ADVANCES IN
AGRONOMY VOLUME 24
CONTRIBUTORS TO THIS VOLUME
PHILIPBECKETT D. S. FREAR P. H. HARVEY R. H. HODGSON JOHNIEN. JENKINS C. S. LEVINGS,I11
W. L. LINDSAY
DONALD J. LISK FOWDEN G. MAXWELL H. M. MUNGER J. L. OZBUN WILLIAML. PARROTT F. N. PONNAMPERUMA R. H. SHIMABUKURO G. G. STILL
D. H. WALLACE E. A. WERNSMAN
ADVANCES
IN
AGRONOMY Prepared under the Auspices of the
AMERICAN SOCIETY OF AGRONOMY VOLUME 24
Edited by N. C . BRADY Roberts Hall, Cornell University, Ithaca, New York ADVISORY BOARD
W. L. COLVILLE W. A. RANEY I. J. JOHNSON J. R. RUNKLES R. B. MUSGRAVE G. W. THOMAS 1972
ACADEMIC PRESS
New York and London
COPYRIGHT 0 1972, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM. WITHOUT PERMISSION IN WRITING F R O M THE PUBLISHER.
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CONTENTS
.......................................... PREFACE.............................................................
CONTRIBUTORS TO VOLUME 24
ix xi
THE ROLE OF EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
P. H . HARVEY. C . S. LEVINGS. 111. I. I1. I11. IV. V. VI.
AND
E. A . WERNSMAN
Introduction .................................................... Composition of the Cytoplasm ..................................... DNA and Its Role in Cytoplasmic Organelles ...................... Plant Traits Influenced by Cytoplasm ............................... Possible Mechanisms Involved in Cytoplasmic Inheritance of Plant Traits Cytoplasmic Diff erences-Possible Origin and Ramifications . . . . . . . . . . . . References .....................................................
1
3
7 10 18 21
24
THE CHEMISTRY OF SUBMERGED SOILS
F. N . PONNAMPERUMA I. I1 I11 IV. V VI. VII
. . . .
Introduction ................................................... Kinds of Submerged Soils ........................................ Characteristics of Submerged Soils ................................ Electrochemical Changes in Submerged Soils ........................ Chemical Transformations in Submerged Soils ...................... Mineral Equilibria in Submerged Soils ............................ Perspectives ................................................... References ....................................................
29 30
34 48
58 80 87 88
PHYSIOLOGICAL GENETICS OF CROP YIELD
D. H . WALLACE. J . L. OZBUN.AND H . M. MUNGER I. I1. I11. IV. V.
VI.
Introduction .................................................... Identification of Genetic Variation ................................. Genetics and Heritability ......................................... Relative Importance of Physiological Components . . . . . . . . . . . . . . . . . . . Using Genetic Differentiation for Elucidation of Physiological and Biochemical Pathways ........................................... Summary and Applications in Plant Breeding ........................ References ..................................................... V
97 99 123 132 136 138 142
vi
CONTENTS
ZINC IN SOlLS AND PLANT NUTRITION
W. L. LINDSAY
............................. .................
111. Availability of Zinc to Plants
..........
147 158
.............. VI.
Summary and Future Research Needs References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RESISTANCE
OF
181
PLANTS TO INSECTS
FOWDENG. MAXWELL, JOHNIE N. JENKINS,
AND
WILLIAML . PARROTT
.......................... I. Introduction . . . . . . . . . . . . . . . . . IT. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Insect Resistance in Selected Field Crops . IV. Horticulture Crops . . . . . . . V. Forest Trees . . . . . . . . . . . . VI. Miscellaneous . . . . . . . . . . .
187
VII. Problems Associated with Breeding for Resistance to Insects . . . . . . . . . . . . . . . 249 VIII. Utilization of Resistant Var 250 IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
TRACE METALS I N SOILS, PLANTS, AND ANIMALS
DONALD J . LISK I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TI. The Soil-Plant Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Aquatic Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Continuing Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
267 268 299 309 311
BEHAVIOR OF HERBICIDES I N PLANTS
D. S. FREAR,R. H. HODCSON,R. H. SHIMABUKURO, A N D G . G . STILL
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzoic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Dinitroanilines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Triazines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.
328 328 337 342
CONTENTS
V. VI. VII. VIII. IX . X.
Heterocyclics .................................................. Diphenylethers ................................................ Substituted Ureas .............................................. Carbamates ................................................... Anilides ...................................................... Summary ..................................................... References ....................................................
vii 351 355 358 363 368 371 372
CRITICAL CATION ACTIVITY RATIOS
PHILIPBECKETT
I. I1. I11. IV.
Introduction .................................................... Cation Activity Ratios in Relation to Nutrient Uptake of Plant Growth . . . Experimental Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Threshold Ratios ................................................ References .....................................................
AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
379 380 385
393 408
413 444
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CONTRIBUTORS TO VOLUME 24 Numbers in parentheses indicate the pages on which the authors’ contributions begin.
PHILIPBECKETT (379), Soil Science Laboratory, Department of Agricultural Science, University of Oxford, Oxford, England D. S. FREAR(327), Agricultural Research Service, U S . Department of Agriculture, Metabolism and Radiation Research Laboratory, Fargo, North Dakota P. H. HARVEY( l ) , Department of Crop Science, North Carolina State University, Raleigh, North Carolina R. H. HODGSON(327), Agricultural Research Service, U S . Department of Agriculture, Metabolism and Radiation Research Laboratory, Fargo, North Dakota JOHNIE N . JENKINS (1 87), Departments of Entomology and Agronomy, Mississippi State University, and United States Department of Agriculture, Boll Weevil Research Laboratory, State College, Mississippi C. S. LEVINGS,I11 (1 ), Department of Genetics, North Carolina State University, Raleigh, North Carolina W. L. LINDSAY(147), Department of Agronomy, Colorado State University, Fort Collins, Colorado DONALD J. LISK (267), Pesticide Residue Laboratory, New York State College of Agriculture and Life Sciences, Cornell University, Ithaca, New York FOWDEN G. MAXWELL ( 187), Departments of Entomology and Agronomy, Mississippi State University, and United States Department of Agriculture, Boll Weevil Research Laboratory, State College, Mississippi H. M. MUNGER(97), Department of Plant Breeding and Biometry, Cornell University, Ithaca, New York J. L. OZBUN(97), Department o f Vegetable Crops, Cornell University, Ithaca, New York WILLIAM L. PARROTT ( 187), Departments of Entomology and Agronomy, Mississippi State University, and United States Department of Agriculture, Boll Weevil Research Laboratory, State College, Mississippi F. N . PONNAMPERUMA (29), The International Rice Research Institute, Los Bafios, Laguna, Philippines R. H . SHIMABUKURO (327), Agricultural Research Service, U.S. Department of Agriculture, Metabolism and Radiation Research Laboratory, Fargo, North Dakota G. G. STILL(327), Agricultural Research Service, U.S. Department of Agriculture, Metabolism and Radiation Research Laboratory, Fargo, North Dakota D. H. WALLACE (97), Departments of Plant Breeding and Biometry, and Vegetable Crops, Cornell University, Ithaca, New York E. A. WERNSMAN ( l ) , Department of Crop Science, North Carolina State University, Raleigh, North Carolina ix
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PREFACE
Two pressing human problems continue to receive prominent attention by soil and crop scientists: The mounting concern for the quality of our environment and the need to provide food for an ever-expanding world population. Most of the papers contained in this volume address themselves directly or indirectly to these two problems. Some man-made chemicals, such as pesticides and fertilizers, have helped revolutionize commercial agriculture. Other such chemicals, used sparingly in agriculture, find their way into human and animal foods and thus affect, at least indirectly, the agricultural supply system. In any case, the introduction of these chemicals into our environment has had considerable ecological consequences. Where these consequences are bad they pose a threat to the continued use of man-made chemicals. Where they are good they encourage further exploration in attempts to improve production and marketing efficiency. Soil and crop scientists are helping to identify such consequences and, just as importantly, they are seeking means of avoiding them. Three papers in the volume are concerned directly or indirectly with chemicals. One, addressed to the behavior of pesticides in plants, complements a soil-related article in the same subject area. The second paper reviews attempts to increase host resistance to insects and also suggests an important method of biological control as a means of reducing the need for chemical pesticides. In the third paper, we are further alerted to the potential dangers of -adding to soils-intentionally or otherwise-toxic metals which are becoming increasingly ubiquitous in our environment. The article on the chemistry of submerged soils and the review on rice nutrition in Volume 23 remind us that rice is the major food staple for most of the people living in tropical and semitropical areas, where population pressures are greatest. The other four articles address themselves to critical aspects of soil or crop science, each having a bearing on crop production. Factors affecting the availability d essential elements from the soil and genetic and physiological factors affecting plant growth and development are covered. In each case, the authors have critically analyzed our current state of knowledge in their respective subject areas.
N. C. BRADY Ithaca, New York August, 1972
xi
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THE ROLE OF EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING' P. H. Harvey, C. S. Levings, Ill, and E. A. Wernsman Departments of Crop Science and Genetics, North Carolina State University, Raleigh, North Carolina
I. Introduction ..................................................... 11. Composition of the Cytoplasm ..................................... A. Cytoplasmic Organelles and Macromolecules .................... B. Extracytoplasmic Inclusions (Pathological Inclusions). . . . . . . . . . . . . . 111. DNA and Its Role in Cytoplasmic OrganeIles.. ..................... A. Chloroplasts ................................................. B. Mitochondria................................................. IV. Plant Traits Influenced by Cytoplasm ............................... A. Direct Cytoplasmic Control ................................... B. Other Unclassified Effects ..................................... V. Possible Mechanisms Involved in Cytoplasmic Inheritance of Plant Traits .......................................................... A. Genetic System of the Organelle ............................... B. Viral Effects ................................................. C. Nuclear Genetic Systems Which Mimic Extrachromosomal Inheritance ....................................................... D. Undiscovered Effects ......................................... VI. Cytoplasmic Differences-Possible Origin and Ramifications ............. References ......................................................
I.
