJ, Slippers B, Roux J, Wingfield BD (2001) Worldwide movement of exotic forest fungi especially in the tropics and Southern Hemisphere. Bioscience: 51, 134–140. Wingfield MJ, Swart WJ, Abear BJ (1989) First record of Cryphonectria canker of Eucalyptus in South Africa. Phytophylactica 21, 311–313. Yuan ZQ, Mohammed C (2000) The pathogenicity of isolates of Endothia gyrosa to Eucalyptus nitens and E. globulus. Australasian Plant Pathology 29, 29–35. Yuan ZQ (1999) ‘Fungi associated with diseases delected during health surveys of eucalypt plantations in Tasmania. A report on the project funded by the FWPRDC Fellowship.’ (School of Agricultural Science, University of Tasmania: Hobart)

K. Sivasithamparam, K.W. Dixon & R. L. Barrett (eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 307–335. © Kluwer Academic Publishers.

Chapter 12 MICROBIAL CONTAMINANTS IN PLANT TISSUE CULTURE PROPAGATION

Eric Bunn Kings Park & Botanic Garden, Science Directorate, Botanic Gardens and Parks Authority, West Perth 6005, Western A u s t r a l i a .

Beng Tan Department of E n v i r o n m e n t a l Western Australia.

Biology, Curtin University of Technology, Bentley 6102,

1. Introduction Bacteria, f u n g i , m o u l d s and yeasts are common contaminating microorganisms in tissue culture. Microorganisms and their reproductive structures (e. g., spores) are ubiquitous although their relative abundance may vary considerably with environment and season (Atlas and Bartha 1987). Bacteria, fungi and other microorganisms are found not only in soil, water and air, but also on and inside plants (and animals). In plants they occur as microflora associated with roots (rhizosphere and rhizoplane) leaves (phyllosphere and phylloplane), other plant parts and subliminally as endophytes in plant tissues and vegetative propagules. It would be exceptional for plants growing in nature to be free of both epiphytic and endophytic microorganisms. Whilst some organisms may be pathogens or symbionts, many microbial epiphytes and endophytes associated with plants operate as either saprophytes or asymptomatic parasites. Micropropagation refers to the rapid cloning of desirable plant genotypes through in vitro (tissue culture) propagation of shoots, and mass production of whole plants capable of establishment in soil. Micropropagation is an integrated sequence of preparatory and in vitro

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procedures (see Table 1) typically i n v o l v i n g four stages. Stage 1 involves the initiation of a “clean” shoot starter culture, from which exponential shoot multiplication is obtained through regular sub-culturing of proliferating shoots (stage 2). Stage 3 involves root induction in selected well-developed shoots, and finally deflasking and acclimatisation (stage 4). Where somatic embryos or calluses are induced or initiated, germination and shoot regeneration are prerequisites within the propagation protocol (see Figure 1). Stage 1 is therefore the primary stage of disinfestation, although microorganisms can still re-infest cultures at later stages.

Microorganisms become problematic by virtue of their prolific growth under high nutrient in vitro conditions, hence the need to eliminate them from explants before entering the in vitro cycle. Exclusion of

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microorganisms from potential explant material can be approached in a number of ways. Reducing the abundance of fungi (spores and small hyphal fragments) and bacteria on donor plants before explant material is taken will obviously be beneficial in reducing reliance on harsh surface “sterilisation” (disinfestation) procedures, which often only just avoid killing the explant while causing considerable damage to sensitive tissues. Even where the explant is effectively surface sterilised, tissue injury attributable directly to the decontamination process often leads to irreversible oxidation and phenolic leakage, and eventual death.

The phenomenon of microbial contamination in plant tissue culture has been reviewed by Cassells (1991), Dodd et al. (1992), George (1993), Leifert et al. (1994), Cole (1996), and Herman (1996). Collectively these quite recent reviews provide a broad background on the subject. The intention of this chapter is to focus on practical means of circumvention, recognition and eradication of microbial contaminants in applied plant tissue culture. An aspect that has not been discussed in previous reviews is the use of the broad-spectrum biocide Plant Preservative Mixture (PPM®) (manufactured by Plant Cell Technology Inc., Washington DC. ) in plant tissue culture applications. This proprietary mixture has been available to

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tissue culture researchers only since late 1997 and may be of benefit in some applications. 2. Source, vectors and types of microbial contaminants found in plant tissue culture (PTC) In theory, any microorganism (or its reproductive structures, e. g. spores), that is capable of growing on a plant tissue culture medium or in plant tissue in vitro, is potentially a contaminant. Potential microbial contaminants are those intimately associated with plant tissues and surfaces, as well as spores adhering to plant surfaces or present in the laboratory environment. Resident non-pathogenic bacteria present in the initial explant are a common source of endogenous contaminants (see ‘endophytic contamination’, next section). However, superficial contaminants may be traced to residual surface microflora on explants that have survived ineffectual surface treatment or, in the case of clean established cultures, from chance introduction or crosscontamination due to poor aseptic technique. A faulty autoclave or insufficient autoclaving can be a source of contamination, but this can be identified from the consistent and even distribution of contaminant fungal and bacterial colonies within the body of the medium rather than as surface growths. 2.1. External contaminants Fungal contamination is visually manifest: a filamentous fungus will carpet the tissue culture medium with mycelia within days. Bacterial or yeast contaminants, by contrast, are generally slower growing, developing small discrete pale or coloured colonies. Yeasts can be distinguished from bacteria by cell size (typically versus for bacteria), as well as by the larger non-translucent colony size and fermentation odour (medium fouling). Cole (1996) gives a practical guide to identification of common fungal contaminants at the genus level. Fungal contaminants are generally aerially or dust-borne (e. g. Alternaria, Aspergillus, Botrytis, Candida (yeast), Cladosporium, Epicoccum, Microsporum, Mucor, Penicillium, Phialophora, Rhizopus, Rhodotorula (yeast), Trichoderma) or associated with soil e. g. Fusarium (George 1993, Leifert et al. 1994, Cole 1996). The spores of some (e.g. Fusarium poae) are specifically carried and spread by dust mites (Leifert and Woodford 1997). An outbreak of mite infestation can be deduced from “signature footprints” of initially discrete mycelial colonies, around the periphery and/or across the surface of the medium (Dodd et al. 1992; see also Figure 2). Over 30 genera of bacteria are known to be associated with plants (Bradbury 1988). In theory, any bacterium that is associated with explant

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tissue as an epiphyte, endophyte or pathogen is a potential contaminant in plant tissue culture. Leifert et al. (1994) list in excess of 40 different bacteria which have been isolated as contaminants in tissue and cell cultures of a wide range of plant species. Overall, Gram-positive and Gram-negative bacteria appear equally common. Bacillus spp. and Pseudomonas spp. are well represented among the Gram-positive and Gram-negative isolates, respectively.

In a study of contamination pattern in two different commercial laboratories in England, Leifert et al. (1989) noted a preponderance of Gram-negative bacteria (e. g. Pseudomonas and Enterobacter) when plant tissues were indexed in the early stages of micropropagation, but found that Gram-positive ones (e. g. Bacillus, Staphylococcus and Micrococcus) were more likely to be contaminants of older cultures. This is consistent with the dominance of Gram-negative bacteria in epiphytic bacterial populations in the natural growing environment. In older cultures, contamination tends to stem from faulty aseptic technique, and Gram-positive bacteria found in

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these instances are known to be associated with humans (Weller 1997) or laboratory environments (Leifert et al. 1994). It may also be possible to i n f e r the l i k e l y source for the contamination through identification of the contaminant itself, at the microorganism and generic level (Leifert and Woodford 1997). For instance, the presence of Bacillus spp. is an indication of inefficient sterilisation of media or instruments. Common grey, black and green moulds (e.g. Botrytis, Aspergillus, Alternaria, and Penicillium) and pink yeasts (Rhodotorula spp.) indicate high laboratory air contamination, suggesting faulty laminar flow cabinets or operator error. However, the appearance of Cladosporium mould would suggest i n s u f f i c i e n t protection of the laboratory atmosphere from contamination with outside air, since spores of this fungus are very common in the air outdoors. Positive laboratory air pressure and dual-door entrances significantly reduce contamination from Cladosporium. Plant pathogens are less l i k e l y to be introduced into plant tissue culture if explants are screened for absence of blemishes and disease symptoms or if pre-treatments (e. g. fungicidal and/or antibiotics) are applied to donor plants. The presence of n o n - l a t e n t , pathogenic p l a n t viruses, viroids and mycoplasmas are usually predicated by classical symptoms such as leaf streaking, mosaicism, yellowing or plant stunting. However latent viruses can be introduced through infected but symptomless explant tissue.

2.2. Endophytic contaminants Healthy-looking plants and tissues may be host to non-pathogenic bacterial endophytes - bacteria that are normally associated with plants in the in vivo environment. I n i t i a l entry may be gained through natural openings (e. g. stomata, lenticels) or wounds (e. g. leaf scars, cortical ruptures of lateral roots), or as a result of tissue damage caused by herbivores and insect pests. Some well-known endophytic bacteria (e.g. Enterobacter asburiae, Pseudomonas fluorescens) are thought to engineer entry into intact root and leaf tissues by producing cellulase and pectinase (Quadt-Hallmann et al. 1997). Distribution of P. fluorescens is confined to intercellular spaces (Hollis 1951, Petit et al. 1968, Quadt-Hallmann and Kloepper 1996), or systemically in vascular tissues (Hayward 1974, Gagne et al. 1987, Lamb et al. 1996). Intracellular colonization is generally less common, although it has been recorded for the underground parts of numerous Australian Orchids (Wilkinson et al. 1989, 1994). Colonization, however, is generally “spatially limited and is probably a major factor differentiating endophytic bacteria from plant pathogens” ( H a l l m a n n et al. 1997). Underground vegetative organs are particularly predisposed to internal colonisation by soil bacteria. Healthy potato tuber, for example, is internally colonised by non-pathogenic bacteria originating from the root zone soil

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(Hollis 1951, Sturz 1995). On the basis of bioassays, these bacteria were found to have promotory, retarding or neutral effects on plant growth (Sturz 1995), and it was suggested that these non-pathogenic endophytic bacteria may play an important role in tuber decay, tuberisation and plant growth. Even without fresh colonization, propagation from vegetative organs ensures transmission of resident endophytes from mother to clonal progeny plants. From a micropropagation perspective, endophytes are not eliminated by surface treatment(s), as they are not exposed to the sterilant during treatment. Although normally introduced into plant tissue cultures through endogenously contaminated initial explants, they can also appear in established, previously clean cultures through the careless use of inadequately sterilised instruments in the sub-culturing process (Singha et al. 1987). When endophytic contamination is recognised in established plant cultures, it poses a dilemma for the micropropagator. Should a valuable culture be destroyed, or should efforts be expended in cleansing the contaminated culture (e. g. Taji and Williams 1990). Easy-to-initiate cultures are obviously best disposed of (“when in doubt - out!”) but there is a pragmatic view that endophytic microorganisms can be tolerated, even in commercial propagation, if their presence elicits no symptom and the plants appear to grow and multiply satisfactorily in culture (de Fossard 1990). On the other hand, they pose a real financial risk for commercial operations trading locally or i n t e r n a t i o n a l l y where phytosanitary considerations preclude the transfer of contaminated cultures. Although the infection may be benign, reputation is compromised when contamination is discovered by clients before or after plant weaning, or when a whole consignment of in vitro cultures has to be returned or destroyed when a single contaminated culture is discovered during quarantine inspection. Non-latent bacterial endophytes in surface-treated explants are initially detected from a halo or cloudiness around the base of the explant in the medium. When endophytic bacteria are suspected, although no colony or cloudiness is observable, detection is made by incubating tissue macerate in a bacteriological medium or broth. The assay is non-specific but is useful for indexing plant tissue for cultivable bacteria. Bacterial identification requires biochemical tests using commercially available diagnostic kits, which complement Gram staining and morphological characterisation. The detection of non-bacterial endophytes such as viruses, viroids, mycoplasmas and bacteria-like microorganisms are more involved e. g. ELISA (enzyme­ linked immunosorbent assay), serological tests, fatty acid profiling, DNA/RNA fluorochrome staining, etc. (Cassells 1991, George 1993). Some endophytic bacteria are latent, i. e. producing no visible plant symptoms or growth in the medium through many subculture cycles. In

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contrast, fungal and yeast contaminants normally do not remain latent in plant tissue cultures, with the exception of certain obligate fungal pathogens, e.g. Sclerospora sacchari, the fungus causing downy mildew in sugarcane (Leifert et al. 1994). George (1993) attributes the latency of endophytic bacteria to their need to adapt to conditions in vitro; they may not multiply until the host is transferred to a medium more favourable to their growth. It has also been suggested that poor growth of latent bacteria may be due to insufficient release of essential nutrients by the host (Gunson and SpencerPhillips 1993). Latency ends when the normally latent endophyte becomes pathogenic, or when sub-culturing is unduly delayed; tissue death in old cultures forces endophytic bacteria to survive saprophytically on dying necrotic tissues, revealing themselves as isolated colonies on dead tissues. The physiological effects of bacterial endophytes on in vitro cultured plant tissues appear to depend on the host species, bacteria and strain, and age of the cultures; these can range from severe to mild and non-discernible. For example, potato shoot cultures infected with Clavibacter (Corynebacterium) exhibit leaf tip necrosis, reduced leaf development and thinner stems (Long et al. 1988), and, shoot necrosis and even death was observed in apple shoot cultures infected with a strain of Xanthomonas campestris (Maas et al. 1985). Milder effects include lower plant growth and multiplication rates (Leifert et al. 1989), or reduced rooting (Hanus and Rohr 1987). In contrast, latent infection has no discernible adverse effect in Riesling grape; infected cultures grew as well as non-contaminated ones (Reustle et al. 1988). Whether some endophytic bacteria may confer beneficial effects on the host in an otherwise sterile in vitro environment remains speculative. It is now recognised however, that certain bacteria associated with plants in the natural growing environment can stimulate plant growth indirectly by producing antibiotics against plant pathogens (antibiosis), by competing with pathogens (antagonism), induced resistance or by enhancing the effect of useful microorganisms (synergism) (Hallmann et al. 1997). The eco-pathogenic behaviour of bacterial endophytes can be anomalous. Bacteria that are normally saprophytic in the epiphytic phase can be pathogenic in plant tissue cultures (“vitropaths”), and known pathogens in vivo can become saprophytic in vitro (Herman 1990). Whereas pathogens weaken or kill plants by direct parasitisation, vitropaths reduce growth or kill plants growing in vitro by inducing changes to the growth medium, rather than by direct parasitisation of host tissue (Leifert and Waites 1992). It is assumed that the change in pathogenic behaviour is brought about by a pronounced change in the environmental conditions and the physiology of the plants. For instance, surface sterilisation may have removed natural antagonistic microflora so that surviving bacteria, which

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were once kept in check, can proliferate in the in vitro environment without the competition from other microorganisms, i. e. they can become “vitropathic”. The second factor is nutrient luxuriance of the tissue culture medium compared with what is available on plant surfaces, especially in sugars and organics. Pathogenic bacteria that (in vivo) derive nutrients by killing plant tissues with toxins (or macerating enzymes) may, in a wellnurtured heterotrophic host, switch adaptively to a saprophytic mode. Endophytic bacteria are, in many respects, intermediate between saprophytic bacteria and plant pathogens in their behaviour. This has led Hallmann et al. (1997) to speculate that “they are saprophytes evolving toward plant pathogens, or are more highly evolved than plant pathogens (to the extent that they) conserve protective shelter and nutrient supplies by not killing their host”. 3. Combating contaminants in vivo and in vitro 3.1. In vivo and avoidance strategies Cassells (1991) reports that over 20 species of yeasts, 32 species of bacteria and 6 species of mycoplasma are found on and in the tissues of some common agricultural and or horticultural species. The number and type of microorganisms vary considerably between species, habitats, and seasons, and between individuals according to their age, health and vigour. In the case of wild species, very little is known of the species of saprophytic and or epiphytic microorganisms associated with them. It is therefore largely empirical knowledge and observation that guides disinfestation procedures with wild species. The following strategies represent the basic options open to the tissue culturist in minimising the impact of contaminating organisms. Avoidance strategies are ultimately dependent on (a) selecting the type of explant material, (b) selection of the appropriate stage of development and (c) scope for pre-treatment of the starter material in vivo. 3.1.1. Selection of explant material – type of explant Seed (seedling, seedling tissues, seed embryo) Seed or extracted seed embryos are a good choice if the need for mature clonal material is not crucial. Seeds are generally able to withstand vigorous sterilisation, including sterilisation under vacuum, which usually results in elimination of external contaminants. With care, embryos can be extracted aseptically from most seeds provided the surface sterilisation procedure is effective. The exterior surfaces of some seeds are deeply pitted, striated or hirsute and may have adhering remnants of fruit which can harbour contaminating organisms (particularly with dry seed or seed harvested from the soil). Seeds may have to be thoroughly cleaned or even scrubbed before surface sterilisation can be

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truly effective. In general, the interior of seeds is naturally aseptic unless there is systemic infection, or if seeds are perforated by borers or other insects during the early stages of seed development. With some seeds (e. g. native rush (Restionaceae) and sedge (Cyperaceae) species of Western Australia), the embryos are located at one end of the seed requiring only partial removal or piercing of the testa to facilitate extraction of the embryo (Meney and Dixon 1988). Similarly, with very small-seeded species, aseptic embryo extraction presents a technical challenge but this can be accomplished with adequate practice. Very small or soft seeds may require benign sterilisation protocols; hence it may be better not to use vacuum sterilisation for seeds with a thin testa and delicate endosperm. Shoot tips (and apical meristems) These are the most common explants used to initiate an in vitro culture for a wide variety of plant species. Shoot tips are considered to be genetically stable and where clonal (i. e. genetic) fidelity is required these are the preferred explants (Dixon and Touchell 1995). New shoots are less likely to be contaminated with fungal propagules or bacteria and may be free of invertebrate predation in early stages of development. Such material is ideal for culture initiation as it requires less rigorous surface sterilisation and hence better survival, and a higher percentage of clean explant initials can be obtained compared with older and more heavily contaminated shoot material. The dissection and isolation of apical meristems is technically demanding; however, apical meristems provide superior explant material which are generally free of exogenous and endogenous microorganisms (particularly viruses). Nodes Nodal material may be an alternative where shoots are not available or cannot be used. Nodes are older and therefore potentially more contaminated than young apical shoots, requiring correspondingly more stringent surface sterilisation protocols. However, nodal explants are generally more robust and can survive harsher sterilisation treatments, although in woody species oxidation damage and phenolic leakage are still major concerns. Even if external damage occurs, sub-cuticular axillary buds, which generally sustain less damage, are the primary sites of shoot regeneration in nodal cultures. After sprouting and vigorous growth, the axillary shoots can be removed and, if need be, given a precautionary sterilisation treatment before proceeding to induce shoot multiplication. The original node e x p l a n t can be discarded as it may harbour latent contaminants.