1 3 3 6
7 7 9 10 10
17 18 18 18 20 20 21 24
Introduction
The presence of extrachromosomal inheritance is now generally accepted by biologists. The relative magnitude of importance between extrachromosomal inheritance and that of chromosomal (nuclear genic) is still not well understood. Research on extrachromosomal inheritance has been limited by the lack of adequate techniques for studying inheritance patterns of cytoplasmic traits. The rapid development of genetic and plant breeding techniques during the first half of the twentieth century lead to a very extensive advancement in the knowledge of nuclear gene behavior and its utilization in crop improvement. 'Paper No. 3636 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. 1
2
P . H. HARVEY, C. S. LEVINGS, 111, AND E. A. WERNSMAN
The exploitation of hybrid vigor by the commercial production of first generation crossed populations while making extensive use of the knowledge of nuclear gene behavior also raised the question of cytoplasmic inheritance. The best known example of the use of lirst generation F, hybrids is the widely used hybrid maize (or corn) Zea mays L. Plant breeders could see a practical advantage to a male-sterile line that could be maintained for use in the production of hybrid seed on a very large scale. Rhoades (1931, 1933) reported on a male-sterile maize that was controlled by cytoplasmic factors and was maternally inherited. This strain offered the possibility of producing hybrids on a large scale without requiring emasculation (detasseling) of the female parent strain. Unfortunately, this cytoplasmic male sterile was too easily influenced by different environmental conditions, which often caused it to revert to partial fertility in the production field, thereby requiring the detasseling of the female parent. It was not until Rogers and Edwardson (1952) introduced a male-sterile from GOLDEN JUNE that hybrid seed production using male-sterile lines became common among maize seed producers. This new male sterile is controlled by a cytoplasmic factor which has been designated Tcms (Texas cytoplasmic male sterile). All seed produced using a Tcms line as the female parent are carriers of the Tcms factor and produce only male-sterile plants unless the strain used as source of pollen carries nuclear genes for the restoration of pollen fertility. This role of both the cytoplasmic factor and nuclear genes in controlling male sterility and fertility was ideal for the breeder to manipulate. While other cytoplasmic male steriles were isolated in maize (84 separate discoveries according to Duvick, 1965 ) , Tcms was almost universally used by the United States hybrid maize industry by 1970. Tcms and its restorer genes were working so well that it was estimated that over 90% of the maize crop of the United States in 1970 carried this cytoplasm. Geneticists have for many years been concerned over having too narrow a germ plasm base for crop production. Few had really thought much about the danger of a narrow cytoplasmic base until Villareal and Lantican (1965) reported the susceptibility of maize with Tcms to a pathogen, Helmintlzosporiuin rnaydis. It was not until 1969 when the disease yellow leaf blight, Phyllosticta zeae, was observed by Scheifele et al. (1969) attacking Tcms strains in the corn belt states that maize breeders in the United States became alarmed. Then in 1970 the disastrous epidemic of H . rnaydis (race T) dramatically showed that a narrow cytoplasmic base could also be very detrimental. Although some work on extrachromosomal inheritance had been conducted over the years, most breeders were content to make use of the one proven male sterile, Tcms. Stringfield (1964) stated, “The whole study
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
3
of corn cytoplasms in relation to corn improvement is not currently as acutely needed as are several other studies, but in the long run, the cytoplasm must be given a place of importance. The cytoplasms of exotic corns would seem to offer a most interesting area for further investigation.” The maize disease disaster of 1970 was summarized by Tatum ( 197 1 ) . Tatum stated, “The appearance of two important diseases of corn, for which susceptibility resides in a particular type of cytoplasm, dramatically emphasizes our need of knowledge of cytoplasmic genetics and especially its role in host-parasite interactions.” In a very real sense, the host reaction to Helminthosporium maydis and Phyllosticta zeae are cytoplasmic markers similar to marker traits controlled by nuclear genes. The plant breeder and pathologist can use these traits to gain new insight into the functioning of the cytoplasm and those entities which play a vital role in the transmission of the traits from generation to generation. Interest in cytoplasmic inheritance is by no means confined to maize. The extensive application of cytoplasmic male sterility has been made in many other crop species. We will not present a complete review, but rather will select examples from those crops that seem to best illustrate the role of extrachromosomal inheritance in plant breeding. There have been a number of reviews of various aspects of extranuclear inheritance (Caspari, 1948; Bhan, 1964; Jinks, 1964; Edwardson, 1970). A limited discussion of what is currently known about the cytoplasmic makeup of plants is included to aid the reader in understanding how these factors may play a role in plant breeding. The literature on these factors is rapidly expanding and cannot be fully covered here. Several writers have included discussions on the mechanisms involved in extrachromosomal inheritance. We have presented some of these suggestions of the mechanisms and have tried to relate these to plant breeding procedures. The building blocks for the plant breeding program are the variations present in the species or closely related species and that new variation which can be induced. Much more information is needed concerning different cytoplasms and how differences arise or may be induced. We have summarized some of the most promising suggestions and have discussed their ramifications. II.
A.
Composition of the Cytoplasm
CYTOPLASMIC ORGANELLES AND MACROMOLECULES
The generalized cell of a higher plant is composed of the living protoplasm surrounded by a rigid pectocellulose cell wall. Cell protoplasm con-
4
P. H. HARVEY, C. S. LEVINGS, 111, AND E. A. WERNSMAN
sists of the cell membrane surrounding the cytoplasm with its paraplasmic inclusions (such as vacuoles) and the cell nucleus. The plasma membrane or plasmalemma surrounding the cytoplasm is of tripartite lipoprotein structure (Robertson, 1959) with osmotic properties of a semipermeable membrane; i.e., permeable to water and some molecules, but impermeable to many other molecules in solution. Continuity between cell membranes of adjacent cells is provided through numerous fine pores in the cell wall, the plasmodesmata. It is tempting to regard plasmodesmata as favored routes of exchange between cells, since plant viruses are commonly assumed to pass through these pores (Esau, 1968). The cytoplasm is delimited into elongated and irregular cavities by a membranous network, the endoplasmic reticulum (Porter et al., 1945). The cavity system of the endoplasmic reticulum is occupied by the aqueous medium of the cytoplasm which contains enzymes, soluble ribonucleic acids and other macromolecules. The endoplasmic reticulum is connected to and continuous with the outer membrane of the double membrane nuclear envelope ( DuPraw, 1969). Numerous pores pierce the nuclear membrane and probably provide avenues of communication between the nucleoplasm and the internal spaces of the endoplasmic reticulum (Feldherr, 1962, 1965). Electron photomicrographs of the cytoplasm of plant cells reveal ribonucleoprotein particles (ribosomes) associated with the endoplasmic reticulum, as well as areas where the ribosomes are free in the cytoplasm ( Frey-Wyssling and Muhlethaler, 1965). Ribosomes associate with a single strand of messenger ribonucleic acid forming a polysome or polyribosome (Risebrough et al., 1962; Clark et al., 1964) in the translational process of protein synthesis. Pea seedling ribosomes were first isolated and purified from cell homogenates by sucrose gradient centrifugation (Ts’o et al., 1956, 1958). These ribosomes contained approximately 60% protein and 40% ribonucleic acid and exhibited a sedimentation coefficient of 80 S. Two classes of ribosomes with sedimentation coefficients of 70 S and 80 S were later found in leaf extracts of clover and spinach (Lyttleton, 1960, 1962; Clark et al., 1964). Boardman et al. (1966) demonstrated that the 70 S ribosomes are derived from cytoplasmic organelles, mainly chloroplasts, whereas the ribosomes of the cytoplasm possessed an 80 S sedimentation coefficient. In addition to the above components numerous membrane-enclosed organelles are carried in the aqueous medium of the cytoplasm by cytoplasmic streaming. These organelles include the plastids or proplastids, mitochondria, dictyosomes, spherosomes, peroxisomes, and glyoxysomes. In meristematic cells proplastids exist as small, colorless, undiff erentiated organelles and may develop into chloroplasts (or etioplasts in leaf cells grown in the dark), amyloplasts or chromoplasts depending on the
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
5
final tissue in which the cell differentiates (Kirk and Tilney-Bassett, 1967). Chloroplasts contain the chlorophyll and carotenoid pigments in the green tissues of leaves and stems, and are responsible for the photosynthetic conversion of carbon dioxide into carbohydrates. Amyloplasts are found in differentiated cells of roots, tubers, cotyledons, and endosperm tissues and serve as starch storage sites. Chromoplasts are found in cells of flowers, fruits, and certain root tissues (carrots), and their carotenoid content is responsible for the red, orange, and yellow colors of these tissues. Mitochondria are numerous in the cell cytoplasm and primarily function in a respiratory capacity. Mitochondria possess all the enzymes and cofactors required in the tricarboxylic acid (TCA) cycle which occurs exclusively in this organelle (Lehninger, 1964). The organelle also contains the electron transfer carriers associated with systems that involve the TCA cycle. Oxidative phosphorylation reactions are coupled to the electron transfer system resulting in the generation of “energy-rich” adenosine triphosphate (ATP); the latter system is located on the membranes of the organelle. Collectively, a number of interassociated dictyosomes in a cell form the Golgi apparatus, a structure whose existence was denied by most cytologists until its demonstration in electron micrographs (Perner, 1958). Cunningham et al. (1966) isolated plant dictyosomes and showed that an individual dictyosome is built up from a pile of five or six platelike membranous structures, cisternae, with its attached tubules. The margins of the cisternae apparently break off to form Golgi vesicles, which grow and migrate to the cell membrane. Mollenhauer and Whaley (1963) have demonstrated that dictyosomes play a part in the shedding of root cap cells of the maize root. Thus, it has been suggested that the Golgi apparatus supplies secretory products which participate in the construction of the pectocellular cell wall (Dalton, 1961; Mollenhauer and Whaley, 1963). Spherosomes are intracellular particles which exhibit an aflinity for lipophilic stains (Frey-Wyssling and Muhlethaler, 1965) are found in abundant numbers in seeds of oil crops (Yatsu, 1965). Present evidence indicates that spherosomes are principal sites of lipid storage in cotyledons (Jacks et al., 1967). In the germination of lipid-containing seeds, stored lipids are converted to sugars by enzymes of the glyoxylate cycle. Briedenbach and Beevers (1967) have shown these enzymes to be associated with a subcellular particle termed glyoxysomes. Tolbert et al. (1968) isolated similar microbodies from leaf tissue which they call peroxisomes. The latter authors suggest that the presence of peroxisomes may be correlated with the phenomenon of photorespiration. However, peroxisomes were also present in leaves of plants that do not exhibit photorespiration (Tolbert et al., 1969).
6
P . H. HARVEY, C . S .
LEVINGS,
111, AND
E. A. WERNSMAN
Both glyoxysomes and peroxisomes contain catalase and glycolic oxidase enzymes and appear to be closely related (Briedenbach et al., 1968), although Ching (1970) has proposed that glyoxysomes in pine seeds disintegrate after germination when the lipid substrates of the seed are exhausted. Animal cells contain additional organelles, Iysosomes, which are biochemically characterized by the presence of acid hydrolytic enzymes. Lysosomes digest unwanted materials and remove damaged organelles from the cytoplasm. Lysosomes, as such, have not been demonstrated in plant cells at this time (DuPraw, 1969). However, lysosomal enzymes and membranous materials have been observed in plant vacuoles (Matile, 1968, 1969; Gahen, 1969; Villiers, 197 1 ) . Villiers suggests, therefore, that the vacuole in a mature plant cell originates and develops from a lysosome in a meristematic cell and through modification assumes vacuolar functions of turgor and storage.
B.
EXTRACYTOPLASMIC INCLUSIONS (PATHOLOGICAL INCLUSIONS)
In addition to normal constituents, plant cells may contain various pathological inclusions; of these only viruses will be considered in the present discussion. The location and effect of a particular virus in a plant is dependent on the virus-host combination rather than the virus itself. Virus inoculation into a host may be restricted or nonrestricted to particular plant tissues for successful plant infection. Esau (1968) cites as examples, beet yellows virus, a virus which is largely dependent on introduction into the plant phloem for successful infections, whereas tobacco mosaic virus is a nonselective virus and may infect any host tissue. Viruses can be widely distributed throughout a cell but are generally considered to be located in the cytoplasm (Gerola and Bassi, 1966; Arnott and Smith, 1967,1968; Esau, 1967). Viral infections can have pronounced effects on plant morphology, inherited genetic characters, and general plant vigor. Nyland ( 1962) suggested that viruses were capable of inducing genetic abnormalities in fruit trees. Sprague et al. (1963) observed deficiencies of marker genes in F, seeds, and distorted segregation ratios in F, ears when the P, pollen parent in a maize cross was infected with barley stripe mosaic virus. These distortions persisted in further backcross generations. Viral infections are known to cause male sterility in some host plants and numerous examples are given in the review by Atanasoff (1964a). However, it appears that these male steriles consist of two types: (1) those instances where the general vigor of the plant is reduced to the point the
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
7
plant is male sterile and ( 2 ) examples where the general morphology and vigor of the plant seem normal but the plants fail to produce viable pollen.
111.
DNA and Its Role in Cytoplasmic Organelles
The presence of extranuclear DNA in the cytoplasm is now well established. In the higher plants DNA has been isolated from chloroplasts and mitochondria (Granick and Gibor, 1967). Tobacco, spinach, lettuce, broad bean, pea, and beet are some of the crop plants from whose chloroplasts DNA has been isolated. Similarly, mitochondria1 DNA (mDNA) has been isolated from tobacco, bean, turnip, onion, sweet potato, spinach, beet, swiss chard, lettuce, and peanut. Furthermore, the possibility cannot be discounted that cytoplasmic genetic systems based on DNA other than those of the chloroplasts and mitochondria will eventually be found. A.
CHLOROPLASTS
The size of chloroplast DNA (cDNA) is approximately that of the bacterial chromosome (Noll, 1970). Clearly, evolution would not allow the preservation of cDNA unless a significant informational content was present. Only recently have studies begun to elaborate on the informational content of the cDNA and it is readily apparent that our knowledge is still meager. A summary of the role of cDNA follows with particular emphasis on crop plants. cDNA has been demonstrated to differ from nuclear DNA (nDNA) in buoyant density, base composition, and renaturation properties. For example, a buoyant density of 1.700 f 0.001 for cDNA and 1.697 zk 0.001 for nDNA was reported in tobacco by Tewari and Wildman (1970). Differences in buoyant density between cDNA and nDNA were not found in spinach, lettuce, broad bean, and tobacco (Whitfeld and Spencer, 1968; Wells and Birnstiel, 1969). A distinction in base composition has been determined between nDNA and cDNA. The base, 5-methylcytosine, comprised between 3.6 and 6.5% of the base composition in nDNA from tobacco, spinach, and lettuce while only 0-1.6% was found in cDNA (summarized by Tewari and Wildman, 1970). Finally, differences in renaturation properties between nDNA and cDNA were found in tobacco, spinach, and lettuce (Tewari and Wildman, 1966; Whitfeld and Spencer, 1968; Wells and Birnstiel, 1969). A difference in renaturation behavior between c- and nDNA has been used as a criterion for distinguishing between DNA’s in the broad bean by Kung and Williams (1969) since similar buoyant densities were found.
8
P . H. HARVEY, C. S. LEVINGS, 111, AND E. A. WERNSMAN
The discovery that chloroplasts have their own DNA would suggest the possibility that they have some capability of directing the synthesis of their own RNA, DNA, and protein. Evidence that chloroplasts direct their own DNA synthesis comes from the finding of a DNA polymerase capable of making chloroplast-like DNA in vitro in tobacco and spinach (Tewari and Wildman, 1967; Spencer and Whitfeld, 1967a). Furthermore, Green and Gordon ( 1966) have demonstrated replication of cDNA with tobacco. Isolated chloroplasts from pea, spinach, and tobacco have been shown to possess an independent system for protein synthesis (Sissakayan et al., 1965; Boardman et al., 1966). A DNA-dependent RNA polymerase has been found in spinach and tobacco chloroplasts (Spencer and Whitfeld, 1967b; Tewari and Wildman, 1969). These polymerases were capable of making in vitro RNA ranging in size from 4 S to 30 S and differed in many properties from the corresponding nuclear polymerase. Aliev et al. (1967) have isolated aminoacyl-tRNA synthetase and tRNA from pea chloroplasts. A distinctive species of ribosomes has been obtained from chloroplasts of clover, spinach, Chinese cabbage, and tobacco. These ribosomes have a sedimentation constant of approximately 70 S as contrasted with 80 S for cytoplasmic ribosomes as previously described. Finally, by hybridization studies Tewari and Wildman ( 1970) have demonstrated in tobacco that ribosomal-, transfer- and messenger-RNA of chloroplasts can be coded by the chloroplast genome. Precisely which enzymes or proteins are coded by plastid DNA remains largely unknown. Kirk and Tilney-Basset ( 1967) have reviewed the evidence from higher plants and algae. It is clear that many enzymes involved with synthesis of chlorophylls and carotenoids and the photosynthetic process are controlled by nuclear genes. However, genes which determine chloroplast ribosomal RNA appear located in the plastid, and there is some evidence that two structural genes for enzymes concerned with chlorophyll synthesis are also in the plastid. Biochemical characterizations of the plastom mutants of higher plants have not progressed to the point where definite information has been determined as to the nature of the genes of cDNA. Mutant phenotypes themselves should provide the most direct method of identifying the corresponding mutant; that is, a mutation in cDNA should affect chloroplast function. This, however, is an oversimplification since it is apparent that the chloroplast and nuclear genome interact and regulate each other. Therefore, it is not easy to associate unique phenotypic changes to either n- or cDNA. Furthermore, the informational content of cDNA is not necessarily similar among different genera. In summary, considerable is known about what functions are not controlled by genes of cDNA, but very little is known about the functions it does control.
EXTRACHROMOSOMAL INHERITANCE I N PLANT BREEDING
9
B. MITOCHONDRIA The DNA of higher plant mitochondria has not been extensively investigated. Where it has been studied, it has been determined to have a molecular weight about 10 times greater than found in mammals and about 3 times greater than in yeast (Ashwell and Work, 1970; Tewari, 1971). It has been suggested in explanation that the size of mDNA decreases as we go to organisms of higher levels of evolution (Borst, 1970). In any event, the larger amount of mDNA in higher plants should permit a greater coding potential than is found in other organisms. Interestingly, the molecular weight of mDNA (140 x lo6 daltons) is of the same order as cDNA (120 X l o 6 ) in higher plants (Tewari, 1971). The presence of mDNA would suggest that mitochondria may have some capability for the synthesis of their own DNA, RNA, and protein. Indeed, studies oriented to test these questions have found that mitochondria contain most of the elements required for a functioning mitochondrial genetic system. These studies have dealt primarily with mitochondria of animals and lower organisms such as yeast, Neurospora, Tetrahymena, and algae, and many excellent reviews are available (Borst and Kroon, 1969; M. M . K. Nass, 1969; Swift and Wolstenholme, 1969; Ashwell and Work, 1970). Tewari (1971) has recently discussed mDNA in higher plants. Briefly, these studies have determined that mDNA has unique structural properties and can be synthesized and replicated within the organelle. Mitochondria can synthesize RNA and contain ribosomes and ribosomal RNA's which differ from those of the cytoplasm as well as specific species of tRNA and aminoacyl-tRNA synthetases. Last, the organelle can incorporate amino acids into protein and this incorporating system differs in sensitivity from the cytoplasmic system with regard to several antibiotics. The presence of mDNA and many components of the protein synthesizing system strongly suggests that mDNA has a genetic role. The exact genetic role of mDNA as with cDNA, is largely undefined. Evidence from Neurospora and yeast suggest that mutations of mDNA cause alterations in mitochondrial protein. The poky mutant in Neurosporu apparently causes alteration of the amino acid composition of mitochondrial structural protein (Woodward and Munkres, 1966) while several instances of missing membrane protein of the mitochondria have been reported from the petite mutant of yeast and the poky mutant of Neurosporu (Work, 1967; Tuppy and Swetly, 1968; Sebald et al., 1968). Evidence that mDNA codes for some ribosomal and transfer RNA's is good and some evidence exists that it codes for its own messenger RNA, and thus, some of its own proteins (Ashwell and Work, 1970). However, the mito-
10
P.
H. HARVEY, C. S.
LEVINGS,
111, AND E. A. WERNSMAN
chondrial enzymes present an interesting contrast since more and more of these enzymes are being found to be under control of nuclear genes. The situation appears complex and analogous to that found with chloroplast; that is, the mitochondrial and nuclear genomes apparently interact and regulate each other. In conclusion, very little is known about the biochemical significance of mitochondrial genes.
IV.
Plant Traits Influenced by Cytoplasm
A.
DIRECTCYTOPLASMIC CONTROL
The developing organism is under the controlling influence of both nuclear factors and cytoplasmic factors, and of the interaction of the two. Most traits are reported to be under mainly nuclear genic control while others (relatively few by comparison) are under mainly cytoplasmic factor control. Among these latter cases are several that are of much concern to the plant breeder. 1. Chlorophyll Deficiencies
The study of variation in chlorophyll within the same plant tissue gave
us the first example of transmission of extrachromosomal traits. Correns (1909) first demonstrated that variegated plants of Mirubilis jalopa (four o’clock) had two kinds of plastids, normal for green and mutant for colorless or white. These types of plastids were passed on through the seed based on the maternal parent of the seed. Similar variation in plastids has now been reported in many species. Rhoades (1943) reported more than 100 cases of chlorophyll variants in maize and two examples of cytoplasmically inherited chlorophyll variegations. Chlorophyll deficiencies have been discussed by Bhan (1964) and Jinks ( 1964). Examples of maternally transmitted variegation were given in Spiragyra triformis (alga), Primula sinensis, Pelargonium zonale, Epilobium, Oenothera, Nepeta cataria, and Zea. For an extensive review of plant variegations caused by environmental, nuclear, and extrachromosoma1 factors, the reader is referred to Kirk and They-Bassett (1967). Chlorophyll variegations in tobacco were summarized by Smith ( 1968 ) . These include those that are cytopiasmically inherited, one case where patterns of variegation arose from the different histogenic layers, and a third type where a nuclear gene interacted to eliminate the cytoplasmic factors. Working with a maternally inherited variegated plant of cotton, G o s ~ y p ium hirsutum L., Kobe1 and Benedict ( 1971) reported the mutant chloro-
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
11
plasts fixed reduced amounts of CO, but utilized acetate-2-14C as effectively as the normal green chloroplasts. Sixteen spontaneous plastogene mutants (resulting in variegated plant types) in poinsettia, Euphorbia pulcherrima Willd. ex Klotzsch., and two in carnation, Dianthus, species have been reported and studied by Stewart (1965). A complex genetic system was suggested for at least 16 different mutants to account for the 16 observed phenotypes. The author summarized: “The data established that the plastogenes of the two species undergo extensive spontaneous mutation including stepwise change, indicating that there are a number of sites capable of change and that at least some of the mutants are the result of change in and not the loss of genetic material. Among the characters controlled by plastogenes are the size and color of chloroplasts, the sensitivity of chlorophyll to light, the rate of division of the mutant proplastid, and the growth and differentiation of tissue derived from the histogenic layer carrying the mutant.” Stroup (1970) reported a chlorophyll change in corn which was induced by a recessive gene cm but once induced was transmitted cytoplasmically. This gene thus acted in a similar fashion to the iojap gene in inducing a change in an extrachromosomalfactor. In ornamental crops the presence of variegated plants may be the objective of the breeder’s research. In this case the breeder clearly wishes to know how the variegated leaf patterns are transmitted so that they may be effectively propagated. Generally, however, variegated plants must be considered detrimental and would be of interest only for fundamental studies. 2 . Male Sterility In a review Edwardson (1970) stated that cytoplasmic male sterility has been reported in 80 species, 25 genera, and six families. He also stated, “Locating sites of sterility factors, determining how they are transmitted, and elucidating mechanisms controlling sterility have received less attention than the use of cytoplasmic male sterility to exploit heterosis.” Edwardson also pointed out that sources of cytoplasmic male steriles may be grouped as: (a) intergeneric, (b) interspecific, (c) intraspecific, and (d) apparently spontaneous. Smith (1968) has reviewed recent publications on the occurrence and inheritance patterns of male-sterility factors in tobacco. A number of male-sterile lines have been developed and studied over the past forty years. Smith stated, “In fact, it now appears to be a widespread phenomenon in the genus; that is, the cytoplasm of one species (A) combined with the partial or complete genome of another species (B) will often produce male sterility, and furthermore, genetic restorers to pollen fertility will be
12
P. H. HARVEY, C. S. LEVINGS, 111, A N D E. A. WERNSMAN