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Non-apical (stem, leaf, floral, root, bulb scales) (i) Stem tissues Stem culture can be used for in vitro propagation of a number of species (George 1993, 1996). As with apical tissues, the younger the stem material, the lower the incidence of surface and endogenous microbial contamination. Occasionally the inner pith tissues, which should be largely contaminant free, can be used providing the epidermis can be effectively sterilised or carefully excised. However, Leifert et al. (1994) and Bove and Gamier (1998) report that certain types of pathogenic microorganisms have been located in vascular tissues of some species, which adds a certain element of risk in using stem explants over apical meristems, for example. (ii) Leaf tissues Leaves have a large surface area to mass ratio. A thin epidermis is generally ideal for rapid penetration of media supplements, particularly growth regulators that mediate rapid direct adventitious shoot production. Young shoots are also sites of very active metabolism relative to tissue mass, and where many young, highly active cells are available to convert to vegetative regeneration. Leaves are also easily damaged by surfactants, sterilising agents and handling. Leaves of many plant taxa accumulate phenolic compounds to combat predation and disease, which may restrict their suitability in culture i n i t i a t i o n . Through insect predation, bacteria and viruses may be introduced in the saliva of sucking insects and further entry may be gained through these wounds by opportunistic fungal and bacterial pathogens. Highly sclerophyllous leaves may also be unsuitable as explants due to their thick, waxy epidermis, as well as their relatively high proportion of structural rather than actively metabolising tissues and cells. The use of leaves as explants is therefore limited to certain plant species, particularly those with fleshy leaves from which plant regeneration has been successful in conventional propagation. As with non-leaf tissues, leaf explants should be young, non-predated, and visually disease-free. Bagging of branches (to exclude light but not air) for a short period of time may induce slight etiolation and reduce phenolic content in the shoots and leaves (see below). (iii) Flowers (and all floral parts) Flowers have been used as explant sources in cases where vegetative material is in short supply, endogenously contaminated (Tan 1995), or is otherwise unsuitable or too difficult to use (Collins and Dixon 1992; Bunn and Dixon 1996). Unopened flower buds should be aseptic internally, provided there has been no predation, disease or mechanical damage. 3.1.2. Age and stage of development of explant material Some prior knowledge of the growth habit and life history of the plant species to be cultured is useful; these contribute towards sampling of non­

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predated explant material in peak physiological condition. Sometimes the choice may be robust nodal material if softer e x p l a n t tissue cannot be decontaminated s u c c e s s f u l l y due to tissue damage or severe oxidation reaction, e. g. many Eucalyptus species (Le Roux and van Staden 1994). Generally, however, juvenile or new apical growth provides the best material with which to i n i t i a t e aseptic shoot cultures in mature plants, whereas microorganisms tend to accumulate in the more mature parts of the plant (George 1993). Shoot material from seedlings or saplings is more likely to retain juvenile characteristics whereas that from mature plants (depending on the type of material and location on the parent plant) may exhibit adult characteristics e. g. precocious flowering. So, while there may be compelling reasons for choosing explant material from mature plants rather than from seedlings or juvenile plants, there is generally a greater risk of microbial contamination.

3.1.3. Pre-treatment of explant material in situ Preliminary decontamination can be initiated on the donor plant growing in vivo with chemical application or physical treatment. Fungicides and/or antibiotics A program of spraying of the donor plant with a fungicide(s) may be undertaken several weeks to a few days prior to taking explant material, depending on the severity of fungal contamination, type of fungicide(s) and concentration(s) used. A n t i b i o t i c sprays can be applied to plants prior to taking material for tissue culture. George (1993) describes several instances where f u n g i c i d e s and a n t i b i o t i c s are combined to reduce external contaminants of cultivated plants prior to sampling for in vitro culture. Whichever anti-microbial compound(s) is used, it should not cause undue stress to the treated plant, or the quality of explant material would otherwise be compromised. However, widespread use of fungicides, biocides and antibiotics in the natural environment should be used with caution as the impacts of these compounds in natural ecosystems is not fully understood. Heat treatment Heat treatment is usually applied to plants suspected of harbouring a viral or a systemic bacterial pathogen (George 1993). Many viruses are killed or inactivated at temperatures as low as 30ºC, while others with higher thermal death points may require higher temperatures for effective inactivation. The upper limits of heat treatment are limited by the plant species or cultivar tolerance to elevated temperatures for extended periods. Hence a regime of alternating periods of high and lower temperatures may be beneficial to allow for the plant’s recovery from heat stress. Details on the production of virus-free plants through tissue culture techniques are reviewed by Horst

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(1988) and Walkey (1985), while virus-indexing techniques are described by George (1993). Irradiation (UV or Gamma-rays) Although used to reduce microbial spoilage of stored fruit and vegetables (Sendra et al. 1996), irradiation requires specialised equipment and facilities and highly trained operators. As a result, irradiation is also expensive in terms of equipment and safety requirements. Irradiation as a routine method for decontamination of plant material is therefore considered impractical. Etiolation The strategy of i n d u c i n g etiolation in donor plants to obtain less contaminated explant material has been successful in a number of cases (Cooper 1987; Murasaki and Tsurushima 1988), even in field-grown plants (Fay et al. 1999). Etiolation is achieved by bagging to exclude light from ends of branches that have been pruned and treated with fungicide to prevent mould development. The bagging material should be porous to allow gaseous exchange and prevent excessive humidity (which is conducive to fungi and moulds). The thin-stemmed, fast-growing etiolated shoots are less likely to harbour microorganisms; bagging excludes a large number of airborne spores from landing on the shoots, and insects which may be vectors for plant viruses or bacteria. A further advantage of etiolation may be a reduction in phenolic compounds in etiolated material (George 1996), which is detrimental to culture initiation. Growth regulators Plant growth regulators can be applied to donor plants to stimulate new shoot growth to provide source material for initiation of tissue cultures. Cytokinin (e.g. kinetin, 6-benzylaminopurine (BAP)) and/or gibberellic acid (GA) can be sprayed on intact plants, or applied in forcing solutions to stimulate new shoot growth from cuttings (Read and Yang 1987). They can also be combined with fungicides and/or antibiotic treatment to further reduce or prevent microbial contamination. Relatively clean explant material can then be taken prior to re-establishment of surface microflora which may result in a reduced need for other sterilisation procedures. 3. 2. In vitro Surface sterilisation treatments for explant material can be applied in a number of ways i. e. as a l i q u i d or (rarely) as a gas and even more rarely as irradiation. The use of gas or irradiation is covered in George (1993) and will not be dealt with here. In most cases sterilisation is performed with liquid chemical sterilant which may be a single or mixture of general biocide(s), fungicides/algicides, or antibiotics. The concentration, duration of treatment, sequential sterilisation, repetition and co-usage of sterilants is highly variable and there are many factors to consider in arriving at the

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optimal sterilising procedure for specific explant material. The effectiveness of a variety of sterilisation protocols employing different chemical sterilants on field-collected native plant material is given in Table 2 and discussed further below. 3.2.1. General biocides George (1993) details the recommended concentrations and exposure times for three chemical biocides ( i . e. sodium and calcium hypochlorite, and mercuric chloride) and the species and type of explant material treated. These sterilants are used at relatively low concentrations (typically 0. 1% for mercuric chloride 0. 5% for sodium hypochlorite (NaOCl) and 3-5% for calcium hypochlorite ranging from a few minutes of treatment for soft shoot material up to 45 minutes for dormant buds and seeds. Several factors need to be considered when choosing an appropriate chemical sterilant, concentration and exposure time. The type of explant available usually predetermines the sterilisation procedure. New apical shoots from disease-free cultivated plants, for example, are soft and relatively microbefree, requiring only minimal surface sterilisation with a dilute but moderately toxic sterilant. At the other extreme, field-collected or older dormant material which is likely to be predated and highly contaminated will require a more rigorous sterilisation protocol, invariably requiring the use of a more toxic sterilant. Chlorine compounds should be effective in most instances but there is a limit to the concentration of NaOCl that can be used on vegetative material before oxidative damage becomes problematic. Mercuric chloride by comparison is highly toxic at a relatively low concentration. This makes a very effective surface sterilant but disposal of spent mercury-containing compounds poses occupational health and environmental hazards. This inevitably constrains the use of to the most difficult-to-sterilise cases, when other chemical sterilants have proved ineffective. The use and efficacy of other surface sterilants are described in the literature (e. g. hydrogen peroxide, benzalkonium chloride, potassium permanganate) but few are reported to be as effective as NaOCl, CaOCl or A different form of chlorine (sodium dicloroisocyanurate) with a low phytotoxic effect has been used successfully as a surface sterilant at high or low concentrations (Parkinson et al. 1996). Many years of practical experience invested on disinfestation of fieldharvested material from a wide range of Western Australian plant species is summarised in Table 2. The data presented illustrates surface sterilisation protocols for field-collected material of a diverse flora. Accumulated experience with local flora is invaluable in the choice of appropriate sterilisation protocols for the explant material. Nearly 70% of the adopted

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procedures resulted in a 50% success rate, which must be considered highly satisfactory for field-collected material. Although it is difficult to make critical comparisons between varied procedures, appears consistently to be the most effective general chemical biocide. Chlorine compounds have also, in many cases, proved efficacious in yielding viable explants that are visibly free of contaminants and these compounds would be preferred. is recommended only in cases of more severe infestation. 3.2.2. Other in vitro disinfestation techniques Thermosterilisation Heat treatment of in vitro cultures is usually aimed at virus elimination in excised shoots and shoot meristems. However it has also been used to rid explants of other microbial contaminants (George 1993). Heat treatment has also recently been combined with salicylic acid treatment (see below). ASA (acquired resistance) The use of salicylic acid (SA) and its derivative acetyl salicylic acid (ASA) as a spray purportedly enhances plant resistance to pathogen attack (i.e. acquired resistance) (Sticher et al. 1997; Mauchmani and Metraux 1998). SA or ASA treatment of plants in vivo may yield explant material that is more tolerant of the sterilisation process and be resistant to infection by surviving contaminating organisms in vitro, but this does not solve the primary problem of ridding the explant of contaminating microorganisms. It is possible that SA- or ASA-treated explants may better tolerate repeated sterilisation before and after establishment in vitro compared with untreated explants. ASA treatment confers greater thermotolerance on potato microplants (Lopezdelgardo et al. 1998) as part of heat treatment for virus elimination. It is possible that this protocol could be adapted to other species, under in vitro conditions, for management of endogenous viral or bacterial pathogens. Sonication Sonication, i. e. the use of ultrasound, can be applied as a pre-treatment to assist in dislodging contaminants and is generally used in conjunction with chemical sterilants (NaOCl, CaOCl or other sterilant) to give the best result (Herman 1997). Electrosterilisation This is a relatively new method of sterilising plant material for tissue culture and is achieved by brief exposure of plant material to a low level of electric current and it has been used with some success for the elimination of virus from potato (Herman 1997).

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3.2.3. Antibiotics Antibiotics are not normally used in plant tissue culture; when they are, they are used ad hoc to prevent, control, or to eliminate persistent bacterial contamination. In theory, an effective antibiotic can be applied prophylactically or therapeutically in plant tissue culture. However, reliance on antibiotics for prophylaxis, as an alternative to asepsis, is questioned as such a practice may encourage the development of antibiotic resistance in contaminating bacteria (Falkiner 1997). Exposures of short duration, e.g. in explant decontamination, appears to be the most appropriate application for antibiotics in plant tissue culture, in instances where surface sterilants alone are not adequate. Antibiotics generally permit temporary control (suppression) rather than complete eradication of bacterial contaminants in infected cultures. Rarely is a single antibiotic effective when several contaminating bacteria are present. Although sensitivity of a contaminant isolate(s) can be determined from its antibiogram (growth inhibition of lawn cultures around paper disks impregnated with different antibiotics), poor tissue absorption and phytotoxicity often compromise antibiotic efficacy. Endosporous bacteria (e.g. Bacillus) tend to persist even when absorption is systemic, as evidenced by reappearance of the same bacterial contaminant when “cleansed” cultures are transferred to a medium without the antibiotic. Gentamycin, rifampicin and cefotaxime are used to treat in vitro cultures of tansy (Tanacetum vulgare L.) infested with several Gram negative bacteria (Keskitalo et al. 1998) with growth retarding effects experienced with both gentamycin and rifampicin, but less so with both gentamycin and cefotaxime. Some antibiotics may have growth enhancing effects such as increased root induction with some genotypes of tansy (Keskitalo et al. 1998). Some endogenous bacteria may have growth enhancing effects, for instance, some Pseudomonas strains have the capacity to produce IAA (indole acetic acid) which promotes rooting and shoot multiplication in Cotoneaster lacteus (Monier et al. 1998), whereas deleterious bacterial strains produced cyanide. Antibiotics in combination with a broad-spectrum fungicide (e. g. benomyl, miconazole) may be useful for decontaminating explants in certain “stubborn” cases. George (1993) and Herman (1996, 1997) cite cases of successful disinfestation with antibiotics (e. g. ampicillin, cefotaxime, rifampicin, and tetracycline) applied either sequentially or in a mixture. The widespread resistance to antibiotics and release (accidental or otherwise) of antibiotic resistant organisms into the environment is a cause for concern. In some cases the use of antibiotics or several antibiotics in sequence or in unison is the only way to get rid of ubiquitous internal

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contaminants in tissue cultures (Herman 1997). Nevertheless the indiscriminate use of antibiotics is not advisable or warranted. Also, some antibiotics are known to be phytotoxic and need to be used with caution (George 1993; Leifert et al. 1994). A prevailing view is that “antibiotics are no substitute for careful aseptic techniques“ (George 1993), and, “it is poor science to develop a micropropagation system which relies on the routine incorporation of antibiotics into the culture medium” (Cole 1996). 3.2.4. Plant Preservative Mixture (PPMTM) In 1996, Plant Cell Technology (PCT), an American plant technology company in Washington USA, introduced a broad-spectrum proprietary biocide (‘PPM’) which it claims can prevent or reduce microbial contamination in plant tissue culture. This biocide contains a mixture of two isothiazolones, methylchloroisothiazolinone and methylisothiazolinone, two widely used industrial biocides (Niedz 1998) which may be beneficial for the control of contamination in in vitro cultures. As the product is relatively new to plant tissue culture applications, much of the technical and scientific information is disseminated through PCT’s Internet website (PCT 1996 onwards), newsletters or via agents. Information on laboratory experience with PPM is exchanged among subscribers to the University of Minnesota’s Plant Tissue Culture Server (PTC 1994 onwards). There has been only one scientific publication on PPM application in plant tissue culture: Niedz (1998) reported on the elimination of contaminating bacteria with PPM “under certain conditions of low inoculum density in citrus protoplasts, callus culture, shoot organogenesis and seed germination”. Isothiazolone biocides (PPM) act by removing protein sulphyryl groups and succinate dehydrogenase, a key respiratory enzyme of bacteria and fungi (Chapman and Diehl 1995). Antibiotics, by contrast, act on microorganisms by inhibiting DNA or protein synthesis, or by altering cell wall or membrane properties (Russell and Chopra 1990). According to the literature from the PTC website, plant tissues are less sensitive to PPM than microorganisms because PPM molecules penetrate bacterial and fungal cells more easily than plant cells owing to differences in their cell wall composition and complexity. Depending on applied concentration and duration of exposure, localised tissue cytotoxicity rather than acute phytotoxicity may be expected in treated plant tissue. Biocidal efficacy can be improved somewhat by judicious reduction of salts and organics (sugars and amino acids) in the culture medium to discourage microbial growth. PCT’s confidence in PPM’s biocidal potency is reflected by its assurance that “PPM-containing culture media can be dispensed outside the laminar flow hood, exposed to ambient air” (PCT 1994 onwards) and that

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membrane-filtering or autoclaving is superfluous for heat-sensitive liquid media containing PPM. However, PCT cautions that PPM is less effective when the explant has a high density of bacteria or fungal spores (i. e. a low ratio of PPM molecules to contaminant microbial cell). A more tangible proof of PPM’s potency is, ironically, not from treated in vitro cultures but in in vivo situations such as direct seed germination on non-sterile filter paper, where spore germination and fungal growth is clearly suppressed (Figure 3). Although many aerobic bacteria, mould and yeast fungi have been reported to be sensitive to PPM (Table 3), its effectiveness against anaerobic bacteria is unknown. The latter may not be relevant, as the anaerobic condition is not typical of the in vitro plant or cell culture environment. The question also arises of whether PPM can become ineffective over time due to mutations in the bacteria and fungi. PCT maintains that “this is unlikely because PPM targets multiple enzymes” (PCT 1996 onwards). The prognosis may be disregarding the occurrence of naturally resistant microbes. A species of Phoma (Figure 4) and several bacteria that appear to be insensitive to PPM have been isolated from explants of kangaroo paws (Anigozanthos spp. ) (B. Tan unpubl. ). The second constraint to PPM’s universal applicability is the sensitivity of some plant species. In Agrobacterium-mediated transformation, for example, exposure to PPM after co-cultivation of host (Ilex and Capsicum) and the bacterium vector revealed detrimental effects on host explant tissue (PTC 1994 onwards, Hu 30/4/98). Freshly prepared protoplasts, for example, are extremely sensitive to very low concentrations of media supplements (Niedz 1998). However, many plant species appear not to be

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adversely affected by PPM at typical recommended concentrations (0. 5-2. 0 ml/L); these include Nicotiana tabacum, Arabidopsis thaliana, Beta vulgaris, Epidendrum spp., Eucalyptus grandis, Nepenthes spp., sweet pepper, Citrus spp. (PTC 1994 onwards). Although procedures for eliminating endophytic bacteria with PPM have been suggested, unpublished results have been mixed or equivocal (PTC 1994 onwards). In kangaroo paws (Anigozanthos spp. ), for instance, it was noted that two four-weekly continuous subcultures on a medium containing PPM (5 ml/L) failed to eradicate certain endogenous bacteria although treated shoots were visibly healthier after they were returned to routine medium (B. Tan unpubl. ). For example, leaf tip necrosis had disappeared and multiplication rate had improved, but leaf tissues were positive when indexed on nutrient agar. It is interpreted that PPM has significantly reduced but not eliminated the endophyte population.

The sterility of explant material after surface sterilisation is critical to successful culture initiation. Residual contamination may be detectable visually or indirectly through indexing. Index-positive or visually contaminated explants are culled at this stage. The prevention of re­ infection (including mite infestation) and maintenance of “clean” cultures at all later stages of propagation should be possible through good hygiene and sound aseptic techniques; in special cases, culture sterility can be maintained by routine incorporation of a prophylactic biocide (such as PPM) in the medium.

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3.2.5. Recovery of contaminated cultures In some cases aseptic material can be rescued from contaminated explants. If the contaminant is external, i. e. has not penetrated into the tissues of the explant, then disinfestation of at least part of the explant is possible, generally the tissues furthest from the site of contamination. A useful method for “rescuing” material that is contaminated is to induce etiolation of apices, axillary shoots or buds so that the sprouting shoot grows rapidly away from the contaminated basal node or shoot stems. This etiolated growth can then be excised (and briefly sterilised as a precaution if desired) and transferred to fresh medium. This method is relatively simple to apply; however, some species do not respond well to etiolation by dark incubation, so there are some limitations (see George 1993). Another method is to combine the above techniques with the use of media supplemented with biocides, fungicides and/or antibiotics or substances like PPM, however, with the removal of the additives the contaminants may arise again. The efficacy of such methods is not well reported. Generally it is better to discard all contaminated cultures but there are some instances, i. e. in the case of rare or extremely valuable clonal material, where attempting to recover contaminated explants is worthwhile. 3. 2. 6. Mite infestation Mite infestation is extremely serious in terms of contamination of previously sterile cultures. Losses due to mite infestation can be devastating if not discovered and appropriate action taken immediately, especially in large commercial operations. Mites can only crawl or be artificially introduced to culture collections, so all means of preventing mites from entering the culture room in the first place and restricting mites crawling from one site to another in the culture room once introduced, is advantageous. Regular cleaning (with a mild disinfectant) of floors is very helpful, as is regular cleaning (with mild disinfectant) of culture racks and shelves. Operators need to ensure all culture containers entering the culture room are clean. Storing media in dusty conditions for long periods of time will risk mite contamination. All contaminated cultures need to be promptly removed from culture rooms and if mites are confirmed or suspected, all associated and adjacent cultures should be quarantined pending disinfestation of the culture shelving and the suspect cultures. If contamination via mites is detected early enough, simple transfer of explants promptly to fresh medium may be sufficient to rescue the explants and maintain asepsis, but a brief disinfestation with a low concentration of CaOCl or NaOCl (and rinsed with sterile water) is recommended as a precaution.

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Prevention of mite infestations is very difficult if strict hygiene standards cannot be guaranteed. Smith (1967) reports the use of the acaracide Kelthane® (active ingredient di-(p-chlorophenyl) trichloromethyl­ carbonol) on cotton wool plugs of test tubes, to kill entering Tarsonemid mites, mite eggs and hatching larvae. However the particular contaminants introduced by the mites still posed a problem, therefore the effectiveness of such chemicals in preventing contaminants from actually entering and proliferating in culture containers must be considered questionable. Nevertheless acaracides could be useful in restricting the spread of mites under conditions of severe infestation. Mites tend to infest the culture containers which offer the least resistance to their entry, which means that Petri dishes, which need to be sealed with plastic films, are generally more likely to acquire mite infestations than more tightly sealed jars and tubes. Mites also appear to have definite media preferences, as plates of potato dextrose agar (PDA) even if sealed with thermoplastic film, still become infested with mites (almost exclusively), in the presence of other plates containing MS (Murashige and Skoog 1962) derived media. It is therefore important to keep plated media separate from other cultures as plates can quickly become a breeding ground for mites. Mite infestations can be managed by strict hygiene, good aseptic techniques, regular spraying of the laboratory and culture room with an appropriate acaracide, separate incubation of plated media (particularly nutrient agars which mites seem to prefer) and constant vigilance to due process. 4. Conclusions Effective disinfestation of plant material for in vitro culture is reliant on many mitigating factors, i. e. type, age, health and predation history of explants, seasonal f l u c t u a t i o n s of surface microorganisms, the type, concentration, toxicity, specificity and duration of sterilant application, and the species and life history of the donor plant. Examples of practical experience with a range of species and sterilisation procedures suggests that effective surface disinfestation of a wide range of field collected material is possible with accumulated knowledge of local flora to draw on. It would appear both from the literature and based on practical experience that common biocides containing sodium or calcium hypochlorite are effective in most circumstances for disinfestation of shoot material. More toxic sterilants such as mercuric chloride are highly effective in many cases but not environmentally friendly and should be used sparingly. Antibiotics are very useful in some cases but not recommended unless absolutely necessary

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owing to hazards of uncontrolled selection for antibiotic resistance in common bacterial and fungal contaminants. Newer sterilising agents such as PPM are still being evaluated and may offer low-cost, easy-to-use preventive measures against accidental infection of sterile cultures. Other sterilising methods have been reported (e. g. sonication, e l e c t r o s t e r i l i s a t i o n ) but these appear to have been used infrequently hence a comprehensive assessment of their efficacy is difficult. Pre-sterilisation treatments such as thermal treatment appear to be useful for limiting viral spread prior to shoot tip culture for some species. The induction of acquired systemic resistance and stress tolerance by compounds such as SA or ASA is an interesting development that may assist with the successful initiation of pathogen-free cultures or maintenance of symptomfree plant propagation material. Maintenance of sterile cultures also relies on many factors, the most important of which are: ensuring good basic aseptic practices at all times in the laboratory and culture room – including preventative measures such as basic cleanliness and spraying regimes for mite control, coupled with vigilance for diagnosing contamination as early as possible, and taking immediate steps to limit the spread. In vitro microbial contamination and control is one of the most important phenomena confronting the tissue culturist. With microorganisms now known to inhabit the inner tissues and intercellular spaces of a large number of plant species, the concept of “sterile culture” appears to be overstated. Leifert et al. (1994) suggests the terms ‘index negative’ or ‘free of detectable contaminants’ if cultures have been indexed for contaminants. Although the number and sensitivity of methods for screening plant tissue cultures for exogenous and endogenous microorganisms is presently considerable and s t i l l expanding, it is the larger laboratories indulging in extensive exporting of tissue cultured plants that will be using such methods on a regular basis, particularly those which require specialist skills. How widespread the practice of disease indexing is in the wider world of tissue culture work is more difficult to quantify. The likelihood is that very little comprehensive in vitro disease indexing is practiced. The reasons for this probably range from the most common, i. e. the tests are too expensive to use (even if facilities are available) for the average tissue culture laboratory, too complicated, or require specialised equipment and technical expertise not readily available; to the fact that many operators just do not see a need for it, especially if their product is used exclusively within the country (or State) of origin, or is for research purposes only. Regardless of the various arguments for or against regular screening/ indexing for microbial contaminants and endpoint usage of the product, it can be argued that unless achievable, practical and uniform standards (national and international) are formulated and that test procedures are truly

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affordable for all operators, there will be a reluctance to embrace in vitro disease indexing. In some cases, e. g. domestic markets, less stringent standards may be acceptable, but exporting tissue cultured plants is very different. Quarantine laws for most countries now encompass requirements for tissue cultured plant material to be free of specified disease-causing microorganisms, and the likelihood is that these laws will become more stringent in the future as countries try to block all avenues for the accidental introduction of exotic plant pathogens. The challenge lies ahead for all plant tissue culturists to be aware of the limitations of their craft in producing truly pathogen-free material and keep abreast of new methods for in vitro disease indexing, particularly if relying on exporting and importing tissue cultured plants. References Atlas RM, Bartha R (1987) ‘Microbial ecology: fundamentals and applications. ’ (Benjamin/Cummings Publishers: Menlo Park) Bove JM, Garnier M (1998) Walled and wall-less eubacteria from plants – sieve-tuberestricted plant pathogens. Plant Cell Tissue and Organ Culture 52, 7–16. Bradbury JF (1988) Identification of cultivable bacteria from plants and plant tissue cultures by use of simple classical methods. Acta Horticulturae 225, 27–37. Bunn E (1994) ‘Micropropagation of recalcitrant Australian plants, with special emphasis on rare and endangered taxa.’ MSc Thesis, Dept. Soil Science and Plant Nutrition, University of Western Australia. Bunn E, Dixon KW (1996) In vitro propagation methods for Blandfordia grandiflora, Hibbertia miniata, Newcastelia chrysophylla and Eucalyptus graniticola (ms.). In (Eds A Taji and R Williams) ‘Proceedings of the Vth International Association for Plant Tissue Culture (Australian Branch) Conference.’ pp. 157–163. Cassells AC (1991) Problems in tissue culture: culture contamination. In (Eds PC Debergh and RH Zimmerman). ‘Micropropagation technology and application.’ pp. 31–44. (Kluwer Academic Publishers Dordrecht) Chapman JS, Diehl MA (1995) Methylchloroisothiazolone-induced growth inhibition and lethality in Escherichia coli. Journal of Applied Bacteriology 78, 134–141. Cole M (1996) Microbial contaminants and aseptic techniques in plant tissue culture. In ‘Tissue culture of Australian plants.’ (Eds A Taji and R Williams) pp. 204–239. (University of New England Press: Armidale, Australia) Collins MT, Dixon KW (1992) Micropropagation of an A u s t r a lian terrestrial orchid Diuris longifolia R. Br. Australian Journal of Experimental Agriculture 32, 131–135. Cooper PA (1987) Advances in the micropropagation of avocado (Persea americana Mill. ). Acta Horticulturae 212, 571–576. Dixon KW, Touchell DT (1995) Cryobiological methods for the conservation of Western Australian plant species. In: ‘Integrated plant conservation in Australia.’ (Ed LD Meredith) pp. 41–46. (Australian Network for Plant Conservation: Canberra) de Fossard RA (1990) ‘Micropropagation.’ (Xarma Pty Ltd: Eagle Heights, Queensland) Dodd H, Taji A, Hayward C, Dodd B (1992) An unrecognised problem in plant tissue culture. Australian Horticulture (Dec.) pp. 46–50. Falkiner FR (1997) Antibiotics in plant tissue culture and micropropagation - what are we aiming at? In ‘Pathogen and microbial contamination management in micropropagation.’ (Ed AC Cassells) pp. 155–160. (Kluwer Academic Publishers: Dordrecht)

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Fay MF, Bunn E, Ramsay MM (1999) In vitro propagation In ‘A colour atlas of plant propagation and conservation.’ (Ed BG Bowes) pp. 97–107. (Manson Publishing London) Gagne S, Richard C, Rousseau H, Antoun H (1987) Xylem-residing bacteria in alfalfa roots. Canadian Journal of Microbiology 33, 996–1000. George EF (1993) ‘Plant propagation by tissue culture. Part 1 The technology.’ (Exegetics Ltd.: Edington) George EF (1996) ‘Plant propagation by tissue culture Part 2 In practice.’ (Exegetics Ltd.: Edington) Goodman RN (1959) The influence of antibiotics on plants and plant disease control. In ‘Antibiotics: their chemistry and non-medical uses.’ (Ed HS Goldberg) pp. 322–421. (van Nostrand: New York) Gunson HE, Spencer-Phillips PTN (1993) Latent bacterial infections: endophytes as contaminants of micropropagated plants. In ‘Physiology, growth and development of plants in culture.’ (Eds PJ Lumsden, JR Nicholas and BJ Davis) (Kluwer Academic Publishers: Dordrecht) Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Canadian Journal of Microbiology 43, 895–914. Harms D, Rohr R (1987) In vitro plantlet regeneration from juvenile and mature sycamore maple. Acta Horticulturae 212, 77–82. Hartmann HT, Kester DE, Davis Jr FT, Geneve RL (1997) ‘Plant propagation: principles and practices’ Edn 6. (Prentice-Hall: New Jersey) Hayward AC (1974) Latent infections by bacteria. Annual Review of Phytopathology 12, 87–97. Herman EB (1990) Non-axenic plant tissue culture: possibilities and opportunities. Acta Horticulturae 280, 112–117. Herman EB (1996) ‘Recent advances in plant tissue culture. Vol. 4. Microbial contamination of plant tissue cultures.’ (Agritech Consultants: New York) Herman EB (1997) ‘Recent advances in plant tissue culture. Vol. 5. New techniques and systems for growth, regeneration and micropropagation 1995-1997.’ (Agritech Consultants: New York) Hollis, JP (1951) Bacteria in healthy potato tissue. Phytopathology 41, 350–367. Horst RK (1988) Production of plants free of virus and prevention of reinfection. Acta Horticulturae 234, 393–402. Keskitalo M, Pohto A, Savela ML (1998) Alterations in growth of tissue-cultured tansy (Tanacetum vulgare) L. treated with antibiotics. Annals of Applied Biology 133, 281–296. Lamb TG, Tonkyn DW, Kluepfel DA (1996) Movement of Pseudomonas aureofaciens from the rhizosphere to aerial plant tissue. Canadian Journal of Microbiology 42, 1112–1120. Leifert C, Morris CE, Waites WM (1994) Ecology of microbial saprophytes and pathogens in tissue culture and field-grown plants: reasons for contamination problems in vitro. Critical Review of Plant Science 13, 139–183. Leifert C, Waites WM, Nicholas JR (1989) Bacterial contaminants of micropropagated plant cultures. Journal of Applied Bacteriology 67, 353–361. Leifert C, Waites WM (1992) Bacterial growth in plant tissue cultures. Journal of Applied Bacteriology 72, 460–466. Leifert C, Woodford S (1997) Laboratory contamination management; the requirement for microbiological assurance. In ‘Pathogen and microbial contamination management in micropropagation.’ (Ed AC Cassells) pp. 237–244. (Kluwer Academic Publishers: Dordrecht) Le Roux JJ, Van Staden J (1994) Micropropagation and tissue culture of Eucalyptus - a review. Tree Physiology 9, 435–477.