found in certain chromosomes of species ( A ) .” Chaplin ( 1964) investigated eight different sources of cytoplasmically inherited male sterility to determine their relative value in production of hybrid tobacco seed. He described six types based on flower structures and their modification. He concluded that type 5 , shortened corollas, with modified petaloid anthers, and protruding stigmas ( N . undulata and N . tabacurn cytoplasm) would probably have the most economic value in production of hybrid tobacco seed, but that hand pollination would be required. As mentioned earlier, cytoplasmic male sterility in maize was extensively studied as a tool in the production of hybrid seed on a commercial scale. Beginning in the mid 1930’s Rhoades worked with a cytoplasmic malesterile and some attempts were made to use the male sterile as a seed parent in hybrid production. This source of male sterility proved to be too easily affected by environmental changes to be a dependable seed parent. Rogers and Edwardson (1952) reported on the successful use of their male-sterile lines in hybrid seed production. This source of cytoplasmic male sterility could be developed in many inbred lines by repeatedly backcrossing the normal inbred pollen to the male-sterile line. Certain inbred lines were found to restore pollen fertility in this cytoplasm (now widely known as Tcms). These restorer lines were useful in the final production of commercial hybrid seed. One important aspect of this male-sterile and restorer program was the stability of the T cytoplasm. The restorer genes Rf,, Rf2, while restoring pollen fertility did not change the cytoplasmic characteristic. Male sterility could be recovered in any generation by the removal of the Rf,,Rf2 alleles and the substitution of rf,rf, and rf2rf2.This stability of the cytoplasm was dramatically demonstrated with the reaction of the two diseases, yellow leaf blight, Phyllosticta zeae (Scheifele et a!., 1969), and southern leaf blight, Helrninthosporium maydis (race T ) (Villareal and Lantican, 1965). These two diseases have attacked the T cytoplasm irrespective of the presence of restorer genes. In addition Pesho et ul. (1969) reported increased susceptibility to leaf feeding by European corn borer, Ostriniu nubilalis Hubner, on maize strains carrying Tcms. Feeding response of the borer was variable in the presence of restorer genes. The role of the mitochondria in corn as a possible explanation for susceptibility to the disease-producing organism ( H . rnaydis) was suggested by Miller and Koeppe (1971). They have observed differential responses to the corn blight pathotoxin on mitochondria from normal cytoplasm corn plants (resistant) compared to mitochondria from T cytoplasm plants (susceptible). Mitochondria from the susceptible hybrid exhibited a different respiration rate depending on the substrate being oxidized. Other differ-
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
13
ences observed included reduced respiratory control and ADP :0 ratios and increased swelling rate of mitochondria in KC1 medium under either passive or active conditions. These differential responses of mitochondria to the pathotoxin might account for the rapid dying of infected plant tissue in T cytoplasm lines. This raises some very interesting points concerning the distinction between T and normal cytoplasm. Are there three or more plasmagenes which are different between the two cytoplasms? The independence of the male sterility from the reaction of the two disease organisms would certainly suggest that these may be multiple changes which are self-reproducing. The cytoplasmic male-sterile factor gave us a new tool for studying the plasmon and for commercial use. The two disease reactions now give us additional markers for studying the plasmon complex and may well help clarify the role of extrachromosomal inheritance. In a comprehensive treatise on cytoplasmic pollen sterility in corn, Duvick (1965) listed five well known corn cytoplasms which induced sterility: Peruvian (Rhoades) ; Argentinian (Edwardson) ; Inbred 33-16 (Josephson and Jenkins) ; U.S.D.A. (Jones from genetic marker stock of iojap X teopod) and Texas (Rogers). The source of 84 separate discoveries of cytoplasmic male sterility was reported as coming from United States dents, sweet, and flint varieties and from genetic tester stocks but especially from Latin American open-pollinated varieties (26 out of 84). Through test crosses with inbreds with and without pollen fertility restorer genes, five sources were shown to be of Texas type and 25 of U.S.D.A. type. Many sources of male-sterile cytoplasms have been observed in breeding programs that have not been reported in detailed publications. Singh and Laughnan (1968) reported on a mutation in (S-type, U.S.D.A.) male-sterile cytoplasm producing a restoration of fertility. Very few back mutations of this type have been reported, and none in the extensively used Texas source. Cytoplasmic male sterility in sorghum functions in much the same manner as in corn. Stephens and Holland (1954) reported that the interaction between milo cytoplasm and kafir nuclear factors produced male-sterile lines. They showed that crossing the male-sterile kafir lines to milo restored pollen fertility and they suggested that more than two factor pairs are operating in restoring fertility. This system of using male-sterile kafir lines as seed parents has been the sole system used in our present day hybrid sorghum commercial seed production. This complete reliance on one cytoplasmic system for a total industry is extremely dangerous, as has been shown in the corn disease problems already discussed. Ross (1971) reported the release of six cytoplasmic male-sterile lines with cytoplasm from five different species. According to Ross these releases were prompted by
14
P. H. HARVEY, C.
S. LEVINGS, 111, AND E. A. WERNSMAN
leaf blight in corn. These may prove to be valuable as sorghum breeders investigate their potential as seed parents and in furthering our knowledge of cytoplasmic control of plant development. Alam and Sandal (1967a) studied six lines of Sudangrass which restored pollen fertility in the male-sterile cytoplasm. They reported one, two, and three pairs of genes interacting with male-sterile cytoplasm to restore pollen fertility. Thus Sudangrass seems to have the same nuclearcytoplasmic interaction of fertility-sterility control as reported in sorghum. Raj (1968) working on male-sterile and male-fertile sorghum reported the degeneration of the male gametes in male steriles. Degeneration took place following meiosis and the first mitotic division of microspores. Alam and Sandal ( 1964) working with Sudangrass reported normal meiosis and tetrads of microspores but degeneration of the pollen grains during anther maturation in cytoplasmic male-sterile lines. Alam and Sandal (1967b, 1969) showed differences in free amino acids in male-sterile and malefertile Sudangrass. Proline was high and asparagine low in prepollen stage anthers in male-sterile plants as compared to male fertiles. They also have shown fewer biochemical components in male-sterile anthers indicating that heritable abnormal metabolic activity was associated with pollen abortion. Breeders have been investigating various methods of producing F, commercial wheat ( Triticunz aestivum L. ) hybrids. While there is still considerable disagreement as to the final value of hybrid wheat to the commercial grain producer, there are enough favorable data to have stimulated a considerable amount of research. Both public and private agencies are engaged in such studies. The report of cytoplasmic male-sterile wheat by Wilson and Ross (1962) gave hopes that a workable plan of seed production could be developed. Since wheat is grown annually on a vast acreage both in the United States and on a world basis, the benefits of any favorable response by hybrids over pure lines could be extended manyfold. Maan and Lucken ( 1971) have given a brief review of nuclear-cytoplasmic interactions and how substituting the genome of one species into the cytoplasm of another introduces male sterility in Triticum. These authors also pointed out the use of such interspecies genome-cytoplasm substitutions as a method of studying the origin of emmer and common wheat. From the plant breeding point of view, the wheat breeding lines are designated (suggested nomenclature) as: A-line has cytoplasmic male sterility; R-line has a gene or genes that restore male fertility to an A-line; B-line is a normal fertile counterpart of an A-line without a restored gene and is used as a pollen parent to maintain the A-line. This system is similar to that referred to in a sorghum except that the origin of the wheat cytoplasmic male sterile resembles more closely that of tobacco.
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
15
A slightly modified type of male sterility was reported by Grun (1970) in the cultivated potato, Solanum tuberosum. The cytoplasmic factor (inr) conditions resistance to the effects of the dominant In gene. The cytoplasmic factor (In*) and the In gene lead to the development of indehiscence of anthers and male sterility. The authors discussed the role of these geniccytoplasmic interactions in relationship to the origin of the cultivated potato. Interest in male sterility in the potato has centered around evolutionary studies rather than as a means of developing F1 hybrid seed, since the potato is reproduced asexually. The use or potential use of cytoplasmic male sterility and restorer gene interactions for the production of hybrid seed is of interest in onion (Jones and Clarke, 1943), cotton (Meyer, 1971); flax (Chittenden, 1927), sugar beets (Theurer and Ryser, 1969), alfalfa (Davis and Greenblatt, 1967), intermediate wheatgrass (Schultz-Schaeffer, 1970), petunia (Edwardson and Warmke, 1967), and several others. Most crop species have genic controlled male-sterile types. These are not a part of this discussion except to point out that they do play a role in plant breeding. In barley Thompson (1970) discusses a “balanced tertiary trisomic concept’’ which uses a genetic recessive male-sterile gene in hybrid seed production. Brim and Young (1971) have reported a genetic male sterile which they are using to speed up recombinations in soybean breeding schemes. 3. Agronomic Characters
Comparatively little is known or reported on the cytoplasmic control of those plant traits which are commonly referred to as agronomic characters. Most such traits are primarily under nuclear control, and the literature is extensive on genic influences of such characters as plant height, number of heads per plant, number of leaves per culm, maturity, and yield of the economic portion of the crop. Some cases of plant traits being influenced by the cytoplasm have been reported. Duvick (1958) working with seven male-sterile hybrids of maize and their normal counterparts reported several examples of significant differences. The six T cytoplasm hybrids yielded less in Iowa but more in Illinois. Other traits showing significant differences between the T cytoplasm and normal hybrids included number of barren plants at high planting rates in Illinois, number of tillers per plant, and in the S cytoplasm hybrid yield and number of barren plants were affected. Moisture in grain at harvest, stalk breaking, and southern leaf blight showed no consistent differences-for either T or S cytoplasm. The lack of consistent differences in response to the southern leaf blight in the two cytoplasms is of real significance when compared to the very striking difference observed in recent years to the new race T of H . rnaydis.
16
P. H. HARVEY, C. S. LEVINGS, 111, AND E. A. WERNSMAN
This lends support to the theory that the disease organism has changed markedly. Fleming et al. (1960) studied the effect of cytoplasm on the genotype of one yellow double cross in maize. The same hybrid was made up using each inbred parent as the cytoplasmic donor. These were then compared in paired plantings in the field. Ear and plant height were significantly different when CI 21 cytoplasm was compared to GA 172. Other comparisons showed differences in number of erect plants and grain yield. These differences were of a relatively low magnitude and were strongly influenced by environment. Six exotic maize cytoplasmic sources when crossed with WF 9 x 38-11 and Oh 45 X C 103 were reported to influence some agronomic traits (Singh, 1966). Number of smutted plants, and ear and plant height were influenced by the source of cytoplasm in half or more of the observed comparisons. Days to mid-silk, number of leaves and number of tillers were affected by the cytoplasms in more than a fourth of the comparisons. Exotic cytoplasms had very slight effects on grain yield, ear length, and moisture content at harvest. Plant growth habit in peanuts, Arachis hypogeue L. (runner vs bunch) is governed by interaction of two cytoplasms and two nuclear loci (Ashri, 1968). The two nuclear genes, Hb, and Hb, are complementary for runner growth habit when in (V,) plasmon. If either locus is lacking a dominant allele, the resulting plant has the bunch growth habit. In the “Others” plasmon, however, the dominant alleles are additive and may be complementary so that three or more dominant alleles in any combination will result in a runner plant while those with 2, 1, or 0 dominant alleles will produce bunch-type plants. Significant maternal effects were observed in peanut crosses by Parker et al. (1970). Characters showing maternal effects were number of leaves, cotyledonary branches (at 15 days of growth), and leaf width (at 18 days). These observations do not establish extrachromosomal influences on these traits. A leaf trait, compact, was reported as being controlled by extrachromosoma1 factor or factors in Chenopodium rubrum by Murray and Craig (1968). This trait was induced by X-ray treatment and the compact phenotype was associated with the curled-leaf (genic controlled) trait. The authors indicated that an extranuclear factor, or factors, involved in the curled-leaf expression had been altered. These examples of agronomic traits which are or may possibly be controlled by extrachromosomal factors illustrate how relatively few traits seem to be outside nuclear gene control. The difficulty of observing differences in cytoplasmic factors may contribute to the scarcity of our present knowledge. In contrast to the seemingly overpowering influence of the nu-
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
17
clear genes on dozens of morphological, physiological, pathological, etc., traits we iind few really well documented cases of characters other than the plastid mutants and male steriles. As mentioned earlier, the renewed interest in extrachromosomal inheritance may well add considerably to our understanding of what factors are present in the cytoplasm which can control the traits we work with as plant breeders. At this time one can only surmise that most of our so-called agronomic traits are influenced primarily by nuclear genes, with only minor interactions with cytoplasmic factors. B.
OTHERUNCLASSIFIED EFFECTS
The role of nuclear gene control over the cytoplasm in general is well accepted by most biologists. We have already mentioned many cases of genome-plasmon interactions. Some of the examples already cited of differences between reciprocal crosses may be the result of a delayed reaction of nuclear genes over cytoplasmic factors. These differences would tend to diminish in advanced generations. Another type of delayed gene action results in those traits that are controlled by maternal tissue in the developing seed. Brim et al. (1968) reported maternal effects on fatty acid composition and oil content of soybeans. All seed produced on a heterozygous plant (e.g., an F,) had similar fatty acid composition and oil content. It is necessary to study progeny of such heterozygous plants to observe segregation in fatty acid composition and oil content. This reaction may delay selection for these traits but also makes selection possible on a whole-plant basis rather than on an individual seed. Many investigators have considered the genetic basis for heterotic responses observed in many F, hybrids. Recently, mitochondria has been shown to have activities that are correlated to growth vigor of corn inbreds and their hybrids (Hanson et al., 1960; McDaniel and Sarkissian, 1966, 1968; Sarkissian and McDaniel, 1967). The function of the mitochondria have been discussed in Sections I1 and 111. They are now known to be extremely important cytoplasmic inclusions. Comparatively little is known about how they are inherited and how they interact with nuclear genes. Sarkissian and associates have discussed several interesting facts about mitochondria from corn scutellar tissue. McDaniel and Sarkissian (1968) demonstrated polymorphism of maize mitochondria. The role of complementation by mitochondria from two inbred lines and its relationship to mitochondrial activity from the F, hybrid has been discussed (Sarkissian and Srivastava, 1967). The authors have shown a direct relationship in most cases between mitochondrial activity (measured as oxidation and phosphorylation rates ) and seedling germination and radicle-elongation
18
P. H.
HARVEY,
C. S. LEVINGS, 1x1, AND E. A. WERNSMAN
rate in maize seedlings of inbreds and their hybrids. They suggested that complementation of mitochondria from two inbreds may provide an operational means of studying the biochemistry of heterosis. In addition, it may be useful in determining potential combining ability of parental lines in a breeding project. McDaniel (1972) proposed the use of mitochondrial complementation as a shortcut in predetermining hybrid vigor in crop varieties, thereby cutting years off the conventional breeding program. This work was related primarily to barley, although work on corn and wheat was mentioned.
V.
Possible Mechanisms Involved in Cytoplasmic Inheritance of Plant Traits
A. GENETICSYSTEM
OF THE
ORGANELLE
Earlier convincing evidence was presented which demonstrated that chloroplasts and mitochondria contain their own DNA with a unique informational content. Consequently, it seems reasonable to assume that many cases of extrachromosomal inheritance do, in fact, involve the genetic system of the organelle. Circumstantial evidence supporting this concept comes from the plastom mutants of higher plants (Kirk and Tilney-Bassett, 1967). However, few molecular studies of organelle DNA's, RNA's, and protein synthesis have been related to genetic analysis, nor, on the other hand, have genetic studies been considered in molecular terms. Recent findings that certain petite mutants of yeast possess structurally altered mDNAs (Mounolou et al., 1966) and the construction of linkage maps for chloroplast genes in Chlarnydornonas (Sager and Ramanis, 1970) and mitochondrial genes in yeast (Coen et al., 1970) have done much to establish the correlation between extrachromosomal inheritance and organelle DNA. Verification in higher plants awaits more refined studies.
B. VIRALEFFECTS Viral infections have been suggested as a possible explanation of some cytoplasmically inherited male sterilities. Furthermore, Atanasoff ( 1964b) has suggested that viral infections could account for all cytoplasmically inherited traits, even though their presence has not been demonstrated. Successful asexual transmission of male sterility through plant grafts has been demonstrated in petunia (Frankel, 1956, 1962, 197 1; Edwardson and Corbett, 1961; Bianchi, 1963) and sugar beet (Curtis, 1967). Petunia grafts of normal fertile scions on cytoplasmic male-sterile stocks exhibited
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
19
complete fertility in the grafted generation, but selfed or sibbed progenies from fertile graft components segregate for male sterility. Curtis observed similar results with sugar beets except that some fertile-sterile grafts when made as seedlings, exhibited male sterility in the grafted scion. Continued backcrossing of these induced male steriles to maintainer lines resulted in all male-sterile progeny and the character continued to behave as a cytoplasmically inherited trait. Attempts to asexually transmit cytoplasmic male sterility by rubbing leaves, inoculation of normal plants with expressed juices from steriles, growing sterile and fertile plants in adjacent plots, as well as fertile-sterile plant grafts have failed in maize (Rhoades, 1933), tobacco, (Burk, 1960; Sand, 1960), pepper (Ohta, 1961), Epilobium (Michaelis, 1964), wheat (Zevin, 1967; Lacadena, 1968), beets (Cleij, 1967), and onion (van der Meer and van Bennekom, 1970). Likewise, heat treatments for the inactivation of viruses have been ineffective in “curing” plants of cytoplasmic male sterility (Shumway and Bauman, 1966; Zevin, 1967). However, many viral diseases are difficult to transfer asexually as shown by Blakeslee’s (1921) classical demonstration that grafting was the only successful means of transmitting the Quercina character (a viral disease causing male sterility and other abnormal morphological phenomena) in Daturu. Although viral infections have not adequately explained all cases of cytoplasmically inherited characters, plant breeders should be cognizant that normal appearing plants may be carriers of latent viruses. Atanasoff (1925) demonstrated that some potato varieties artificially inoculated with Y virus showed slight or no symptoms even though the varieties carried the virus. Johnson ( 1925) and Schultz (1925) transmitted virus diseases from apparently healthy potatoes to susceptible varieties and the potato variety KING EDWARD is a classic example of a masked carrier of viruses (Salaman and Le Pelley, 1930; Salaman, 1932). More recently Isaacs and Lindemann ( 1957) and Isaacs (1961) have demonstrated that the inoculation of organisms with a heat-inactivated virus results in the induction of a proteinaceous factor (interferon) in the host which interferes with this virus and others. Atanasoff (1963, 1964b) suggested that the development of interferon is a universal process among some genotypes of all living organisms and provided numerous examples of viral phenomena in plants that could be explained by interferon development. Under such a hypothesis, infection of a plant variety with a virus could result in the induction of interferon. Provided that the virus is seed transmitted, as many viruses are (Bennett, 1969), or the crop is asexually propagated, this variety would be expected to carry the virus in a latent condition and, although exhibiting no disease symptoms itself, would serve as a source of inoculum to infect susceptible varieties.
20
P. H.
HARVEY, C.
S . LEVINGS,
111,
AND E. A. WERNSMAN
C . NUCLEAR GENETICSYSTEMSWHICHMIMIC EXTRACHROMOSOMAL INHERITANCE The possibility that nuclear genes are responsible for apparent extrachromosomal inheritance cannot be dismissed. Heslop-Harrison ( 1963, 1967) has proposed that cytoplasmic male sterility might be explained by operon-type controls rather than by independent cytoplasmic determinants. Without detailing his explanation, the basic argument is that a specific condition under genic control is propagated in the cytoplasm of female plants which imposes a permanent repression upon the pollen detemining genetic system. Because cytoplasm is passed between female parent and offspring, the progeny of male-sterile plants will also be male sterile. Edwardson (1970) pointed out that extension of Heslop-Harrison’s hypothesis to systems with fertility restorers has not been attempted and would require the formulation of further assumptions. Nevertheless, the hypothesis, although unproved, provides a feasible and interesting alternative. D.
UNDISCOVERED EFFECTS
Episomes or genetic elements are known in bacteria which can be inserted at a particular site and become an integral part of the bacterial chromosome, or may exist and replicate independently of the bacterial DNA (Jacob and Wollman, 1961). The possibility of similar episomal systems between nuclear DNA and DNA of plant organelles or any other cytoplasmic DNA that may exist in higher plants merits discussion. Organelle DNA when initially isolated was considered to differ from nuclear DNA in buoyant density, base ratios, and the apparent absence of 5-methylcytosine in cytoplasmic organelles (Kirk, 1963; Whitfield and Spencer, 1968; Baxter and Kirk, 1969). More recent evidence indicates that differences between nuclear and organelle DNA are probably minimal (Tewari and Wildman, 1970). However, translational products of organelle DNA are essentially unknown in higher plants, and it is difficult to verify that similar information can be coded in either the nucleus or the organelle (Anderson and Levin, 1970; Williams and Williams, 1970). In progenies from petunia grafts of normal fertile scions on cytoplasmic male-sterile stocks, Frankel (1 97 1 ) observed two types of male steriles. The cytoplasmic-type sterile previously reported in petunia grafting experiments (Frankel, 1956) were the most common, but a chromosomally inherited recessive gene for male sterility was also observed. It was definitely shown that the gene for male sterility originated in the grafted generation, and Frankel has suggested that the same hereditary element for male sterility in petunia may exist either in a cytoplasmic or chromosomal state.
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
21
However, this chromosomal element for male sterility could be explained equally well by the occurrence of a spontaneous mutation of a normal gene for fertility to the recessive allele for sterility. Consequently, the generality of such an “episomal-type” phenomenon in higher plants awaits further verification.
VI.