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Long RD, Curtin TF, Cassells (1988) An investigation of the effects of bacterial contaminants on potato nodal cultures. Acta Horticulturae 225, 83–91. Lopezdelgardo H, Dat JF, Foyer CH, Scott IM (1998) Induction of thermotolerance in potato microplants by acetylsalicyclic acid and Journal of Experimental Botany 49, 713–720. Maas JL, Finney MM, Civerolo EL, Sasser M (1985) Association of an unusual strain of Xanthomonas campestris with apple. Phytopathology 75, 438–145. Mauchmani B, Metraux JP (1998) S a l i c y l i c acid and systemic acquired resistance to pathogen attack. Annals of Botany 82, 535–540. Meney KA, Dixon KW (1988) Phenology, reproductive biology and seed development in four rush and sedge species from Western Australia. Australian Journal of Botany 36, 711–726. Monier C, Bossis E, Chabanet C, Samson R (1998) Different bacteria can enhance the micropropagation response of Cotoneaster lacteus (Rosaceae). Journal of Applied Microbiology 85, 1047–1055. Murasaki K, Tsurushima H (1988) Improvement on clonal propagation of Cyclamen in vitro by the use of etiolated petioles. Acta Horticulturae 226, 721–724. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497. Niedz RP (1998) Using isothiazolone biocides to control microbial and fungal contaminants in plant tissue culture. HortTechnology 8, 598–601. Parkinson M, Prendergast M, Sayegh AJ (1996) Sterilisation of explants and cultures with sodium dicloroisocyanurate. Plant Growth Regulation 20, 61–66. Plant Cell Technology (PCT) (1996 onwards) http://www. mktechnology. com/ppmweb2. htm Petit RE, Taber, RA, Foster, BG (1968) Occurrence of Bacillus subtilis in peanut kernels. Phytopathology 58, 254–255. PTC Listserver (1994 onwards) http. //www. agro. agri. umn. edu/plant-tc/listserv/ Quadt-Hallmann A, Kloepper JW (1996) Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in different plant species. Canadian Journal of Microbiology 42, 1144–1154. Quadt-Hallmann A, Benhamou N, Kloepper JW (1997) Bacterial endophytes in cotton ­ mechanisms of entering the plant. Canadian Journal of Microbiology 43, 577–582. Read PE, Yang Q (1987) Novel plant growth regulator delivery systems for in vitro culture of horticultural plants. Acta Horticulturae 212, 55–58. Reustle G, Mann M, Heintz C (1988) Experience and problems with infections in tissue cultures of grapevine. Acta Horticulturae 225, 119–129. Russell AD, Chopra I (1990) ‘Understanding antibacterial action and resistance.’ (Ellis Horwood: London) Sendra E, Capellas M, Guamis B, Felipe X, Mormur M, Pla R (1996) Review. Food irradiation. General aspects. Food Science and Technology International 2, 1–11. Singha S, Bissonette, GK, Double ML (1987) Methods for sterilising instruments contaminated with Bacillus sp. from plant tissue cultures. Horticultural Science 22, 659. Smith R (1967) Control of tarsonemid mites in fungal cultures. Mycologia 59, 600–609. Sticher L, Mauchmani B, Metraux JP (1997) Systemic acquired resistance. Annual Review of Phytopathology 35, 235–270. Sturz, AV (1995) The role of endophytic bacteria during seed piece decay and potato tuberization. Plant and Soil 175, 257–263. Taji A, Williams R (1990) Recovering infested shoot cultures. Australian Horticulture 88, 58–61. Tan B (1995) Flower bud c u l t u r e : a microbe-cleansing technique towards sterile in vitro propagation of kangaroo paws. Australian Horticulture 93, 53–58.

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Walkey DGA (1985) ‘Applied plant virology.’ (Heinemann: London) Weller R (1997) Microbial communities on human tissues; an important source of contaminants in plant tissue cultures. In ‘Pathogen and microbial contamination management in micropropagation.’ (Ed AC Cassells) pp. 245–257. (Kluwer Academic Publishers: Dordrecht) Wilkinson KG, Dixon KW, Sivasithamparam K (1989) Interaction of soil bacteria mycorrhiza fungi and orchid seed in relation to germination of Australian orchids. New Phytologist 112, 429–435. Wilkinson KG, Dixon KW, Sivasithamparam K, Ghisalberti EL (1994) Effect of IAA on symbiotic germination of an Australian orchid and its production by orchid-associated bacteria. Plant and Soil 159, 291–295.

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K.Sivasithamparam, K.W.Dixon, & R.L.Barrett (Eds) 2002. Microorganisms in Plant Conservation and Biodiversity. pp. 337–367. © Kluwer Academic Publishers.

Chapter 13 PHYTOSANITARY CONSIDERATIONS IN SPECIES RECOVERY PROGRAMS Giles E.St.J. Hardy School of Biological Sciences and Biotechnology, Murdoch University, Perth 6150, Western Australia.

K. Sivasithamparam Soil Science and Plant N u t r i t i o n , Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley 6009, Western Australia.

1. Introduction Plant species recovery programs are enacted when naturally occurring diversity of a species or population falls close to or below what is considered to be a sufficient size for the species to continue to exist without human intervention. The purpose of these plans is to ensure the long-term survival of the taxon concerned, and where possible, to re-establish self-sustaining populations in their natural habitat. In such an endeavour to increase plant numbers, there exists a risk that seed, soil, machinery and plant material used in the introduction of plants to native habitats will include the introduction of phytopathogens capable of destroying the very population that we are attempting to increase. Alternatively, changes to environmental conditions created by the activity of the program may favour disease development by native or naturalised pathogens. For these reasons, phytosanitation (literally, plant-health-process) procedures are employed to minimise the threat of pathogens already at a site and, more importantly, to prevent infected or infested material entering a site. Development of diseases within plant populations is a natural process and an inherent part of dynamics within ecosystems (Ingram, this volume). Under natural conditions within an area, interactions between the pathogens and host plants are mediated by environmental conditions including soil, moisture, temperature, other organisms and commonly occurring

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disturbances (such as fire in the Australian biota) (Gill et al. 1981; Palti 1981). Hazardous levels of disease capable of seriously reducing plant numbers can arise when the balance w i t h i n a system is modified by introducing hosts or changing environmental factors within the range of an indigenous (or n a t u r a l i s e d ) pathogen or when an exotic pathogen is introduced into a new area (Chesson 2000; Burgess and Wingfield, this volume). This chapter primarily focuses on the interaction of phytopathogens and hosts in an environment and various methods that can be employed to help prevent the development of diseases in species recovery programs. There are three main scenarios to be considered: Firstly, a plant host species being introduced into the present distribution of a phytopathogen. For example, the rust disease of eucalypts newly introduced into South America (see Burgess and Wingfield, this volume). This rust disease does not occur in the region of origin of the eucalypts. Secondly, the introduction of an exotic phytopathogen into an environment. The two most notorious examples of this being the chestnut blight caused by Cryphonectria parasitica and the destruction of susceptible native f l o r a of south Western Australia by Phytophthora cinnamomi (Cook and Baker 1983). The destruction of west European elms by Ophiostoma novo-ulmi (Brasier 1986) is yet another significant event in which the disease onslaught resulted in the removal of a dominant tree species allowing the emergence and replacement by less competitive species. Thirdly, a change in environmental conditions (often caused by human activity) that brings about a disease outbreak in a native plant population by a native phytopathogen. For example, the Cryptodiaporthe canker of Banksia coccinia both of which are native to the south west of Western Australia (Shearer 1994). This disease is currently threatening all natural stands of the host. Although the belated quarantine regulations enacted by authorities world-wide cannot restrain the localised spread of major pathogens previously introduced, regulations will however be most useful in reducing the incidence of new introductions. Equally important are the monitoring and/or avoidance of disturbances that lead to serious disease outbreaks in natural vegetation. Human endeavours have caused major changes to environments around the world. These include intensive and extensive farming, forestry, mining, and clearing for urbanisation and associated transport networks. This constant demand for space has resulted in many detrimental changes to the landscape. These include salinity, rising water tables, eutrophication, and

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pollution. These activities have put many plant species under threat of total or local extinction both directly and indirectly. Consequently, there is a need for species recovery programs to ensure the long-term survival of plant species that are in decline. In developed areas, it is possible to argue that any remaining natural bush land, heath land, forests or woodlands should be treated and managed as a recovery program. Improper management could not only affect the success of such programs but also contribute to the destruction of flora outside the managed areas. Consequently, phytosanitation should be a major factor in all management activities associated with the long-term maintenance of these landscapes. Large-scale conservation programs are not only difficult to manage but are also likely to be plagued by limitation of funds. A species recovery program however could be considered at a much more localised level. These include the (i) protection of a plant or plants in a natural ecosystem that are under threat from an introduced pathogen or pest, (ii) introduction of plants by seed or vegetative material into denuded areas such as rehabilitated mine sites, ex-farm sites, areas impacted by salinity or water-logging or (iii) production of plants in production nurseries. Whatever the reason, it is important to consider the role of plant disease management to ensure effective and sustainable recovery programs. Plant diseases must be managed to ensure that they do not spread from pathogen infested into noninfested areas. In addition, the ecosystem being managed must be treated in such a way that environmental factors such as receding or rising water tables, salinity or fire do not predispose plants in that ecosystem to attack by infectious agents. Plant diseases pose great threats to plant conservation. In many i n s t i t u t i o n s and enterprises dedicated to the conservation of rare and endangered flora, diseases threaten not only in the propagation of plant material in the laboratory and glasshouse but also in the field introduction of the cultured plantlets. Thus methods have to be developed to minimise disease hazards both during and after recovery of such threatened flora. Importance of these strategies is relevant to horticulture and forestry e.g. propagation of rare plant species with potential for large-scale commercialisation of the material for floriculture and tree plantations. Such recovery could also be relevant to propagation of native species for the rehabilitation of disturbed land such as mined sites. 1.1. Leaf and stem diseases Leaf and stem diseases of plants cause considerable damage to herbaceous and woody plants. In comparison to agricultural and horticultural crops relatively little is known of the diseases of rare and endangered plants. For

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instance, the examination of herbarium material of Western Australian orchids revealed the existence of several unrecorded rust diseases of Australian orchids, some of which affected certain endangered orchid taxa (Nichol et al. 1988). More recent monographs (e.g. on fungi recorded on Eucalyptus spp., see Crous 1998) should enthuse future studies on diseases of such rare taxa. Recent studies also indicate the existence of several canker fungi which have a significant latent (endophytic) phase in woody plants which could make its diagnosis difficult at quarantine points world wide (see Burgess and Wingfield, this volume). These examples indicate that the lack of accurate disease records from countries of origin and the existence of asymptomatic infections in plant materials imported for species rehabilitation purposes may become sources of invasions by exotic pathogens with undetermined host ranges. 1.2. Historical disasters Two pathogens that have caused considerable damage to native flora of Western Australia are Phytophthora cinnamomi and Cryptodiaporthe semiperda. P. cinnamomi was apparently introduced with horticultural plants (Zentmyer 1980). Phytophthora species in general thrive under conditions that prevail in production nurseries (Hardy and Sivasithamparam 1988). The infected plants and/or infested soil could be sources of spread of the fungi which can then be distributed to a wide range of geographical regions (Shearer and Tippett 1989). The introduction of P. cinnamomi has caused large scale destruction of a wide range of susceptible flora as the native species were not naturally selected for resistance to this exotic pathogen. C. semiperda on the other hand appears to be endemic to Western Australia causing major destruction only of Banksia coccinia in natural stands in the lower South West of the state (Shearer 1994). As it does not appear to have a wide host range, the origin of C. semiperda is not clear, nor why the epidemic occurred only recently. Since this pathogen spreads by rain splash and wind, it is difficult to control. There is some suggestion that the use of fire could be used to reduce or eradicate the inoculum potential of diseased stands as the inoculum forms in dead branches of the host. This in turn would reduce spread between infested and non-infested stands and possibly in burnt stands with seedlings or resprouting plants (Shearer 1994). 1.3. Factors affecting species recovery – disease and disturbance 1.3.1. The disease triangle In order to consider the development of a disease outbreak in an ecosystem and its subsequent control, it is useful to consider the potential impact of disturbance on the role of environment in the plant disease triangle (Figure 1). All major disease outbreaks result from an interaction of three factors,

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(1) the presence of susceptible hosts, (2) a pathogen that is capable of infecting the host, (3) environmental conditions that favour infection and subsequent reproduction and spread of the pathogen (Agrios 1978). The disease is invariably most severe when there is inadequate time for the host to respond to the pathogen while sufficient for the pathogen to reproduce.

Major disease outbreaks result if all three factors are favourable for disease development, such as an abundance of susceptible hosts, the presence of a virulent, aggressive pathogen and environmental conditions that favour the rapid multiplication, spread and invasion of the host plant by the pathogen. Consequently, a disease can often be managed by changing the dynamics of one or all of these factors. In order to manipulate the triangle to manage a disease, it is critical that the biology, ecology and pathology of the pathogen and how it interacts with its hosts and the environment are well understood (van der Plank 1982). Without this basic and fundamental knowledge, management or control of the disease cannot be undertaken effectively, whether in natural ecosystems or in rehabilitated sites. 1.3.2. Susceptible hosts Susceptible plants are those that provide a food source for the pathogen. In recovery programs, plants are often grown in large numbers in a limited space in plant production nurseries. Some plant species are more susceptible to disease than others. Plants can also vary in their susceptibility to a pathogen according to their age and physiological status. For example, new growth may be more susceptible to a pathogen than older growth. In a

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species recovery program, it is therefore useful to determine what pathogens the plants are susceptible to at particular growth phases and whether they are likely to come in contact w i t h these pathogens in the nursery or once established in the field. 1.3.3. Capable pathogens Pathogens are l i v i n g organisms that cause disease. A pathogen must be present for disease to occur. The quantity of the pathogen and its ability to cause disease (virulence) directly affects the occurrence of a disease and its severity. Some pathogens such as Botrytis cinerea (grey mould) or Phytophthora cinnamomi (root and collar rot pathogen) can cause disease in many different kinds of plants (Cook and Baker 1983; Ristaino and Gumpertz 2000). Other pathogens such as powdery mildews tend to be host specific (Agrios 1978). Once present, pathogens have to be able to enter the host, reproduce, and disseminate their infective propagules to other plants. Some pathogens are spread in water splash while others produce spores that are wind-borne (Agrios 1978). Of note is that some pathogens can be spread during propagation in or on plant parts during nursery operations (Baker 1962; Cook and Baker 1983). 1.3.4. Favourable environment The environment is made up of all the factors and conditions that affect the growth and development of l i v i n g organisms including the host plants and the pathogens. Relevant factors include light, temperature, nutrition, moisture and physical and chemical characteristics of the soil or substrate in which the plant is growing. Other factors include insects, animals and the management activities of people. All of these factors influence both the pathogen and the host plant and how they interact with each other. However, where a host has no resistance to a particular pathogen, a wider range of environmental conditions are conducive to the disease than when the host has some level of resistance (van der Plank 1982). Temperature influences the growth and development of both the host plant and the pathogen. Some pathogens are most pathogenic during cool weather, while others many be active at high temperatures. Many pathogens develop during periods conducive for disease when temperatures are moderate, the occurrence of rainfall is frequent and plant growth is rapid (Agrios 1978). Moisture in the form of h u m i d i t y , rain or irrigation (in the case of container grown nursery plants) is critical in the development of most infectious diseases. There is an optimum amount of water required for healthy plant development. Increasing or reducing the amount of water

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available to the plants tends to predispose them to disease (Handreck and Black 1989). 1.3.5. Sufficient time Disease development can be strongly influenced by time. For example, the longer plants remain in containers in a nursery, the more likely that all conditions required for disease development w i l l occur simultaneously. All pathogens require time to infect and develop in a host to a stage that allows it to spread. The longer conditions remain favourable for the pathogen (fungal spread us generally seasonal), the more severe will be the disease resulting from the progressive build-up of inoculum (Baker 1962). 1.4. Disturbance Disturbances are processes that change an ecosystem beyond those conditions considered normal. For the purposes of this chapter we will consider those disturbances that tend to leave the ecosystem permanently changed. Disturbances are discrete or gradual processes that change ecosystem characteristics beyond what is normal within a human timeframe. Disturbances are commonly classified as natural or anthropogenic although this line can be quite blurred as in the case of forest fires (human intervention in burning some areas and preventing fires in other areas are both disturbances). Both natural and anthropogenic disturbances occur on a variety of scales with the overall impact of the disturbance including a time component and whether the disturbed ecosystem will ever fully recover from the process. All disturbances change at least one of the three factors considered above in the disease triangle, and thus affect all three factors as they are inter-linked. Some of the most profound changes to ecosystems that have occurred w i t h i n a human timeframe have been due to the introduction of new plant or pathogen species (see Burgess and Wingfield, this volume). Natural disturbances (which by default illogically assumes humans are not part of all ecosystems) are those that appear to not be directly attributable to human activity. These occur on a wide range of scales from small individual tree falls in a forest to volcanic destruction, with fire (common in Australia) being somewhere in between. In some cases, ecosystems have adapted to a relatively frequently occurring natural disturbance (within a timeframe long enough for natural selection to have occurred). An example of such an adaptation is the adaptation of flora of mediterranean environments to periodic fire events (Gill et al. 1981). In extreme cases, preventing these disturbances w i l l change the ecosystem beyond what is normal and can therefore be considered a disturbance in itself! A good example of this is the natural / aboriginal fire regime in some Australian

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ecosystems, where seed germination requires fires, without which the species composition shifts (Bell et al. 1984). As diseases are a natural part of ecosystems, it is possible for major disease outbreaks of naturally occurring diseases on native plants to result from natural disturbance. Anthropogenic disturbances include obvious destructive disturbances such as logging, clearing, pollution, rising water tables and the like. In a similar manner to natural disturbances, anthropogenic disturbances occur on a variety of scales, i n c l u d i n g bush-walking, bee keeping and wildflower picking. What is not so obvious in this is that while logging appears more destructive, in the longer term and with careful management the forest can potentially regenerate. Where as while hiking appears to be a lower scale disturbance, it is possible that the introduction of a disease spread on hiking boots can be more destructive in the long term. This is not to say that logging is good and h i k i n g bad, but rather as an example of the innocuous nature of plant pathogens in a wider vision of disturbance and conservation. It also points out how critical the need for phytosanitation is in preventing large scale, permanent disturbances in an environment. 1.5. Species conservation and disturbance Mining operations often cause large shifts in the composition of plant species native to the site. This may be an outcome of changes in the environment but also in the flora and fauna associated with the environment prior to the disturbance. Disturbance can shift interactions of host, pathogen, environment or all three. Hutton et al. (1997) found that Western Australian members of Ericaceae (Epacridaceae) took about twelve years to re-establish by natural means in the soil environment following mining operations. This delay in reestablishment is considered to be due to the time taken for the mycorrhizal associates to re-colonise the site. In Western Australian sites where Eucalyptus marginata and associated flora are severely affected by Phytophthora cinnamomi, affected areas are often dominated by rushes and sedges (Websdane et al. 1994) especially where the death of large trees results in the opening up of the canopy which can further encourage the activities of the pathogen. In Victoria, Australia, the death of the overstorey species results in the exposure of ground infested by P. cinnamomi to sunlight which render the soil warm and conducive for the saprophytic activities of the pathogen (Weste and Monks 1987). In either case, ecosystems as a unit can be remarkably resilient in response to disturbances. This in itself can be a problem as a decline in host numbers can be balanced by an increase in another species tolerant of the changes. Ecosystem disturbance can be the result of either direct human activity or natural processes. Human instituted disturbances to an ecosystem may be minimal (e.g. bush-walking, bee keeping and wildflower picking) or may be

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considerable such as those resulting from broad-scale agriculture, selective logging or clear f e l l i n g of timber, or mining. The effect of such localised disturbances can be m i n i m i s e d by developing management plans that quarantines the affected areas (Shearer and Tippett 1989) and the reduced need for b u i l d i n g roads in and around national parks and reserves. Whatever the level and source of disturbance, the dynamics of the ecosystem will change as a result of the disturbance. Disturbances at a minor scale can at times lead to a major catastrophe. For example, as mentioned above, bush walkers in the montane vegetation of the Stirling Ranges National Park in the southwest of Western A u s t r a l i a probably transported the soil-borne Phytophthora cinnamomi on their boots into the upper reaches of the Park. Consequently, over 70% of the park is now infested by the pathogen. This has had a profound and devastating affect on the many susceptible plant families such as the Proteaceae, Ericaceae (Epacridaceae), Xanthorrhoeaceae and others. There is now a change in the flora in this park from woody perennials to rushes and sedges (Wills 1993). The building of firebreaks and the use of contaminated water for fire control can also inadvertently spread P. cinnamoini through vegetation in this region. Stands of many susceptible plant species such as Banksia brownii that are endemic to the region face extinction as all known populations or i n d i v i d u a l s are only known to occur in infested areas (S. Barrett, pers. comm.). The conservation of such stands represents a substantial challenge in recovery programs. Again, using P. cinnamomi as an example, the erection of a road through the Fitzgerald R i v e r National Park, one of the most biodiverse regions in the lower south-west of Western Australia using heavy equipment is known to have resulted in the contamination with P. cinnamomi into an otherwise disease-free area ( W i l l s 1993). The pathogen is spreading slowly but passively along this road and into the native vegetation that consists of many susceptible plant species. The pathogen can move by root to root contacts, by the movement of animals or alternatively passively with water movement for very long distances (Shearer and Tippett 1989). Therefore, what may appear initially as a small disturbance with potentially little impact has the potential to result in major structural changes to a plant community. The need for recovery programs and appropriate phytosanitary practices in this situation is considerable. Currently, there are no completely effective treatments that can stop this spread, and in the long-term large areas of the park w i l l be impacted. This situation again poses considerable challenge to land managers who wish to conserve those species being impacted directly and indirectly by the pathogen. In such situations when a pathogen is first detected and while the extent of its impact is minimal it may be necessary to consider extreme measures in attempts to eradicate the pathogen. For example, is it feasible to remove and burn all plants in the affected area and

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in an extensive buffer zone surrounding the infested area? Could the bare soil possibly be fumigated, and/or solarised, and/or treated with chemicals or bio-fumigants, or kept bare for a number of years? Such questions (probably never previously addressed) need to be considered as an initially extreme measure over a relatively small area. Though costly and risky, such a strategy could help the eradication of the pathogen. By making no attempt at all, we would allow the pathogen to eventually move passively over a large region and have considerable effect on plant species, especially those that are endemic to an area and susceptible to the pathogen. Although a disturbance may i n i t i a l l y appear m i n i m a l , it might under optimum e n v i r o n m e n t a l c o n d i t i o n s in the presence of an introduced or endemic plant pathogen result in profound changes to the native plant communities. In undisturbed natural ecosystems, there tends to be stability in the relationship between the resident pathogens, non-pathogenic microorganisms associated with plant surfaces (Andrews and Harris 2000) and their plant hosts and therefore major plant disease outbreaks are rare (see Ingram, this volume). It is not until activities such as logging, mining, intensive agriculture, or other disturbances to the environment that disease related problems become e v i d e n t . The need for a species recovery program is usually the result of one or more of the above.

2. Development and spread of new strains – an example of human disturbance creating new disease or severe disease outbreaks The movement of infested soil or plant material whether knowingly or inadvertently can result in the development of new and virulent species or hybrids. It is d i f f i c u l t to monitor entry of exotic pathogens that may be bought in passively in soil or in asymptomatic plant hosts. Entry of novel strains of extensive pathogens can also cause serious problems. New combinations can develop that may create hitherto unrecorded virulence spectrum in common pathogens (see also Burgess & Wingfield, t h i s volume). A good example of this is the alder hybrid Phytophthora which has been shown to be comprised of a range of heteroploid species hybrids (Brasier et al. 1999). A common standard hybrid occurs across most of Europe (Brasier 2000). Evidence based on ITS profiles indicates that the DNA signatures of these isolates are of more than one species, and the likely parents are P. cambivora and P. fragariae (Brasier 2000). These virulent and aggressive hybrids are capable of attacking and causing disease in the riparian Alder (Alnus glutinosa) and the horticultural and woodland alders, i n c l u d i n g A. incana and A. cordata in England, Scotland and Europe (Brasier 2000). This pathogen can move rapidly in waterways and will potentially have a major impact even on the integrity of rivers and streams due to the loss of the riparian Alder that holds the river banks together by an

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extensive root system. The current loss of Alder along UK rivers is estimated to be c. 2% per annum (Gibbs et al. 1999). Cryphonectria parasitica, the causal agent of Chestnut blight in the USA is another interesting example where a strain avirulent on an Asian chestnut species was introduced inadvertently on plant material into the New York Botanic Gardens. It spread rapidly along the eastern states of the US destroying much of the native species of chestnuts (Anagnostakis 1982). Current molecular methods help in the determination of strain characteristics of pathogens from various geographical regions of the world. Despite these advances it is likely that massive world-wide movements of infected plant material prior to the enforcement of quarantine regulations, and centuries, would make it difficult to trace the especially in the origin and genetic development of strains of cosmopolitan fungi. 3. Components of species recovery programs Recovery programs require careful planning and cover monitoring and implementation of a number of steps. Field sites in the program should be secure and protected by fencing or by the choice of an isolated locality. The sites should be free of other growth limiting factors such as salinity, and have secure tenure. It should be within a healthy ecosystem without significant threats from weeds and be large enough to sustain a stable genetic community. These sites could be located either in undisturbed or disturbed locations. If the recovery in the initial test sites appear to be successful, further propagation and introduction of promising provenances could be carried out. The aim of these recovery programs should be clear. For instance, recreating a niche is not only expensive, but can be beset by practical problems related to environments. This can be particularly challenging in the rehabilitation of mined sites. Ecological restoration of the flora may be adequate if the level of diversity and sustainability of the ecosystem is maintained. Therefore, in the choice of planting materials, consideration needs to be given to maximising the genetic diversity and the plants should be appropriate ecotypes or provenances tolerant of resident pathogens and free of disease themselves. Following planting out, there is a need for monitoring to follow performances of the various components of the flora. This would help in developing strategies to expand the area in the program or to find alternative ones that may contain components of the introductions that failed initially. 4. Management of diseases in species recovery programs Management practices for minimising disease hazards require surveillance and application of treatment procedures from the early stages of propagation

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in the nursery to the stage where the plants continue to be protected in the field through to reproductive maturity. Pathogens can be introduced to a natural environment in a variety of ways. At production phase, these include pathogens carried in seed or other propagation material, through entry into nurseries of infested container grown plants, through movement of infested soil, sand, infested bark or wood wastes used as potting mixes for mulches, infested water from dams or other water bodies, and other inadvertent human activities where suitable hygiene or quarantine procedures break down (Sivasithamparam and Goss 1981). The majority of nursery diseases are caused by fungi, including species of Alternaria, Colletotrichum, Cylindrocladium, Fusarium, Mycosphaerella, Pythium, Phytophthora, Rhizoctonia and Botrytis cinerea. These diseases can occur any time from germination (damping-off) through to the hardening-off stage, immediately prior to their dispatch for planting out. Prophylactic sprays can be expensive and sometimes ineffective unless the diseases and the life cycle of specific pathogens are understood. Protecting large trees can also make effective application of pesticides difficult. Low volume aerial sprays with phosphite using light aircraft have been successfully used to contain Phytophthora root rot of Banksia coccinea in natural stands in Western Australia (Komorek et al. 2001). 4.1. Seed Seeds may be passive carriers of bacterial, viral, fungal and nematode pathogens (Neergaard 1977). Pathogens present in seed can spread with seed into new areas and can be a major source of pathogen introduction. Seed-borne pathogens can cause damping-off, blight, wilt, anthracnose, leaf spot, and cankers (Maude 1996). Consequently, it is important to ensure that seed is pathogen-free when used for recovery programs, whether initially to start nursery stock or for their in situ establishment. There are many fungi that can destroy seed during its development on the plant, during storage, after sowing or during germination (Anderson and Miller 1989; Maude 1996). The site of infection/infestation in a seed is largely determined by the source of the pathogen. Some are a result of superficial contamination from soil, while certain pathogens that are systemic in a host find it easy to lodge themselves in the embryo. Control of seed-borne pathogens can be considered in terms of exclusion and elimination of inoculum (Neergaard 1977). Exclusion strategies include the isolation of seed production areas and breeding for resistance to seed-borne infections (such as in the cases of virus infections). Application of these two strategies in recovery programs should be restricted as they limit or modify the genetic integrity of the species. Elimination strategies include the control of organisms by treatment with fungicides of seeds and other plant propagules. Where there are

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responses, systemic or non-systemic fungicides may be applied to the seed prior to sowing. Observation of appropriate hygiene during harvesting and processing and the use of low temperatures and low humidity environments during storage best control seedborne fungi. It is beneficial to collect seed whilst it is still held in capsules or within fruits on the plant, as once on the ground, the seed becomes readily contaminated by soil-borne pathogens. It is also advisable to check the seed before sowing and reject infected seed prior to sowing or treat with seed protectants such as benomyl, carboxin, triforine, chlorothalonil, thiram and captan (Chalermpongse 1987). Hot water treatments (50°C for 5-20 minutes), surface disinfection (10% sodium hypochlorite or 33% hydrogen peroxide for 1, 2 or 4 minutes) and fungicide application (1% captan) have been used to restrict fungal development on eucalypt seed (Donald and Lundquist 1988). While these methods are adequate to minimise seedling deaths the use of very sensitive detection methods such as enzyme-linked immunosorbent assays or PCR based methods are required to clear seed or other propagules of the presence of exotic quarantinable diseases. 4.2. Nursery practices favouring diseases Plant production nurseries are a key step for the production of seedlings or plants from cuttings in many recovery programs, consequently the control of plant diseases in the nursery are critical. Cultivated plants are usually more susceptible to disease outbreaks than their wild relatives. This is partly due to the large numbers of the same species or clones being grown densely packed together within propagation areas. Under these conditions, pathogens can often establish themselves, sporulate and spread rapidly. As already mentioned (Figure 1), plant diseases are a result of interactions between pathogens, hosts and the environment. In the nursery, severe stresses may be imposed on a host because of a space limitation, use of extensive monocultures, the ability of the grower to increase productivity beyond normal limits through water, temperature and nutrient management and due to poor or inadequate hygiene. Losses of planting stock in the nursery and subsequently after ‘planting-out’ can be severe if basic hygiene practices and environmental conditions that predispose plants to root or foliar pathogens are not managed. Nurseries can provide conditions that are ideal for the development of major disease outbreaks. These include inadequate physical, chemical and biological characteristics of the container substrate(s) used, the use of pathogen infested water, inadequate air flow, poor hygiene and poor quarantine practices or from the introduction of pathogens from surrounding areas or from soil, seed or vegetative planting material introduced into the

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nursery (Sivasithamparam and Goss 1980). Nursery diseases are best avoided, however, to do this a thorough understanding of the ecology of the pathogens and their commensal microorganisms is required. Implicit in this understanding is the impact of environmental stresses on both the pathogen and the host, as well as stresses on and imposed by other microorganisms. Intensive nursery production of plants does impose its own set of stresses on all three components of the host-pathogen-environment disease triangle. 5. Integrated disease management The disease triangle (Figure 1) illustrates the importance of deploying a wide range of management practices to prevent disease or keep it at minimal levels (Agrios 1978). There is no one practice that controls all plant diseases in a nursery program. By adopting an integrated approach and utilising a number of control strategies it is possible to (1) reduce the overall incidence and severity of diseases, (2) reduce the chances of major disease outbreaks, (3) improve the quality of plant material being distributed for the recovery program, (4) minimise the need for chemical treatments, and (5) reduce the chances of developing chemical-resistant pathogens. Implementing the above also increases the efficiency of the nursery and subsequently its profits. Good sanitation and cultural practices carried out stringently and consistently w i l l have some effect on diseases of plants grown in the nursery. It is also important to be aware of symptoms and signs of disease in the nursery. This involves monitoring for disease visually by focusing on plant health and understanding the nature of each of the plant species involved. Observations should be made on all aspects of plant health. For example, general plant vigour and growth rates, leaf colour and size. The presence of leaf spots and blights, collar rots and wilt should be noted. Where necessary plant and container substrates should be analysed for pH, cation exchange capacity and nutrient levels to enhance plant growth and vigour necessary for defence against pathogens. Suspect samples of roots or leaf material should be sent to diagnostic laboratories for analysis so that appropriate treatments can be applied as rapidly as possible. Concurrently, thorough written and photographic records should be maintained for each species. A historical database can be developed where symptoms and signs of plant disease and their causative agents are linked with cultural practices and with environmental factors such as rainfall, temperature and humidity. It then becomes possible to l i n k predisposing cultural practices such as fertiliser regimes, fungicide and pesticide applications, container substrates, and changes in environmental factors with disease outbreaks. The manager then becomes aware of the pathogens that cause major problems and the plant species affected. Patterns and timing of disease development within

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the nursery can be established that can be related to the factors that are associated with severe disease problems. 5. 1. Wood and bark mulches The use of wood and bark mulches whether in propagation or for weed/moisture control in planting out needs to be carefully considered for the following reasons. Such material can contain a range of soil-borne or air­ borne pathogens that could adversely affect recovery programs. Material that is not composted or adequately aged can cause nitrogen ‘draw down’ or in the release of phytotoxic compounds that are detrimental to plant establishment and growth. Wood and bark wastes should be adequately composted to ensure that any potential pathogens such as species of Phytophthora, Pythium, Armillaria etc are killed. Composting is a complex process, that combines thermal eradication of a portion of microflora in the organic waste with certain physical and chemical properties of the compost that are valuable in disease control (Hoitink and Fahy 1986; Hardy and Sivasithamparam 1991). Composting involves placing mulched material in windrows, maintaining adequate moisture levels and applying nitrogen and phosphate to facilitate microbial a c t i v i t y . For rapid and appropriate composting, the material should be maintained at between 38°C and 55 °C and turned-over regularly. In the presence of adequate moisture and temperatures, the composting process results in the disinfestation of the material. 5.2. Exclusion A highly effective way of maintaining a disease-free nursery is the exclusion of pathogens. Exclusion is a very effective method of disease control of fungi that are not airborne and have a limited host range. It is a very costeffective practice. The regulatory inspection and certification of disease-free plants and plant parts (cuttings, tissue cultures, seed, bulbs and corms) are a critical component of a disease management program for fungal diseases. 5.3. Sanitation strategies An effective sanitation program should be considered as the most important management practice in a nursery. It should ideally be effective against all major diseases. In particular, it is invaluable for the control of root and stem diseases. Sanitation i n v o l v e s all practices that are aimed at reducing or e l i m i n a t i n g the amount of i n o c u l u m present in the nursery. Sanitation cannot be observed in a haphazard fashion. This will subsequently prevent the movement of disease out of the nursery into disease-free areas and into recovery programs. Without a sound sanitation policy, no nursery will remain disease-free for long (Baker 1962).