Cytoplasmic Differences-Possible
Origin and Ramifications
Convincing evidence has accumulated supporting the theory that plastids and mitochondria originated as prokaryotes which found shelter within primitive eukaryotes and finally established themselves as permanent symbiotes (S. Nass, 1969; Raven, 1970; Taylor, 1970). In the course of evolution it has also been postulated that organelles have lost various functions to the dominant nucleus. Lewis (1970) has suggested that the transfer of all but the bare necessity of DNA from mitochondria and chloroplasts to the safety of the nucleus could provide a selective advantage. The relative small amount of mDNA found in the highly evolved mammals as compared with the large amount found in fungi and higher plants supports this claim. Although the precise nature and location of cytoplasmic factors which control extrachromosomal inheritance in higher plants is unknown, the above theory allows for interesting speculation on their origins. In higher plants speculation on the origin of cytoplasmic male sterility and fertility restoring genes merits consideration because of their importance to plant breeders. If it is assumed that some factors controlling male fertility reside on organelle DNA, then an alteration of organelle DNA might give rise to cytoplasmic male steriles. The alteration might be a point mutation or an extensive loss of DNA inasmuch as both types of events have been observed in organelle DNA of fungi and algae. Since strict maternal inheritance of organelles is more common in crop plants, a nonMendelian mode of inheritance would be observed. Nuclear genes which restore fertility to cytoplasmic male-sterile plants are very common (Edwardson, 1970). Fertility restoring genes might represent the assumption of a function by the nucleus which had been lost from an organelle. The change in site from the organelle to the nucleus might have evolved even before the functional loss occurred in the organelle. In this event, once the function was firmly integrated into the dominant nucleus, a redundancy occurred which was subject to elimination by loss from the organelle. The studies of Ledoux and Huart (1969) suggest that the transfer of DNA from organelle to nucleus is feasible. They have shown that DNA of the bacterium Micrococcus lysodeikticus can be incorporated into the nuclear DNA of barley seedling. In this connection, the
22
P. H. HARVEY, C. S . LEVINGS, 111, AND E. A. WERNSMAN
exchange of DNA between organelles was demonstrated in the genetic recombination studies with chloroplast as well as mitochondria1 genes in Chlamydomonas and yeast (Sager and Ramanis, 1970; Coen et al., 1970). Thus, the transfer of function from organelle to nucleus could actually be a physical transfer of DNA. It is, however, not necessary to invoke a physical transfer of DNA, since evolution at the nuclear level could account for the addition of a new function. Perhaps physical transfer and independent evolution both play roles in the acquiring of functions by the nucleus which were previously solely controlled by organelle DNA. Taxonomic plant classification is based on the assumption that species within a genus as well as closely related genera originated from common ancestors. Close relationships between species generally assume that the evolutionary distance to common ancestors is not great. Consequently, species within a genus might be assumed to evolve from ancestors with a common completent of plastids and mitochondria. Since a redundancy of genetic information could have existed between organelle DNA and nuclear DNA, a portion of this information in one site (organelle) could be lost without deleterious effects of the species as long as the same information was retained by the other (nucleus). The reciprocal situation is possible also, but it is proposed that the nucleus retained the dominant role of information coding. Related plant species would ultimately evolve independently from their common ancestor. The loss of information from the organelle with the retention of this same information by the nucleus in one species could be paralleled by the reciprocal situation in a second close relative. Under these assumptions interspecific hybridization between two species followed by appropriate backcrossing to establish the nucleus of one species into the cytoplasm of a second, or the reciprocal, would result in nuclear cytoplasmic combinations, in which coded information would be absent in cytoplasmic organelles as well as the nucleus. Such combinations would be expected to exhibit nuclear-cytoplasmic interactions and might account for some lethalities and more subtle effects observed in derivatives of interspecific hybrids. Although much remains to be learned concerning extrachromosomal inheritance in higher plants, evaluation of its importance to plant breeders merits consideration at this time. When the informational content of the nucleus and cytoplasm is compared, extrachromosomal inheritance must be relegated to a minor role. Certainly a plant breeder can alter more characteristics by manipulation of nuclear genes than by plasmogenes. Nevertheless, in the minority of cases where cytoplasmic factors do exercise control over a trait, they have been, and will continue to be, useful. Currently, cytoplasmic male sterility in crop plants is the most valuable extrachromosomally inherited trait. Perhaps other useful traits will be found under the
EXTRACHROMOSOMAL INHERITANCE IN PLANT BREEDING
23
control of plasmogenes, particularly those that would benefit from maternal inheritance. Deleterious traits also occur which are ascribable to cytoplasmic factors. The corn blight which afflicts plants with T cytoplasm has dramatically illustrated this point. Interestingly the widespread use of T cytoplasm has uncovered a deleterious cytoplasmic trait, susceptibility to leaf blight diseases, which in all probability would have never been discovered. With the discovery that certain disease organisms were especially virulent on the Tcms inbreds and hybrids, and especially since the widespread occurrence of southern corn leaf blight in 1970, pathologists and plant breeders have seriously questioned the wisdom of using one cytoplasm over as much as 85% of the United States corn acreage. Similar situations exist in other hybrid crops, especially sorghum. Because of this near disaster in 1970, many dternative methods of commercially producing hybrid corn seed are being used or explored. Much of the hybrid corn seed produced in 1970 and the major portion of the 1971 seed crop was produced by mechanically detasseling normal cytoplasm seed parent inbreds and single crosses. The corn seed industry is to be commended for the very rapid changeover in their production methods (at great expense and inconvenience to those concerned) to ensure a supply of resistant and adapted hybrid corns for the nation’s corn producers. It is easy to say that the seed industry should have diversified its methods of seed production to avoid the serious disease outbreak of 1970. However, when one looks at the other side of the picture, the Tcms material and the restorer genes had worked marvelously for nearly 20 years. In hindsight it can now be said we should have been better prepared for such an emergency as the southern corn blight. Plant breeders and geneticists do have a wealth of germ plasm under investigation in most major and minor crop species. Work previously cited illustrates that in our laboratories, both private and public, many different cytoplasms exist and are being actively investigated. It is not too much to hope that from this reemphasis on cytoplasmic factors in corn and other crops will come new knowledge which can make the commercial seed production in several crops more convenient and efficient. Producers of seed and general grain farmers are in business to make an economic return of their investment. Both are going to use their best judment in using methods and seed of those varieties they believe best for their area. This means that when a new superior variety is introduced, most growers will quickly adopt it, and in a short period most of the acreage will be in the new variety. Thus, a very narrow base of a crop species is achieved. Again, this emphasizes the need for a wide genetic base of materials to be maintained and investigated by our research program. Be-
24
P. H. HARVEY, C. S. LEVINGS, 111, AND E. A. WERNSMAN
cause of our standardized marketing system, it is not practical to keep a broad-based genetic population in our commercial programs. Therefore, it makes our dependency on maintaining genetic diversity by research groups mandatory if future disasters are to be avoided. REFERENCES Alam, S., and Sandal, P. C. 1964. Proc. N. Dak. Acad. Sci. 18, 72-73. Alam, S., and Sandal, P. C. 1967a. Crop Sci. 7, 668-669. Alam, S., and Sandal, P. C. 1967b. Proc. N . Dak. Acad. Sci. 21, 188-189. Alam, S., and Sandal, P. C. 1969. Crop Sci. 9, 157-159. Aliev, K. A., Filippovich, I. I., and Sisakayan, N. ?vl. 1967. Mol. Biol. 1, 240-248. Anderson, L E., and Levin, D. A. 1970. Plant Physiol. 46, 819-820. Amott, H. J., and Smith, K. M. 1967. J . Ultrastruct. Res. 19, 173-195. Amott, H. J., and Smith, K. M. 1968. Virology 34, 25-35. Ashri, A. 1968. Genetics 60, 807-810. Ashwell, M.. and Work, T.' S. 1970. Annu. Rev. Biochem. 39, 251-290. Atanasoff, D. 1925. Phytopathology 15, 170-177. Atanasoff, D. 1963. Phytopathol. 2. 47, 207-214. Atanasoff, D. 1964a. Z. Pflanzenzuecht. 51, 197-214. Atanasoff, D. 1964b. Phytopathol. Z. 50, 336358. Baxter, R.,and Kirk, J. T. 0. 1969. Nature ( L o n d o n ) 222, 272-273. Bennett, C W. 1969. Advan. Virus Res. 14, 221-261. Bhan, K. C. 1964. Bot. Rev. 30, 312-332. Bianchi, K. 1963. Genen en Phaetien 8, 36-43. Blakeslee, A. F. 1921. J. Genet. 11, 17-36. Boardman, N. K., Francki, R., and Wildman, S. G. 1966. J. Mol. Biol. 17, 470-489. Borst, P. 1970. Symp. SOC. Exp. Biol. 24, 201-226. Borst, P., and Kroon, A. M. 1969. Int. Rev. Cytol. 26, 107-190. Briedenbach, R. W., and Beevers, H. 1967. Biochem. Biophys. Res. Commun. 27, 462-469. Briedenbach, R. W., Kahn, A,, and Beevers, H. 1968. Plant Physiol. 43, 705-713. Brim, C. A., and Young, M. F. 1971. Crop Sci. 11, 564-566. Brim, C. A., Schutz, W. M., and Collins, F. I 1968. C r o p Sci. 8, 517-518. Burk, L. G. 1960. J . Hered. 51, 27-29. Caspari, E. 1948. Adivan. Genet. 2, 1-66. Chaplin, J. F. 1964. Tob. Sci. 8, 105-109. Ching, T. M. 1970. Plant Physiol. 46, 475-482. Chittenden, R. J. 1927. J. Hered. 18, 337-343. Clark, M. F., Matthews, R. E. F., and Ralph. R. K. 1964. Biochim. Biophys. Acts 91, 289-304. Cleij, G. 1967. Euphytica 16, 23-28. Coen, D., Deutch, J., Netter, P., Petrochilo, E., and Slonimski, P. P. 1970. Symp. SOC. Exp. Biol. 24, 449-496. Correns, C. 1909. Z. Vererbungslehrc 1, 291-329. Cunningham, W. P., Morri, D. J., and Mollenhauer, H. H. 1966. J. Cell Biol. 28, 169-179. Curtis, G. J. 1967. Euphytica 16, 419-424.
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Dalton, A. J. 1961. In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 2, pp. 603-619. Academic Press, New York. Davis, W. H., and Greenblatt, I. M. 1967. J. Hered. 58, 301-305. DuPraw, E. J. 1969. “Cell and Molecular Biology.” Academic Press, New York. Duvick, D. N. 1958. Agron. J . 50, 121-125. Duvick, D. N. 1965. Advan. Genet. 13, 1-56. Edwardson, J. R. 1970. Bot. Rev. 36, 341-420. Edwardson, J. R., and Corbett, M. K. 1961. Proc. Nut. Acad. Sci. U S . 47, 390-396. Edwardson, J. R., and Warmke, H. E. 1967. J. Hered. 58, 195-196. Esau, K. 1967. Annu. Rev. Phytopathol. 5, 45-76. Esau, K. 1968. “Viruses in Plant Hosts.” Univ. of Wisconsin Press, Madison. Feldherr, C. M. 1962. J . Cell Biol. 14, 65-72. Feldherr, C. M. 1965. J . Cell. Biol. 25, 43-53. Flemming, A. A., Kozelnicky, G. M., and Browne, E. B. 1960. Agron. I. 52, 112-115. Frankel, R. 1956. Science 124, 684-685. Frankel, R. 1962. Genetics 47, 641-646. Frankel, R. 1971. Heredity 26, 107-119. Frey-Wyssling, A., and Miihlethaler, K. 1965. “Ultrastructural Plant Cytology.” Amer. Elsevier, New York. Gahan, P. B. 1969. Biochem. J. 111, 27P. Gerola, F. M., and Bassi, M. 1966. Caryologia 19, 13-40. Granick, S., and Gibor, A. 1967. Progr. Nucl. Acid Res. Mol. Biol. 6, 143-186. Green, B. R., and Gordon, M. P. 1966. Science 152, 1071-1074. Grun, P. 1970. Evolution 24, 188-198. Hanson, J. B., Hageman, R. H., and Fisher, M. E. 1960. Agron. J . 52, 49-52. Heslop-Harrison, J. 1963. Brookhaven Symp. Biol. 16, 109-125. Heslop-Harrison, J. 1967. Annu. Rev. Plant Physiol. 18, 325-348. Isaacs, A. 1961. Sci. Amer. 204, 51-57. Isaacs, A., and Lindemann, J. 1957. Proc. Roy. Soc., Ser. B 147, 258-267. Jacks, T. J., Yatsu, L. Y., and Altschul, A. M. 1967. Plant Physiol. 42, 585-597. Jacob, F., and Wollman, E. L. 1961. “Sexuality and the Genetics of Bacteria.” Academic Press, New York. Jinks, J. L. 1964. “Extrachromosomal Inheritance.” Prentice-Hall, Englewood Cliffs, New Jersey. Johnson, J. 1925. Wis.,Agr. Exp. Sta., Res. Bull. 63. Jones, H. A., and Clarke, A. E. 1943. Proc. Amer. Soc. Hort. Sci. 43, 189-194. Kirk, J . T. 0. 1963. Biochem. J . 88, 45P. Kirk, J. T. O., and Tilney-Bassett, R. A. E. 1967. “The Plastids.” Freeman, San Francisco, California. Kobel, R. J., and Benedict, C. R. 1971. Crop Sci. 11, 486-488. Kung, S. D., and Williams, J. P. 1969. Biochim. Biophys. Acta 195, 434-445. Lacadena, L. R. 1968. Euphytica 17, 439-444. Ledoux, L., and Huart, R. 1969. J. Mol. Biot. 43, 243-262. Lehninger, A. L. 1964. “The Mitochondrion.” Benjamin, New York. Lewis, D. 1970. Symp. Soc. Exp. Biol. 24, 497-501. Lyttleton, J. W. 1960. Biochem. J . 74, 82-90. Lyttleton, J. W. 1962. Exp. Cell Res. 26, 312-317. Maan, S. S., and Lucken, K. A. 1971. J. Hered. 62, 149-152.