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The prevention or minimisation of disease is a much more effective strategy than reacting to a disease after it is established, which in any case is often too late. Any sanitation program must be designed to reduce the amount of inoculum in the nursery by removing dead and dying plants or plant parts and to reduce any potential carryover inoculum between crops. There are a number of fungal pathogens such as Phytophthora cinnamomi, Rhizoctonia solani, and Sclerotium spp. that are not airborne and are therefore unlikely to be introduced into a nursery in air currents. Therefore, their presence in a nursery w i l l be due to poor nursery hygiene practices. Stopping the movement of such pathogens out of the nursery will reduce the distribution and incidence of the disease (Hardy and Sivasithamparam 1988). Air-borne or splash-borne diseases can be a threat at production or plantation levels (see Burgess and Wingfield, this volume). Management of these diseases is economical only through the use of disease resistant host material. Fungicides are expensive and may be ineffective. For effective sanitation practices to be undertaken it is critical that the nursery managers and personnel understand the scientific principles behind effective disease management. Most important is the exclusion of pathogens and in particular root and stem pathogens that tend to be soil-borne. These pathogens appear in the nursery from known sources. These include (1) infested container substrates, (2) infested water sources, (3) poor or inadequate hygiene practices during propagation, (4) media and containers being placed on the ground in contact with soil and (5) the use of infested seed, cuttings, bulbs or other plant material. Ultimately, sanitation involves a series of different but linked functions w i t h i n the nursery production chain. 5.4. Cultural practices Growers must concentrate on providing conditions for the optimal growth and development of their plants and at the same time avoid conditions that favour pathogens (Palti 1981). The majority of pathogens can only penetrate plants when their surfaces are wet. Consequently, many diseases, especially foliar diseases caused by pathogens such as Botrytis cinerea and Cylindrocladium spp. can be managed by avoiding the use of overhead irrigation, or irrigating at times when the plant foliage can dry rapidly. Many opportunistic pathogens can only infect plants when they are wounded or injured in some way, so avoiding injuries will reduce disease. Preventing the build-up of humid micro-climates in seedling trays or closely spaced pots as plants grow, by increasing spacing and airflow will dry out wet foliage more quickly, reduce humid microclimates and reduce the chances of foliar pathogens causing major problems. Finally, the removal and destruction of diseased plants or plant parts will reduce inoculum sources and subsequent disease outbreaks.

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5.5. Container substrates The use of appropriate container substrates is vital (Handreck and Black 1989). Container substrates vary in form depending on cost and on availability. These include sphagnum peat, composted or aged hardwood or softwood barks and sawdust, coarse sand, perlite, plastic beads, coconut coir and many other substrates. All of these substrates will perform adequately as long as they have the right physical, chemical and biological characteristics for plant growth for the life of the plant whilst in the pot, and are pathogen free. If any one of these characteristics is less than optimum, plants can be predisposed to disease, through waterlogging, drought or inadequate or excessive supply of nutrients. From a physical point of view, the substrate must provide anchorage for plant support and it must regulate the supply of oxygen and water to the roots. In nurseries plants tend to be grown in small, shallow multi-celled trays or containers that create physical constraints to plant growth. Firstly, the volume of substrate and water available to the plant in these containers is small. Secondly, since the substrate is contained in a shallow layer, a ‘perched’ water table is created (Handreck and Black 1989; Bunt 1982). This prevents adequate drainage and makes the substrate wetter than it should be immediately after irrigation. There is less oxygen available after every irrigation or rainfall event. In addition, under dry conditions shortage of water can occur rapidly especially during periods of high evapo­ transpiration. Plants can be predisposed to foliar and root diseases as a result of these rapid wetting and drying events. Consequently, it is important to formulate a container substrate that has a total pore space of approximately 85%, airspace between 25-30%, easily available water of 25-30%, and a water buffering capacity of around 10% (De Boodt and Verdonk 1972). Consequently, particle size of container substrates is critical. The physical and chemical characteristics of pine and other bark substrates are affected by age of the material and by how the material is handled during mixing of the ingredients. When using wood or bark based products it is important to have a C:N ratio of approximately 30:1 to avoid nitrogen draw-down. However pine bark, even when composted, may not have a C:N ratio this low due to it being mainly composed of lignin, not cellulose. The chemical characteristics of container media are also a critical consideration in the healthy production of plants. The optimal range for organic potting substrates is pH 5.0 to 6.0. Cation exchange capacity should be between 6-15 milli-equivalents (meq) per (Handreck and Black 1989). All handling and storing of each of the ingredients of the container media should be undertaken with proper care. The media should be stored

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on sloping concrete surfaces above soil level, to prevent surface water “runoff” from surrounding areas contaminating the media. The media should never be mixed on the ground, and the machinery used to carry and mix the media should be clean. If containers are recycled they should be cleaned and sterilised prior to being reused. The container growing areas should be constructed in such a way that containers never come into contact with contaminated soil. Above ground benches, or coarse gravel beds allow for rapid drainage of free water. They also reduce the chances of soil splash and thereby reduce the chances of disease spread. Some nurseries use tightly woven plastic ground cloth directly over soil surfaces, which are an adequate surface as long as the soil underneath rapidly drains. If ponding of water occurs on the surface of the cloth, then the chances of water-borne fungi coming into contact with the plants is greatly increased. 5.6. Disinfestation of pathogens from container substrates The removal of potential pathogens from substrates that are to be used as ingredients in container substrates is essential. Substrates such as washed river sand, sawdust, and crushed bark all carry a risk of containing pathogenic organisms such as species of Pythium, Phytophthora, Fusarium and Rhizoctonia. The removal of potential pathogens from these substrates is a major cost in nursery production. Common methods involve the use of soil fumigants, steam pasteurisation or composting. Microorganisms are more susceptible to heat or fumigants when they are in an active metabolic state, however, many pathogens can form resistant resting structures such as oospores, chlamydospores, or sclerotia in dry and cool substrates. Therefore, effective removal of inoculum is best undertaken when the container substrates are warm and moist. Composting of potting materials may be useful in the destruction of certain pathogens (Hoitink and Fahy 1988). 5.7. Steam pasteurisation Heating soil or container substrates to approximately 60°C with aerated steam for 30-60 minutes and held at 60°C for 30 minutes kills most pathogens without creating a biological vacuum (Baker 1962). Beneficial organisms that form resistant spores survive the pasteurisation. This leaves the soil in its naturally suppressive state with its complement of saprophytic microflora. 5.8. Fumigation Fumigation involves infiltrating a substrate with the vapour of a volatile chemical. Chemicals used include metham sodium, methyl bromide, methyl

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bromide-chloropicrin mixture, chloropicrin, dazomet, ethylene dibromide, and 1, 3-dichloropropane-dichloropropene mixture (Jarvis 1992). Not all of these are registered pesticides in many countries. Methyl bromide is the most widely used fumigant in nurseries, however it is progressively being banned from use in many countries due to its adverse effects on the ozone layer. Fumigation shows varying levels of effectiveness depending on the pathogen(s) being controlled and can leave residues that can have adverse side effects in terms of phytotoxicity and residues that can progress through the food chain.

5.9. Soil solarisation Soil solarisation (Katan 1981) utilises heat from the sun to raise the temperature of the substrate being treated to levels that are sufficient to kill pests, diseases and many weed seeds. The soil or container substrate is layer of moistened to field capacity and then covered with a thin transparent polythene. Care is taken to minimise air spaces between the polythene and substrate to prevent an insulating effect of the air space. The longer the substrate is treated at high temperatures, the more effective the control. 5.10. Disinfestation of irrigation water Water moulds such as Pythium and Phytophthora are readily carried in water bodies such as dams, water tanks and shallow bore holes. This problem can be increased substantially if the water is recycled, such as in hydroponic systems. Water can be effectively disinfested by filtration, irradiation, chlorination, bromination or ozonation. For effective filtration, filters with pore sizes between must be used. If the water contains particulate matter it is necessary to have a series of larger filters to remove the particulate matter. The filter membranes need to be cleaned regularly. Irradiation uses ultraviolet (UV) light in the 200-280 nm range to disinfect water. However, a number of parameters such as flow rate, the duration of exposure, level of particulates in the water, and the strength of the UV lamp must be considered when using this process. Chlorination is the most common and cheapest disinfestation method. The process uses either c h o r i n e gas, sodium hyphochlorite or calcium hypochlorite. To be effective, a minimum concentration of 5 ppm for 20 minutes is recommended. It is necessary to filter out organic particulate matter prior to chlorination. Particulates reduce the effectiveness of the treatment. The water pH should be around pH 6-7.

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5.11. Fungicides Fungicides are used prophylactically by most container or bare root nurseries to control a range of soil-borne or foliar plant pathogens. Many fungicides are fungistatic rather than fungicidal to a range of plant pathogens, especially in their dormant or resting stages. These pathogens express themselves after the fungistatic effects of the fungicides have disappeared on the cessation of their use. It is critical to assess the use of fungicides on plants that are being used in recovery programs and special attention must be given to soil-borne pathogens such as Phytophthora species. Total reliance on fungicides such as phosphite (phosphonate) that do not necessarily kill the pathogen but induce a resistant host response which contains but does not kill the pathogen must be avoided. It is advisable that plants to be used in recovery programs grown in plant nurseries be produced under stringent hygiene measures. These would include the use of disease-free container substrates that have been adequately and stringently composted, or steam pasteurised, or produced from materials such as polystyrene and perlite. The water source must be pathogen-free, containers must be kept off the ground on raised, free-draining benches and environmental conditions such as temperature, relative humidity and air flow must be conducive to plant growth and not to plant pathogens. The accreditation of nurseries that adopt strict and regularly monitored hygiene and quarantine practices must be encouraged and preferably legislated for. 5.12. Ecological restoration The aim of ecological restoration is to reach a sustainable and diverse flora which may or may not be identical to that which existed prior to disturbance. This approach not only requires a sound knowledge of the environment of the affected site and the nature of the suitable flora but also funds to support a large labour force necessary to carry out the program. Although a challenge, such programs have been successfully carried out. A good example is that of Grevillea scapigera, which was at the point of extinction and has recently been restored ecologically through the efforts of community and conservation groups in Western Australia (Bunn and Dixon 1992; Touchell et al. 1992; Rossetto et al. 1995; Krauss et al. 2002). 5.13. Planting out Planting out may not simply be a routine procedure. Timing of planting, land preparation (including soil amendments and irrigation during establishment) needs to be considered carefully in addition to prophylactic measures to reduce disease hazards. Hygiene is an important component of any species recovery program. This includes keeping plants disease-free when planted out and minimising

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the spread of a pathogen into non-infested areas if a recovery program is being undertaken on disease infested sites. For example, the control and management of P. cinnamomi in natural ecosystems raises considerable challenges in terms of reducing spread and the impact of the pathogen in recovery programs in diverse plant communities. There are a number of strategic control procedures that can be used by managers involved in recovery programs (Colquhoun and Hardy 2000). These include: Producing reliable up-to-date maps and field demarcation of disease affected areas. This involves having trained interpreters who have a good understanding of which plants are P. cinnamomi susceptible ‘indicator’ species. The interpreters must be able to discount other factors that can cause plant death such as drought, insects, fire, and other plant diseases. The information on disease affected areas to be stored on a Geographical Information System. Once mapped, appropriate operations planning, scheduling and implementation involving high-risk activities of infested and adjacent non-infested areas must be undertaken. Planning high-risk operations such as road building, forestry activities and mining in diseased areas when conditions optimum for the spread of P. cinnamomi are minimal, such as during hot and dry periods. Restricting the movement of vehicles from disease affected to diseasefree areas. This can be achieved by blocking tracks to stop their use, erecting gates and signs, removing roads and by ensuring road construction through disease-free areas uses non-infested materials. Preventing the movement of infested water moving into disease-free areas. The zoospores of P. cinnamomi are readily transported in water so any surface water movement between infested and non-infested areas needs to be minimised. Thoroughly cleaning vehicles and equipment to remove all adhering soil or plant debris before moving between infested and non-infested areas to minimise the risk of spreading infested soil into disease-free areas. Wash-down stations must be designed so that once washed the vehicles are unlikely to be re-contaminated by passing through wash water that contains propagules of P. cinnamomi. Training all field personnel and planners in good hygiene management and operations to reduce the spread of the pathogen. Increasing the awareness of the general public of P. cinnamomi. This is achieved by information leaflets, newspaper articles, displays at public functions, television interviews, classroom teaching materials and information signs. Similar strategies can be developed for other soil-borne and foliar plant pathogens. In conclusion, phytosanitary protocols should be established for each step or process involved in the conservation and propagation of plants.

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This requirement needs to be most stringently observed in programs relating to the conservation of rare and endangered plants. 5.14. Roots Most root pathogens are necrotrophic and tend to have, with few exceptions, wide host range. The most common examples of these are species of Phytophthora and Pythium, Many of these species occur world-wide, although it is likely that pathogens such as P. cinnamomi were spread world­ wide through movements of horticultural plants. Certain necrotrophs such as the formae speciales of Fusarium oxysporum have specific host ranges, but can easily be spread with non-host plant species. These pathogens can be effectively managed only by the exploitation of host resistance. With exceptions of diseases caused by oomycetes and certain nematodes, root-disease causing pathogens can only be eliminated by pre-planting soil fumigation or drenches. This means that it is possible to introduce perfectly healthy plants of the species you have targeted into an area, with latent infections of fungal pathogens that then cause the loss of other plant species in the area. 5.15. Disease-free planting materials Seed, transplants, and cuttings can be major sources of inoculum in nursery production of plants. There are three main ways to obtain pathogen-free plant material. Firstly, it is essential to ensure that the planting material is derived from a disease-free area and stock. Secondly, a culture-indexing program whereby multiplicative propagation is done only from healthy plant parts such as stem tips or meristems (or even through micropropagation using protoplasts), can eradicate pathogens. Thirdly, once healthy material has been obtained, basic hygiene and quarantine procedures should be maintained by the grower to minimise the chances of reinfection. There are a number of pathogens, particularly fungal pathogens, that can be missed by existing quarantine protocols. For example, there are fungi that can exist for extended periods as endophytes (Saikkonen et al. 1998). These do not express themselves until some stress factor or change in environmental conditions predisposes the host to damaging pathogenic activities of the pathogen. It is therefore critical that any plant material being used in recovery programs that has been imported from areas other than where the plants will be planted is screened for the presence of pathogens. It is critical that this material is disease-free. The use of indexing, serology, ELISA (enzyme linked immuno assorbant assay) and PCR-based molecular tools are critical to ensure the absence of potential pathogens. The use of fungicides that impose stasis on fungi in planta must also be considered. If fungicides have been used prior to plants being

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subjected to quarantine then the duration of the quarantine period should be extended to allow time for dormant propagules to germinate once fungistatic effects of fungicides have dissipated. It could be argued that in some instances systemic fungicides should not be allowed for use in nursery production. An example for this would be oomycete fungicides that have the potential to mask the presence of a pathogen such as P. cinnamomi. The distribution of outwardly ‘healthy’ but infected plants previously treated with fungicides should be avoided. Infested plants introduced into disease-free areas can have potentially catastrophic effects.