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McDaniel, R. G. 1972. “ARS Seed Quality Symposium, Seed Science and Technology,” Vol. 1, No. 1 (in press). McDaniel, R. G., and Sarkissian, I. V. 1966. Science 152, 1640-1642. McDaniel, R. G., and Sarkissian, I. V. 1968. Genetics 59, 465-475. Matile, P. 1968. Planta 79, 181-196. Matile, P. 1969. Bioclrem. I. 111, 26P. Meyer, V. G. 1971. 1. Hered, 62, 77-78. Michaelis, P. 1964. Z. Pflanzenzuecht. 52, 333-353. Miller, R. J., and Koeppe, D. E. 1971. Science 173, 67-69. Mollenhauer, H. H., and Whaley, W. G. 1963. 1. Cell Biol. 17, 222. Mounolou, J. C., Jakob, H., and Slonimski, P. P. 1966. Bioclrern. Biophys. Res. Commun. 24, 218-224. Murray, B. E., and Craig, I. L. 1968. Can. 1. Genet. Cytol. 10, 876885. Nass, M. M. K. 1969. Science 165, 25-35. Nass, S. 1969. Int. R e v . Cytol. 25, 55-129. Noll, H. 1970. Symp. Soc. Exp. Biol. 24, 419-447. Nyland, G. 1962. Science 137, 598-599. Ohta, Y. 1961. Seiken Ziho 12, 35-43. Parker, R. C., Wynne, J. C., and Emery, D. A. 1970. Crop Sci. 10,429-432. Perner, E. S. 1958. Protoplasma 49, 407-446. Pesho, G . R., Russell, W. A., and Dicke, F. F. 1969. Iowa State J. Sci. 44, 165-184. Porter, K. R., Claude, A., and Fullum, E. 1945. I. Exp. Med. 81, 233-246. Raj, A. Y. 1968. Indian I. Genet. Plant Breed. 28, 335-341. Raven, P. H. 1970. Science 169, 641-646. Rhoades, M. M. 1931. Science 73, 340-341. Rhoades, M. M. 1933. J . Genet. 27, 71-93. Rhoades, M. M. 1943. Proc. Nut. Acad. Sci. U.S. 29, 327-329. Risebrough, R. W., Tissieres, A., and Watson, J. D. 1962. Proc. Nut. Acad. Sci. U.S.48, 430-436. Robertson, J. D. 1959. Biochein. SOC.Symp. 16, 13-37. Rogers, J. S., and Edwardson, J. R. 1952. Agron. J. 44, 8-13. ROSS,W. M. 1971. News Release. Kansas and Nebraska Agr. Exp. Sta. and Plant Sci. Res. Div. A.R.S., US. Dept. of Agriculture, Beltsville, Maryland. Sager, R.,and Ramanis, Z. 1970. Symp. SOC. Exp. Biol. 24, 401417. Salaman, R. N. 1932. Proc. R o y . SOC.,Ser. B 110, 186-224. Salaman, R. N., and Le Pelley, R. H. 1930. Proc. R o y . Soc., Ser. B 106, 140-175. Sand, S. A. 1960. Science 131, 665. Sarkissian, I. V., and McDaniel, R. G. 1967. Proc. N a t . Acad. Sci. U.S. 57, 1262-1266. Sarkissian, I. V., and Srivastava, H. K. 1967. Genetics 57, 843-850. Scheifeie, G. L., Nelson, R. R., and Koons, C. 1969. Plant Dis. Rep. 53, 656-659. Schultz, E. S. 1925. Science 62, 571-572. Schultz-Schaeffer, J. 1970. Crop Sci. 10, 204-205. Sebald, W.,Biicher, T., Olbrick, B., and Kandewity, F. 1968. FEBS Lett. 1, 235-240. Shumway, L. K., and Bauman, L. F. 1966. Crop Sci. 6, 341-342. Singh, A., and Laughnan, J. R. 1968. Genetics 60, 226. Singh, M. 1966. Indian J. Genet. Plant Breed. 26, 386-390. Sissakayan, N. M., Filippovich, I. I., Svetalio, E. N., and Aliev, K. A. 1965. Biochim. Biophys. Acta 95, 474-485. Smith, H. H. 1968. Adran. Genet. 14, 1-55.
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Spencer, D., and Whitfield, P. R. 1967a. Biochem. Biophys. Res. Commun. 28, 538-542. Spencer, D., and Whitfield, P. R. 1967b. Arch. Biochem. Biophys. 121, 336-345. Sprague, G. F., McKinney, H. H., and Greeley, L. 1963. Science 141, 1052-1053. Stephens, J. C., and Holland, R. F. 1954. Agron. J . 46, 20-23. Stewart, R. N. 1965. Genetics 52, 925-947. Stringfield, G. H. 1964. Advan. Agron. 16, 102-138. Stroup, D. 1970.1. Hered. 61, 139-141. Swift, H., and Wolstenholme, D. R. 1969. In “Handbook of Molecular Cytology” (A. Lima-de-Faria, ed.), pp. 972-1046. North-Holland Publ., Amsterdam. Tatum, L. A. 1971. Science 171, 1113-1116. Taylor, D. L. 1970. Int. Rev. Cytol. 27, 29-64. Tewari, K. K. 1971. Annu. Rev. Plant Physiol. 22, 141-166. Tewari, K. K., and Wildman, S. G. 1966. Science 153, 1269-1271. Tewari, K. K., and Wildman, S. G. 1967. Proc. Nat. Acad. Sci. US.58, 689-696. Tewari, K. K., and Wildman, S. G. 1969. Biochim. Biophys. Acta 186, 358-372. Tewari, K. K., and Wildman, S. G. 1970. Symp. SOC. Exp. Biol. 24, 147-179. Theurer, J. C., and Ryser, G. K. 1969. Crop Sci. 9, 610-612. Thompson, R. K. 1970. Proc. Int. Barley Genet. Symp., 2nd, 1969 p. 319-322. Tolbert, N. E., Oeser, A., Kisaki, T., Hageman, R. H., and Yamazaki, R. K. 1968. J . Biol. Chem. 243, 5179-5184. Tolbert, N. E., Oeser, A., Yamazaki, R. K., Hageman, R. H., and Kisaki, T. 1969. Plant Physiol. 44, 135-147. Ts’o, P. 0.P., Bonner, J., and Vinograd, J. 1956. 1. Biophys. Biochem. Cytol. 2, 451-465. Ts’o, P . 0 . P., Bonner, J., and Vinograd, J. 1958. Biochim. Biophys. Actu 30, 570-582. Tuppy, H., and Swetly, P. 1968. Biochim. Biophys. Actu 153, 293-295. van der Meer, Q. P., and van Bennekom, J. L. 1970. Euphytica 19, 430-432. Villareal, R. W., and Lantican, R. M. 1965. Philipp. Agr. 49, 294-300. Villiers, T. A. 1971. Nature New Biology (London) 233, 57-58. Wells, R., and Birnstiel, M. 1969. Biochem. 1. 112, 777-786. Whitfield, P. R., and Spencer, D. 1968. Biochim. Biophys. Acta 157, 333-343. Williams, G. R., and Williams, A. S. 1970. Biochem. Biophys. Res. Commun. 39, 858-863. Wilson, J. A., and Ross, W. M. 1962. Wheat Inform. Serv. 14, 29-31. Woodward, D. O., and Munkres, K. D. 1966. Proc. Nut. Acad. Sci. U.S. 55, 872-880. Work, T. S. 1967. Biochem. J . 105, 38-40. Yatsu, L. P. 1965. I . Cell Biol. 25, 193-199. Zevin, A. C. 1967. Euphytica 16, 183-189.
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THE CHEMISTRY OF SUBMERGED SOILS
. N . Ponnamperuma
F
The International Rice Research Institute. Lor Boiior. Laguna. Philippines
I. Introduction ................................................... I1. Kinds of Submerged Soils ....................................... A . Waterlogged (Gley) Soils ..................................... B . Marsh Soils ............................................... C. Paddy Soils ................................................ D . Subaquatic Soils ............................................ 111. Characteristics of Submerged Soils ................................. A . Absence of Molecular Oxygen ................................. B . Oxidized Mud-Water Interface ............................... C. Exchanges between Mud and Water ........................... D. Presence of Marsh Plants ................................... E . Soil Reduction .............................................. IV. Electrochemical Changes in Submerged Soils ....................... A . Redox Potential ............................................. B . pH ........................................................ C . Specific Conductance ......................................... V. Chemical Transformations in Submerged Soils ....................... A . Carbon .................................................... B. Nitrogen ................................................... C . Iron ....................................................... D. Manganese ................................................. E . Sulfur ..................................................... F. Phosphorus ................................................. G. Silicon ..................................................... H . Trace Elements ............................................. VI. Mineral Equilibria in Submerged Soils ............................. A . Redox Systems .............................................. B . Carbonate Systems .......................................... VII. Perspectives .................................................... References .....................................................
I.
29 30 30 31 32 33 34 34 35 35 37 38 48 48
51 56 58
59 65
71 73 74 76 79 80 80 80 85 87 88
Introduction
The chemistry of submerged soils is a subject of unusual scientific and ecological interest. Its scientific interest springs from its applications in geochemistry. pedology. agriculture. limnology. oceanography. and pollution 29
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control. Its ecological importance needs no emphasis, for 72% of the earth‘s surfaqe is covered by submerged soils or sediments. The chemical changes in these submerged materials influence ( a ) the character of the sediment or soil that forms, ( b ) the suitability of wet soils for crops, (c) the distribution of plant species around lakes and streams and in estuaries, deltas, and marine flood plains, ( d ) the quality and quantity of aquatic life, and (e, the capacity of lakes and seas to serve as sinks for terrestrial wastes.
II.
Kinds of Submerged Soils
The Glossary of Soil Science Terms (Anonymous, 1965) defines soil in two ways: ( a ) “The unconsolidated mineral material on the immediate surface of the earth that serves as a natural medium for the growth of land plants”; and ( b ) “The unconsolidated mineral matter on the surface of the earth that has been subjected to and influenced by genetic and environmental factors of: parent material, climate (including moisture and temperature effects) , macro- and microorganisms, and topography, all acting over a period of time and producing a product-soil-that differs from the material from which it is derived in many physical, chemical, biological, and morphological properties and characteristics.” The first definition limits submerged soils to soils carrying dryland crops that undergo temporary waterlogging, for permanently waterlogged soils support marsh plants, not land plants. The second definition embraces waterlogged soils, marsh soils, paddy soils, and lake and ocean sediments. In this chapter, I use the wider definition. A.
( GLEY) SOILS WATERLOGGED
Waterlogged soils are soils that are saturated with water for a sufficiently long time annually to give the soil the distinctive gley horizons resulting from oxidation-reduction processes: (a) a partially oxidized A horizon high in organic matter, (b) a mottled zone in which oxidation and reduction alternate, and (c) a permanently reduced zone which is bluish green (Robinson, 1949). Because the soil is intermittently saturated with water, oxidation of organic matter is slow and it accumulates in the A horizon. In the second horizon, iron and manganese are deposited as rusty mottles or streaks if the diffusion of oxygen into the soil aggregates is slow; if diffusion is fast, they are deposited as concretions (Blume, 1968). While some rusty
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mottles consist of goethite and lepidocrocite (Iwasa and Kamoshita, 1961 ) the deposits are rarely pure oxides. Because iron and manganese form coprecipitates, the concretions are mixtures or complex oxides (Hem, 1964). They also contain small amounts of zinc, copper, nickel, and cobalt (Jenne, 1968). The zone of permanent waterlogging is bluish green because ferrous compounds are present. In this zone, secondary minerals, such as hydrated magnetite, pyrite, marcasite, siderite, vivianite, and ferrous silicates, may be present (Ponnamperuma, 1972). Saturation with water may be due to impermeability of the soil material, the presence of an impervious layer, or a high water table. Waterlogged soils occur in almost any climatic zone from the tundra to the desert or humid tropics (Soil Survey Staff, 1960), usually as the poorly drained members of drainage catenas. Robinson ( 1949), Joffe (1949), Russell (1961), Rode (1962), and Ponnamperuma (1972) have discussed the influence of waterlogging on soil genesis. This influence is so great that wetness has been used as a differentiating characteristic at the suborder level in classifying all soils except aridosols and histosols (Thorp and Smith, 1949; Soil Survey Staff, 1960, 1967). Robinson (1930), Van’t Woudt and Hagan (1957), Grable (1966), and Stolzy and Letey (1964) have reviewed the effects of waterlogging on crop plants. Humphries (1962) has described its effects on perennial grasses, and Potsma and Simpendorfen ( 1962), on pine trees.