6. Case studies 6.1. Phosphite fungicides Phosphite, the anionic form of phosphonic acid has been used to manage many plant diseases caused by Phytophthora species in horticulture and in agriculture. It is effective even at concentrations in planta that only partially inhibit pathogen growth in vitro (Guest and Bombeix 1984; Guest and Grant 1991; Wilkinson et al. 2001a). Phosphite is a systemic fungicide that is translocated in both the xylem and the phloem (Ouimette and Coffey 1989). In the phloem, phosphite is trapped and therefore translocated through the plant in association with photoassimilates in a source-sink relationship (Saindrenan et al. 1988; Ouimette and Coffey 1990; Guest and Grant 1991). The phosphite concentration in plant tissues is directly related to its application rate (Smillie et al. 1989). Phosphite treatment induces a strong and rapid defense response in the challenged plant (Guest and Bompeix 1990; Smith et al. 1997). These defense responses stop pathogen spread in a large number of hosts. Phosphite exhibits a complex mode of action, acting directly on the pathogen and indirectly in stimulating host defence responses to u l t i m a t e l y i n h i b i t pathogen growth (Guest and Grant 1991). In Western Australia, phosphite is currently applied to native plant species as an injection to the trunks of trees or large shrubs, as a conventional foliar application to run-off or as an ultra-low volume mist (Komorek et al. 1997, 2001; Barrett 1999; Hardy 2000; Tynan et al. 2001). The latter is applied by aerial application, usually to communities of high conservation value which contain rare and threatened plant species. It is used routinely in the S t i r l i n g Ranges to protect rare and endangered plant species that are growing in infested areas. Without the use of phosphite, a number of plant species would no longer exist in the wild. Consequently, phosphite is a vital short to medium term ‘tool’ currently utilised in some species recovery programs.

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6.2. Costs Aerial application of phosphite as an ultra low-volume mist costs approximately this includes the cost of the fungicide and aircraft hire. Additional costs are involved in the set-up of targets, in particular for mountain areas, where personnel must be on site to ensure wind conditions are adequate for application. Rates of phosphite application applied as a low-volume mist range from using 40% phosphite sprayed at The rate is applied in two separate sprays 4-6 weeks apart to minimise phytotoxicity. Conventional backpack sprayers or trailer mounted sprayers can be used to apply phosphite to run-off at rates of between 0.5-1% phosphite. At higher rates, phytotoxicity can become a major problem (Hardy 2000). Feasibility of conventional spraying by backpacks and trailers is usually restricted to small areas of approximately 1 ha or less. These include spot infestations or small areas of remnant bushland. Injecting trees is only viable for large trees in areas where their loss would have a visual impact, although in some instances volunteer groups have treated whole reserves by injection trees and spraying the understory to run-off (I. Colquhoun pers comm.). It costs approximately AUD$0.50 cents to treat a medium size jarrah (E. marginata) tree by injection. The best time to inject a tree is during spring and summer in the morning when the tree is actively transpiring. It is critical to add an adjuvant when applying phosphite as a foliar application. In Western Australia, Synertrol Oil (Organic Crop Protectants Pty Ltd), based on food grade canola oil (832 gL) is used. Synertrol increases spray coverage by droplet spreading, promotes spray retention, reduces spray drift, evaporation and wash-off (Organic Crop Protectants Pty Ltd). 6.2.1. Control of P. cinnamomi Injections using 50, 100 and phosphite have controlled P. cinnamomi in a number of species for up to 5 years (Shearer and Fairman phosphite increased the time 1997a,b). Foliar application to run-off of to 50% mortality of three species of Banksia growing along a P. cinnamomi disease-front by an average of 2-6 years depending on the species treated (Shearer and Fairman 1997a). Aberton et al. (1999) stated that foliar application of phosphite to Xanthorrhoea australis prevented deaths for at least two years in P. cinnamomi infested vegetation. Pilbeam et al. (2000) demonstrated that foliar applications of 2, 5 or phosphite effectively restricted the colonisation by P. cinnamomi of stems of three native species. In an operational program using ultra-low volume applications of phosphite to a native plant community in the Fitzgerald River National Park,

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percentage survival of Banksia baxteri and Lambertia inermis plants growing along a dieback front at two years post-spray was 68% and 78% compared with 31% and 54% for non-sprayed plants, respectively (Barrett 1999). Therefore, at the rates of phosphite applied to native plants, P. cinnamomi colonisation is contained or reduced in plant tissue but the pathogen is seldom killed (Ali et al. 1998; Pilbeam et al. 2000; Shearer pers comm). This obviously has implications with regard to the continued spread of the pathogen under optimum environmental conditions. In a study conducted on 1-2 year old E. marginata in a rehabilitated mine site, Wilkinson et al. (2001 b) showed that phosphite, when applied as a foliar spray, contained lesions caused by P. cinnamomi, but sporangial production and zoospore release were not prevented from diseased tissue, although they were reduced in numbers. Consequently, phosphite may slow down or prevent deaths of plants in natural plant communities but not necessarily prevent the spread of inoculum into non-infested areas. Trials in native plant communities to determine whether applications to affected areas could help to prevent the spread off the pathogen to unaffected areas are required to confirm this observation. However, until this is undertaken, good hygiene practices in and around infested areas that have been treated with phosphite must still be a priority. 6.2.2. Phytotoxicity Phytotoxicity in native plant species has been observed (Komorek et al. 1997; Aberton et al. 1999; Fairbanks et al. 2000; Pilbeam et al. 2000; S. Barrett, pers. comm.). In some cases leaf scorching has occurred on plants specifically sprayed for protection against P. cinnamomi. Consequently, there is a fine balance between the rates of phosphite applied, phytotoxicity symptoms and the control of P. cinnamomi. Generally, as the rates of phosphite applied increase, so do the concentrations of phosphite in plant tissue. However, above as a spray to run-off or as a mist or low-volume application, phytotoxicity symptoms increase substantially in a large range of species from different genera and families. It is likely that in whatever natural ecosystem phosphite is applied, it will, even at recommended rates, cause phytotoxicity in some plant due to differences in uptake between species and their sensitivity to the fungicide. However, it is necessary to offset this phytotoxicity with the benefits of phosphite, in terms of P. cinnamomi containment (Hardy et al. 2001).

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6.2.3. Detrimental effects (on plant reproduction, plant growth, tolerant isolates and mycorrhizas) There have been limited studies on the effects of phosphite on plant reproduction. However, Fairbanks et al. (2000) showed that recommended rates of phosphite reduced the reproductive fitness of some annual and perennial understory species from the Eucalyptus marginata (jarrah) forest. Phosphite reduced pollen fertility in a native annual species when sprayed in the vegetative stage and of three others when sprayed at anthesis. Seed germination from treated plants was reduced by phosphite in two species when the plants were sprayed in the vegetative stage. Consequently, more work is required, but it does appear that it would be strategic to m i n i m i s e the use of phosphite when a n n u a l s are growing in plant communities, or to apply phosphite in early autumn prior to germination. Phosphite also affected sexual reproduction of three perennial jarrah forest species (Fairbanks et al. 2001). In Pterochaeta paniculata (Asteraceae), pollen fertility was reduced by phosphite for up to 60 weeks after spraying in autumn, and 35 weeks in spring. Whilst in Trymalium ledifolium (Rhamnaceae), pollen fertility was reduced for up to 38 weeks after spraying with phosphite in spring, and up to 61 weeks after spraying in autumn. 6.3. Effects on mycorrhizal fungi Mycorrhizal fungi are symbionts that confer benefits to their host plant and at the same time obtain a niche and nutrients from their host. Many plants can fail to establish or thrive in the absence of their mycorrhizal symbionts. Consequently, the use of fungicides has the potential to be detrimental to mycorrhizal fungi. Preliminary glasshouse studies on the effects of phosphite on mycorrhizal fungi have shown that phosphite applied as a foliar spray to Eucalyptus globulus, E. marginata and Agonis flexuosa had no effect on ectomycorrhizal (ECM) formation, whilst vesicular-arbuscular mycorrhizal (VAM) colonisation increased four-fold in Agonis flexuosa (Howard et al. 2000).

6.4. Phosphite tolerant P. cinnamomi isolates When using fungicides on plants in recovery programs, it is important to consider the likelihood of the pathogen becoming resistant or tolerant to the fungicide. There is some evidence of tolerance of P. cinnamomi to phosphite in treated plants among isolates from native vegetation which have not previously been exposed to phosphite (Hardy et al. (2001). This observation is of concern, especially in native ecosystems that are being treated regularly w i t h phosphite to save ‘critically endangered’ species. Regular spraying could provide a selection pressure for these more phosphite

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tolerant isolates and could pose additional problems to managers in the future. 6.5. Conclusion on phosphite use in recovery programs Phosphite appears to be effective in keeping susceptible plants in natural ecosystems alive in the short to medium term in areas impacted by Phytophthora. It has particular application to plant communities where rare and endangered plant species are threatened with extinction. Phosphite may have some detrimental effects such as reduced reproductive capacity in some species. However, if a susceptible species is threatened by extinction then phosphite could applied u n t i l some better alternative becomes available. In these cases, phosphite provides managers with the time to develop alternative control strategies such as placing endangered species into cryo­ preservation or developing other conservation strategies. However, appropriate hygiene practices must not be reduced or stopped since phosphite used as a foliar application or injection often will only contain the pathogen in the plant and not kill it. Therefore, under optimum moist and warm conditions it is possible for sporulation to occur. These studies on phosphite in natural plant communities clearly indicate that fungicides can have a role in protection of natural plant communities. However, there is the need to consider the impact of phosphite or other fungicides on other components of the plant community in addition to their effects on the pathogen. 7. Conclusions Prevention is clearly better than cure. Effective conservation efforts aimed at avoiding or preventing disease outbreaks can be far more beneficial than intensive management of affected areas. Options for intensive management of destructive diseases, especially in natural ecosystems, are few, expensive and may at best be only temporary measures to manage the problems until more effective and permanent solutions are found. Recovery programs can succeed only if adequate research is carried out on the ecosystem of the region and the potential for resident and/or exotic pathogens to the restored area. Emphasis must also be made on the employment of appropriate hygiene practices at production and planting-out phases. Several major questions remain from this chapter. Are devastation events such as those of Castanea in north America and other susceptible native flora devastated by introduced pathogens rare events in history? Or is it that they happened at a time and in places were researchers were ready to monitor them? Could they have happened throughout evolution and time? Could they be happening currently in ecosystems in countries too poor or too

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distracted to monitor them? Answers to these questions may need extensive and possible expensive research which is unlikely to be funded.

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INDEX

studies 116 276 2-partner symbioses 58,63,66-7, 108, 113, 128 Acacia melanoxylon 211 32, 155, 158 Acaulospora 25 denticulata ACC deaminase 87, 99 Acclimatisation 308 Acetylene reductionassay 66 328 Achromobacter parvulus 274 Acremonium 245 lolii 51, 56 Actinomycete 51, 53 Actinorhizal Adenostoma fasciculatum 25 59 Aeschynomene 49, 52, 63, 228, 231, Africa 234-5, 295-7 243 Agaricales 362 Agonis flexuosa 62, 242 Agrobacterium 88-9 radiobacter 258 tumefaciens 328 Alcaligenes faecalis 2 Algae 51-2, 56,60, 66-7, Allocasuarina 108, 128 51-4, 56, 108, 128, 277 Alnus 346 glutinosa 122, 126 rubra 310, 312, 348 Alternaria AM fungi 116, 125, 127-8, 132, Ch 6 106, 131 Amanita muscaria 167 Amaranthaceae 46,49 Anabaena 326-7 Anigozanthos 324 viridis ssp. terraspectans 324 Anthericaceae 21 Anthropogenic acidification

Antibiotic 318 Aotus 66 Aponogeton hexatepalus 324 Aponogetonaceae 324 Arabidopsis thaliana 327 Arachis 60 Arbutus 232 Archaea 2,4 Archaeospora 155, 187 Arctostaphylos 126 Argentina 52, 296 Armillaria 287, 351 luteobubalina 288-9, 304 211 mellea Artemisia californica 29 25 tridentata 257 Ascochyta caulina Ascomycetes 107 Ascomycota 243-4, 246, 249, 254 Asia 47, 50-2, 210, 295 347 Asian chestnut Aspergillus 272, 274, 310, 312 niger 328 oryzae 328 Asteraceae 57, 107 Asterolasia drummondii 325 grandiflora 325 nivea 325 228-9 Astroloma xerophyllum 255 Atkinsonella hypoxylon 328 Aureobasidium pullulans Australia 1, 5, 7, 11,45,47,49, 51-2,

63, 66,91, 105, 107, 119-20, 128, 131, 151 ,167, 195, 200, 202, 212, 214, 217, 227-8, 231, 234-5, 250, 255-6, 271, 277, 286-97, 299-300, 337,343-4 210 Autricularia polytricha 23, 29 Avena 51 Azolla 62 Azorhizobium 46, 81, 86 Azospirillum

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46, 86 Azotobacter 80-1, 88, 311-2, 322 Bacillus 83, 328 cereus 88 subtilis Balaustion microphyllum 324 66, 291, 348, 360 Banksia baxteri 361 brownii 345 338, 340 coccinia Basidiomycetes 107, 198, 243, 246 243, 246 Basidiomycota Basidiospores 288 211 Beilschmiedia Beta vulgaris 327 128 Bettongia tropica 106, 108, 210 Betula Betulaceae 52-3, 108 276 Biological control 203 Bletilla striata Boletales 243 Boletinellus merulioides 119 210 Boreal forests 325 Boronia adamsiana 66 Bossiaea 287 Botryosphaeria dothidea 298 rhodina ribis 289 310, 312 Botrytis 258, 342, 348, 352 cinerea Botyrosphaeria dothidea 294 33 Bouteloua gracilis 47 Bowenia Bradyrhizobium 46, 62-3 Brassicaceae 167 Brazil 58, 295-6 328 Brevibacterium ammoniagenes 31 Bromus hordeaceus madritensis 29 Bunts 246 88 Burkholderia cepacia reduction 66 or physiology 23

57-9, 61 Caesalpinioideae Caladenia 202, 206 arenicola 208-9, 217 19, 21-2, 25, 27-31, 34, California 36, 52, 193 Calluna 127, 228, 232 vulgaris 233-4 Calonectria quinquiseptatum 291 Calytrix breviseta ssp. breviseta 324 Candida 310 albicans 328 Canker 249, 286 Capsicum 326 Carex arenaria 253 Caryophyllaceae 167 Cassiope 107-8 Cassiopoideae 228 Casuarina 51-3, 55-6, 60, 108 Casuarinaceae 52-3, 55, 64, 108 232 Cataula Ceanothus 51-3 Cenococcum 109 geophilum 119 Central America 91, 102, 295 Cephalanthera austiniae 210 198 Ceratobasidium cornigerum 200, 202 Ceratocystis 11, 260, 291, 303 fimbriata 296, 304 198, 200, 224 Ceratorhiza Ceratozamia 47 Chaetomium globosum 328 Chenopodiaceae 167 Chenopodium 257 Chestnut blight 301, 347 Chigua 47 Chromista 273 Cryphonectria parasitica 250 Chytridiales 243 Chytridiomycetes 243-4 Chytridiomycota 243 Citrus 327

Index

272, 310, 312, 328 Cladosporium resinae 328 Clamp connections 246 Clavibacter 242, 314 89 xyli 254-7 Claviceps purpurea 46, 57 Clostridium enrichment 22, 25, 30, 121 266 Co-evolution 324 Colchiaceae 245, 298, 348 Colletotrichum 298 gloeosporioides Commelinaceae 167 Competition 170 Comptonia 52, 56, 108 297 Coniothyrium zuluense 295 228 Conostephium pendulum 324 Conostylis misera 324 wonganensis Convention on biological diversity 271 48 Coralloidroot 202, 206 Corallorhiza 210 maculata 200, 210 trifida Coriariaceae 53 210 Corybas cryptanthus 314 Corynebacterium 23 Crassulacean Acid Metabolism 294 Cryphonectria cubensis 286, 338, 347 parasitica 338 Cryptodiaporthe 340 semiperda 3 Cyanobacteria 47 Cyanophyta 47 Cycadaceae 47 Cycads 291, 298, 348, 352 Cylindrocladium 167, 316 Cyperaceae 286 Cypresscanker 319 Cytokinin Cytospora eucalypticola 287, 291

371

Dactylorhiza 202, 212, 214 aristata 200 purpurella 199 Danthonia spicata 255 Datiscaceae 52-3 Daviesia atrophylla 324 speciosa 324 Debilitators 254 Deuteromycetes 246 Didymoplexis 210, 213 Die-back 292 Dillwynia 66 Dioon 47 Diplolaena andrewsii 325 Dipterygeae 58 Disease suppressing bacteria 88 suppressive soils 91 -free planting materials 358 Disinfestation 354-5 Disturbance 10, 12, 112, 160, 174,

212, 343-4

212, 332

Diuris micrantha 217 purdiei 214, 217 DNA/RNA fluorochrome staining 313 Dothideales 243 Douglas fir 119, 122, 124, 253, 265 Downy mildew 248 Drechslera teres 90 Drummondita ericoides 325 Dryas 51-2, 56, 108 Dutch elm disease 11, 250, 254-5, 275

Ebola virus 8 Ecological restoration 347, 356 specificity 186 Ecosystem dynamics 9 restoration 129, 173 Ectomycorrhizas Ch 5 Electrosterilisation 321 Elaeagnaceae 52-3, 55, 60 Elaeagnus 51-2 Elythranthera 212 Empetraceae 232

372

Microorganisms in Plant Conservation and Biodiversity

228 Empetreae 232 Empetrum 47, 68 Encephalartos 322 Endosporous bacteria 295 Endothia eugeniae 287-8, 298 gyrosa 287-8, 291 Endothiella England 223, 233, 311, 346 88-9, 311-2, 328 Enterobacter 328 aerogenes agglomerans 88 312 asburiae cloacae 89 Entrophospora 155 232, 281, 344-5 Epacridaceae Epacridoideae 228 254 Epichloe typhina 310 Epicoccum 196, 222, 327 Epidendrum floridense 210 Epigonium 23 Epilobium 202, 212, 223-5 Epipactis Epiphytes 154 Epiphytic orchids 222 213 Epipogium 198, 200 Epulorhiza 324 Eremophila resinosa Ergot 257 232 Erica Ericaceae 108, 129, 206, 215, 227-30, 232-5, 344-5 227, 229 Ericales Ericoid fungi 227 Ericoid mycorrhizas Ch 8 Ericoideae 228 Eriogonum fasciculatum 29 Erwinia 242 254 amylovara 243, 245, 249, 254 Erysiphales 210 Erythromyces crocicreas 210 Erythrorchis ochobiensis 328 Escherichia coli

Etiolation Eucalyptus

319 66, 107-8, 112, 119, 120, 128, 130, 250, 255, 277, 297, 299, 318, 340 diversicolor 289 dolorosa 324 globulus 106, 123, 362 grandis 294, 296, 327 324 graniticola impensa 324 48, 291, 295, 344 marginata nitens 290 Eukaryotes 2 Euphorbia 23 Europe 21, 51-2, 120, 131, 168, 200, 202, 212, 250, 252, 255, 260, 286, 346 European forest 122 Ex situ conservation 209, 260 Exobasidiales 243, 245 Explant 324-5 324 Fabaceae Fire 212 Flavobacterium suaveolens 328 Fluorescent siderophores 90 Fomes 210 mastoporus 211 112, 115 Forestry activities 248 Formae speciales 46, 51, 55-7, 68, 277 Frankia -based actinorhizal species 60 Fraxinus 119 Fungal propagules 111 succession 123 Fungi imperfecti. 233 Fungicides 318, 352, 356 Fungus-feeding nematodes 158, 161 Fusarium 254, 270, 272, 274, 348, 354 89, 91, 257, 358 oxysporum 310 poae 91 wilt of banana Galearis 202

Index

206 Galeola 210 altissima 210 hydra 211 septentrionalis Galling 249 243 Ganodermatales Gastrodia 198, 206, 213, 215 211 cunninghamii 211 elata 211 javanica 211 Gastrodia minor 211 sesamoides 324 Gastrolobium hamulosum 127, 230, 232 Gaultheria 230, 233 shallon 322 Gentamycin 52 Geographical distribution Geographical Information System 357 Germplasm 209 258 Gibberella fujikuroi 28, 34, 155 Gigaspora 328 Gliocladium fimbriatum 151, 155 Glomales 34, 155, 158 Glomus 28 aggregatum 25 etunicatum 25 intraradices 28 leptotichum 28 occultum 28 tenue 258 Genetically modified crops 47 Gondwana Goodeniaceae 107, 324 202, 212 Goodyera 200, 203 repens 242, 322 Gram negative bacteria 57 Gramineae 212 Grazing 324 Grevillea dryandroides 356, 364, 366-7 scapigera 245 Guignardia 50-1, 54, 68 Gunnera

373

Gunneraceae 50 Gutierrezia sarothrae 30 Gymnosperm 105 Gymnostoma 51-2 Habitat destruction 196 324 Haemodoraceae Haplophase 248 Hartig net 105-6, 111 Hawaiian plants 173, 185 Hebeloma cristuliniforme 119 Hemiandra gardneri 324 rutilans 324 Hemibiotrophs 245 Hemigenia exilis 324 Heterobasidion annosum 257 Holobiotrophic 248 Holobiotrophs 245 Homobasidiomycetidae 243 Hormonal action 85 Horticultural trade 196 Host-fungus 130, 222 Hydnangium 131 carneum 293 Hymenoscyphus ericae 233-4 Hypersensitive flecking 249 116-7, 163-4 Hyphae Hyphal anastomosis 246 digestion 198 grazing 110, 158 networks 9, 21 proliferation 111 responses 28 Hypholoma 116 Hypochytriales 243 Hypochytriomycetes 243 Hypochytriomycota 243 Hypocreales 243 Hysterangium 131 326 Ilex In situ conservation 261 210-1, 295 Indonesia Indonesian archipelago 286 273 Ingoldian fungi

374

Microorganisms in Plant Conservation and Biodiversity

360 Injectingtrees Inoculation 129-30, 277 159, 183, 186 Inoculum Introduced pathogens 286, 291 Irradiation (UV or Gamma-rays) 319 Jacksonia 66, 108 250, 255 Jarrah die-back 167 Juncaceae 127, 232 Kalmia 66 Kennedia 107-8 Kobresia Laboulbeniomycetes 272 Labrinthulales 243 Labrinthulomycetes 243 243 Labrinthulomycota 272 Lacazia 124, 131 Laccaria 293 laccata 324 Lambertia echinata 361 inermis 324 orbifolia 324 Lamiaceae Laurasian 47 339 Leaf and stem diseases 324 Lechenaultia pulvinaris 232 Ledum Legumes 108 210, 225 Lentinula Leoteales 243 47 Lepidozamia peroffskyana 49 Leporella fimbriata 206 211 Leptospermum scoparium 228 Leucopogon conostephioides Linanthus 23, 31 196 Logging 23 Lolium 211 Lycoperdon Lyophilization 271 231 Lysinema ciliatum 47, 66 Macrozamia communis 49 fraseri 67

Macrozamia riedlei 48, 66 Maize 258 Malay 60 Marasmius coniatus 210 Marsupials 129, 161 Meiosis 244 Melaleuca 108, 113, 118, 128 uncinata 211, 214-6 Melampsora lini 253 Meliolales 243 Menyanthaceae 324 Mercuric chloride 320 Mesozoic 47 Methyl bromide 355 Mexico 214, 296 Microascales 243 Microbial communities 33, 335 diversity 12 synergists 87 Microcycas 47 Micrococcus 311 Microorganism conservation 8 Micropropagation 307, 309 Microsporum 310 Microtis parviflora 200, 223 Middle East 257 Mimosoideae 57, 59, 61 Mineral nutrition 204 Mining operations 344 Mites 161, 311, 329-30 Moniliopsis 200 Monocalyptus 293 Monotropa 126, 206, 215 Monotropoideae 126, 129, 215 Moulds 328 Mucor 243, 272, 310, 328 Mucor rouxii 328 Mucorales 243, 272 Mycelia 110 Myco-heterotrophic orchids 205, 210 Mycology 304 Mycorrhizal colonisation 197 community dynamics 27

Index

110 22 277 245, 287, 290, 297-9, 348 290 leaf blotch disease 324 Myoporaceae 51-3, 55-6, 108 Myrica 52-3, 108 Myricaceae 128, 294-5, 324 Myrtaceae 243 Myxomycota 31 N + P availability 46 N-containing molecules 21-2, 27-9 N eutrophication 46, 63-4, 67-9 association 9, 79 N-fixing microbes 65 N-limitingconditions 55 N starvation 20 N transformation 118 Narrow host range fungi 343 Natural disturbances 91 suppressiveness 249 Necrotic spotting 245, 248 Necrotrophs 211 Neottia 327 Nepenthes 1, 7, 50, 52, 131, New Zealand 210-11, 228, 239, 250 327 Nicotiana tabacum 120 Nitrogen deposition 31 Nitrogen oxides North America 11, 51, 119, 122, 128, 131, 168, 200, 210, 233, 250, 252, 286 128 Northern Bettong 46, 49- 51 Nostoc 108, 131, 210-11 Nothofagus 63 Nutrient acquisition strategies 278 Nyctalis 90 O-antigenic side chain 46, 55, 60 251 Obligate parasites 285 Ocotea whitei 233 Oidiodendron

Mycorrhizal exchange functioning syntheses Mycosphaerella

375

Old roots 158-9 Olpidiopsidales 243 Oomycetes 243, 270, 273 Oomycota 243 Ophiostoma himal-ulmi 11 novo-ulmi 250, 254-5, 275, 338 ulmi 11, 286 Ophrys 209, 212 Orchid rhizoctonias 198 symbionts 277 Orchidaceae 57, 126, 195, 199, 215, 218 Orchids 195-6, 200-1, 204, 206, 211, 312 Orchis 209, 212, 214 Ordovician 155 Ozone 31, 162 Pacific seaboard 33 Pangaea 47 Papilionoideae 57-9, 61 Papua New Guinea 210, 286 Paraglomus 155 Parasponia 53-4, 57, 60, 68 Pascopyrum smithii 33 Patagonia 228 PCR/DNA fingerprinting 62 PCR-based molecular analyses 49 Peloton isolation 213 Penicillium 245, 272, 274, 310, 312, 328 funiculosum 328 variable (glaucum) 328 Peronosporales 243, 248, 254 Pests 264-5 pH 60, 87, 94, 115, 122, 162, 167, 172, 204, 228, 350, 353, 355 Phaseoleae 59-60 Phellinus weirii 11 Phenology 203, 212, 334 Phialophora 274, 310 325 Philotheca wonganensis 326 Phoma 328 herbarum (pigmentivora) Phosphite 359, 362-3

376

Microorganisms in Plant Conservation and Biodiversity

232 Phyllodoce 61, 236, 303 Phylogeny Phytophthora 11, 92, 254, 273, 346, 348, 351, 354-6, 358-9, 363 263 cambivora 250, 255, 291-2, 338, cinnamomi 340, 342, 344-5, 352 361 Phytotoxicity Pinus 22, 106, 108, 123, 131, 210 123 banksiana 109, 119, 124, 131, 293 Pisolithus tinctorius 109, 119, Pisonia 108, 118 122 grandis Pithomyces chartarum 245 324 Pityrodia scabra 259 Plant pathogen diversity Plant Preservative Mixture 309, 323 Plantago 23 Platylobium 66 Plasmodiophora brassicae 254 Plasmodiophorales 243 Plasmodiophoromycetes 243 Plasmodiophoromycota 243-4 Platanthera 200, 212, 214 285 Platypodium elegans 278 Pleurocatena Pneumocystidales 272 Pneumocystis 272 Pollination 49, 212 Pollution 120, 167 Polygonaceae 167 144 Polygonum Populus 31, 106, 108, 118, 127-8 Poriales 243 Positive laboratory air pressure 312 Powdery mildew 249 Pre-coralloid root 48 Propagation of rare plants 172 Proteaceae 167, 324, 345 Proteus vulgaris 328 Protosteliales 243 Protosteliomycetes 243

Prunus 285 Pseudomonas 81-2, 87, 92, 242 aeruginosa 328 88, 103, 311, 322 aureofaciens cepacia ‘Gibraltar’ 328 chlororaphis 90 fluorescens 312, 328 oleoverans 328 syringae pv. lachrymans 91 Pseudotsuga 108, 119 menziesii 126 Psidium guajava 295 Pterochaeta paniculata 362 Pterospora 215 202 Pterostylis acuminata 200, 223 sanguinea 217 Puccinia psidii 294-6 Pyrenophora tritici-repentis 90, 101 Pythiales 243 Pythium 252, 254-5, 257, 273, 285, 305, 348, 351, 354-5, 358 oligandrum 92, 282 sylvaticum 253 torulosum 83 ultimum 89-90 Pyxidiophora asterophorae 278 Quercus rubra 127 Radioactive carbon 205 Rainfall 229 Ralstonia solanacearum 89 Ralstonia 89, 242 rDNA internal transcribed spacer (ITS) 109 rDNA sequencing 3 Recovery of contaminated cultures 329 Red-list macromycetes 278 Rhamnaceae 52-3, 55, 60, 108, 362 Rhinosporidium 272 Rhizanthella gardneri 206, 211, 213-6, 221, 223, 225 Rhizobium 46, 57, 62-3

Index Rhizobium-based symbioses 50 -Parasponia symbiosis 60 198-9, 205, 2 1 1 , 214, Rhizoctonia 254, 348, 354 200, 203, 352 solani 203 solani (AG8) 310 Rhizopus 328 stolonifer 127, 230, 232 Rhododendron 310, 312 Rhodotorula Rhodotorula rubra 328 243 Rhytismatales 314 Riesling grape 52-3, 55, 73, 334 Rosaceae 166 Rushes 210 Russula sp 246 Rusts Rutaceae 325 Saccharomyces cerevisiae 328 108, 118, 128, 143, 210 Salix Salmonella typhosa 328 Salvia mellifera 29 Santalum acuminatum 67 245 Saprotrophs 328 Sarcina lutea 142, 215 Sarcodes 249 Scab 124, 148 Scleroderma 293 verrucosum Sclerospora sacchari 314 Sclerosporales 243, 248 245 Sclerotinia 245, 352 Sclerotium 346 Scotland 167 Scrophulariaceae 25, 28, 34, 155 Scutellospora 25 calospora 198 Sebacina 166 Sedges 276 Seed coat 208 germination 348 -borne pathogens

377

Seiridium cardinale 286 Septoria 245 Serapias 209, 212 Sesbania 59 Shigella sonnei 328 Shoot growth increases 82 Smuts 246 Soil-borne spores 157 disturbance 112, 168, 174 -dwelling arthropods 160 hyphae 117, 159, 164 nutrient availability 140 organic matter 115 Solanaceae 325 Sonication 321 South Africa 7, 47, 50-1, 68, 295, 297-8, 300 South America 50-1, 63, 294-5, 338 South East Asia 295 Southern Hemisphere 50, 287, 293 Sowerbaea multicaulis 324 Spatial variability 220 Species conservation 344 Specificity 83, 118 Sphagnum 228 Spiranthes sinensis 200 Spores 112, 158-9, 168 Sporocarps 117 Stangeria 47 Stangeriaceae 47 Staphylococcus 311 aureus 328 epidermidis 328 Starch reserves 49 Steam pasteurisation 354 Stereales 243 Streptococcus pyogenes 328 80, 242, 249 Streptomyces Stylidiaceae 107, 325 Stylidium scabridum 325 Sub-culturing 308 Succession 132, 174 Swartzieae 58

378

Microorganisms in Plant Conservation and Biodiversity

Symbioses 5, 68, 276 Symbiotic associations 218 seed germination 212 Symonanthus bancroftii 325 Synchytrium endobioticum 254 Synertrol Oil 360 Tanacetum vulgare 322 Taphrinales 243, 245 330 Tarsonemid mites Tasmanian bluegum 287 210 Telephoraceae Teliomycetes 243, 246 Temperature 342 Tetratheca deltoidea 325 Thailand 296 Thanatephorus 198, 211, 214, 224 200 Thelephora tomentella Thelymitra manginii 214, 217-8 321 Thermosterilisation Thraustochytriales 243 202, 211 Tipularia Tissue culture Ch 12 Topsoil removal 174 Topsoil 162, 174 121 Toxic metal pollution 210 Trametes Translocation 213 Trema 60 Tremandraceae 325 92, 272, 310 Trichoderma Trichophyton mentagrophytes 328 Trophic 245 Trymalium ledifolium 362 Tsuga heterophylla 137 Tuber melanosporum 129 Tulasnella 198 calospora 202 108, 128 Uapaca Ulmaceae 53, 60 United States 21, 91, 252 Unopened flower buds 317 Uredinales 243, 245, 248, 254, 272 Uruguay 296

Ustilaginales 243, 245, 248, 254, 272 Ustilago maydis 258 Ustomycetes 243, 246 UV light 90 Vaccinioideae 228 Vaccinium 232 Vascular wilt 249, 254 Venezuela 295 Verticillium 254, 274 Verticordia albidia 324 jamesonii 324 sp 324 Vesicles 55, 159 Villarsia calthifolia 324 Viminaria 66 Wash-down stations 357 Waterlogging 115, 162 Weeds 166 Woollsia pungens 233 Wurmbea tubulosa 324 Xanthomonas 242 campestris 314 Xanthorrhoea australis 360 Xanthorrhoeaceae 345 Xerotus javanicus 211 Xylella 242 Yeasts 310, 328 Yoania australis 211 Zamia 47 furfuracea 49 pumila 49 Zamiaceae 47 Zoosporic fungi 272 Zululand 296 Zygomycetes 107, 151, 155, 243 Zygomycota 243, 244