B. MARSHSOILS Marsh soils may be defined as soils that are more or less permanently saturated or submerged. Freshwater marsh soils occur on the fringes of lakes and the networks of streams that feed them (Joffe, 1949). Saltwater marshes are found in estuaries, deltas, and tidal flats (Guilcher, 1963). The outstanding features of these soils are the accumulation of plant residues in the surface horizon and the presence of a permanently reduced G horizon below it. In freshwater marshes, the G horizon is blue or green (Joffe, 1949); in marine marshes it is green if iron silicates are present and dark gray if pyrites are the main iron minerals (Pons and Van der Kevie, 1969). Joffe (1949) and Rode (1962) have classified freshwater marshes according to their origin into upland, lowland, and transitional. Upland marshes receive mainly rainwater and are therefore poor in bases and have pH values of 3.5-4.5. Lowland marshes are saturated or submerged with water-carrying bases and have pH values of 5.0-6.0. Ruttner (1963) has described the transition from lowland to upland and the accompanying changes in vegetation. But there is no niche for these organic soils in the
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classification of histosols proposed by the U.S.D.A. Soil Survey Staff (1968), in which the emphasis is on the kind of plant residue, not the water regime. Pearsall (1938), Misra (1938), Pearsall and Mortimer (1939), Pierce (1953), and Armstrong and Boatman (1967) have shown an association between the chemical properties of the soils, especially their oxidation-reduction state, and the distribution of natural vegetation in marshes. Gorham (1953) found that in passing from the relatively inorganic lake muds through semiaquatic soils to raised bog peats, soil acidity increased, base saturation decreased, and humus nitrogen content fell. These changes were reflected in the nutrient status of the plants. Saltwater marsh soils cover over 20 million hectares of flat land, chiefly in the deltas of the tropics. When submerged and anaerobic they are neutral in reaction and support salt-tolerant marsh plants. But when the land is elevated or when the water recedes, air penetrates the soil and oxidizes the pyrites present in it to basic ferric sulfate and sulfuric acid, producing an acid sulfate soil. I have previously reviewed the genesis of these soils (Ponnamperuma, 1972). Moorman ( 1963), Nhung and Ponnamperuma (1966), and Pons and Van der Kevie (1969) have suggested methods of reclaiming them for crops, especially, rice. C . PADDY SOILS Paddy soils are soils that are managed in a special way for the wet cultivation of rice. The management practices include: ( a ) leveling of the land and construction of levees to impound water; ( b ) puddling (plowing and harrowing the water-saturated soil); (c) maintenance of 5-10 cm of standing water during the 4-5 months the crop is on the land; (d) draining and drying the fields at harvest; and ( e ) reflooding after an interval which varies from a few weeks to as long as 8 months. These operations and oxygen secretion by rice roots lead to the development of certain features peculiar to paddy soils. During the period of submergence, the soil undergoes reduction (Section 111, E ) and turns dark gray. Iron, manganese, silica, and phosphate become more soluble and diffuse to the surface and move by diffusion and mass flow to the roots and to the subsoil. When reduced iron and manganese reach the oxygenated surface, the surface of rice roots, or the oxidized zone below the plow sole (De Gee, 1950; Koenigs, 1950; Mitsui, 1960; Kyuma and Kawaguchi, 1966), they are oxidized and precipitated along with silica and phosphate. Sandwiched between the oxidized surface layer and the zone of iron and manganese illuviation is the root zone of rice with reddish-brown streaks along root channels. When the land is
THE CHEMISTRY OF SUBMERGED SOILS
33
drained at harvest, almost the entire profile above the water table is reoxidized, giving it a highly mottled appearance. Precipitation in the plow layer is not pedologically of any consequence because plowing and puddling redistribute the deposits. But the downward movement of iron and manganese means that these two elements are permanently lost from the topsoil. The eluviated iron and manganese, along with some phosphate, are deposited below the plow sole to produce an iron-rich Bi horizon overlying a manganese-rich B,, horizon. Kyuma and Kawaguchi ( 1966) regarded reduction eluviation and oxidative illuviation as the soil forming processes characteristic of paddy soils and have proposed the new term “Aquorizem” at the Great Soil Group level to define soils which have the sequence of reductive eluviation/oxidative illuviation. A well developed paddy soil has the horizon sequence A,,/A,,,/B rg/ B,,/G. Kanno (1957) has described these horizon sequences and their variation with duration of waterlogging, and has proposed a classification of paddy soils based on the depth of the permanent water table. Brinkman (1 970) has recently drawn attention to another soil forming process associated with alternate oxidation-reduction which he calls “ferrolysis.” During submergence and soil reduction, the cations displaced from exchange sites by Fez+migrate out of the reduced zone and are lost. When the soil is drained and dried, the reduced iron is reoxidized and precipitated, leaving H+ions as the only major cation. The soil is acidified and the clay disintegrates.
D. SUBAQUATIC SOILS These soils are formed from river, lake, and ocean sediments. I justify the use of the term “soil” to describe the uppermost layers of unconsolidated aqueous sediments on the following grounds: (a) the sediments are formed from soil components; (b) typical soil-forming processes such as hydrolysis, oxidation-reduction, precipitation, synthesis, and exchange of matter and energy with the surroundings proceed in the uppermost layers of subaquatic sediments; (c) even deep sea sediments contain organic matter and a living bacterial flora (Goldschmidt, 1958); (d) the bacteria in lake and ocean sediments are similar to those in soils (Hutchinson, 1957; Kaplan and Rittenberg, 1963); (e) the metabolism of subaquatic sediments is similar to those of submerged soils; (f) the uppermost layers show a horizon differentiation distinct from physical stratification (Goldschmidt, 1958) ; and (g) sediments differ in texture, composition, clay mineralogy, organic matter content, and oxidation-reduction level (Rankama and Sahama, 1950; Kuenen, 1965) as soils do. Goldschmidt (1958) defined
34
F. N. PONNAMPERUMA
soil as the habitat for living organisms in the uppermost part of the lithosphere and proposed the inclusion of subaquatic soils in the pedosphere. Mortimer (1949) regarded lake sediments as soils of a special type and called them “underwater soils.” The uppermost layers of unconsolidated river, lake, and ocean sediments may be regarded as permanently submerged cumulative soils. The composition of sediments is so variable (Mortimer, 1949; Kuenen, 1965) that they are best studied from the metabolic standpoint, as proposed by Mortimer. Mortimer (1949), Ruttner (1963), and McKee et al. (1970) have reviewed the metabolism of lake muds; Kaplan and Rittenberg (1963) and Martin (1970), the chemistry and metabolism of marine sediments.
Ill.
A.
Characteristics of Submerged
Soils
ABSENCEOF MOLECULAR OXYGEN
When a soil is submerged, gas exchange between soil and air is drastically curtailed. Oxygen and other atmospheric gases can enter the soil only by molecular diffusion in the interstitial water. This process, according to the figures given by Lemon and Kristensen (1960) and by Greenwood (1961 ), is 10,000 times slower than diffusion in gas-filled pores. Thus the oxygen diffusion rate suddenly decreases when a soil reaches saturation by water (Taylor, 1949; Lemon and Kristensen; 1960; Kristensen and Enoch, 1964). Within a few hours of soil submergence, microorganisms use up the oxygen present in the water or trapped in the soil and render a submerged soil practically devoid of molecular oxygen. Both direct and indirect tests for oxygen, in the laboratory and in the field, have shown this. Evans and Scott (1955) noted that the concentration of oxygen in the water used for saturating a soil decreased to one-hundredth of its initial value in 75 minutes. Takai et al. (1956) found no oxygen in three soils 1 day after submergence. Turner and Patrick (1968) could detect no oxygen in four soil suspensions within 36 hours of withdrawal of the oxygen supply. Yamane (1958) reported the absence of oxygen in two flooded rice fields at five sampling times in 3 months. Yunkervich et al. (1966) and Armstrong and Boatman (1967) found no oxygen in bogs with stagnant water. Mortimer (1941, 1942) could not detect oxygen 1 cm below the surface of submerged lake muds. And Scholander et al. (1955) reported the absence of oxygen in a mangrove swamp. Also, comparison of oxygen consumption rates by lake and ocean muds (2 X 10-lo to
THE CHEMISTRY OF SUBMERGED SOILS
35
2X g cm-2 sec-I) given by Hutchinson (1957), Pamatat and Banse (1969), and Howeler and Bouldin (1971) with oxygen diffusion rates in saturated soils (1 X g cm-2 sec-I) shows that submerged soils and lake and ocean muds are anoxic below the soil-water interface. And respiration studies by Greenwood (1961) indicate that saturated crumbs have no oxygen at the center. The low oxidation-reduction potentials reported by Hutchinson (1957) for lake muds, by Zobell (1946) and Bass-Becking et al. (1960) for fine ocean sediments, and by me for saturated and submerged rice soils (Ponnamperuma, 1965) are further proof of the absence of molecular oxygen in waterlogged soils and sediments. But coarse sediments low in organic matter, in shallow water, may be well supplied with oxygen (Zobell, 1946). B.
OXIDIZEDMUD-WATERINTERFACE
A submerged or saturated soil, however, is not uniformly devoid of oxygen. The concentration of oxygen may be high in the surface layer which is a few millimeters thick and in contact with oxygenated water. Below the surface layer, the oxygen concentration drops abruptly to practically zero (Mortimer, 1941, 1942; Patrick and Sturgis, 1955; Greenwood and Goodman, 1967). The brown color of the oxygenated layer, its chemical properties, and its oxidation-reduction potential undergo a similar abrupt change with depth in submerged soils (Pearsall and Mortimer, 1939; De Gee, 1950; Alberda, 1953; Howeler, 1970), in lake muds (Mortimer, 1942; Hayes and Anthony, 1958), and in sea sediments (Friedman et al., 1968; Friedman and Gavish, 1970). The chemical and microbiological regimes in the surface layer resemble those in aerobic soils.
c.
EXCHANGES BETWEEN MUD AND WATER
The presence of this oxygenated surface layer in lake and ocean muds is of the utmost ecological importance because it acts as a sink for phosphate and other plant nutrients (Hutchinson, 1957; Armstrong, 1965; Mortimer, 1969; Harter, 1968; McKee et al., 1970; Fitzgerald, 1970) and as a chemical barrier to the passage of certain plant nutrients from the mud to the water. The surface operates efficiently in this way only so long as the lake or ocean bottom is supplied with oxygenated water by turbulence due to wind or by thermal movements, and the oxygen supply exceeds the demand at the interface. But these conditions are not always present: the surface may use up oxygen faster than it receives it, undergo reduction, and release large amounts of nutrients from the lake mud into the water (Mortimer, 1941, 1942).
36
F. N. PONNAMPERUMA
In summer, some lakes undergo thermal differentiation into three layers: the epilimnion, the thermocline, and the hypolimnion. The epilimnion is the surface layer of warm water 10-20 m deep (Mortimer, 1949), which, because of mixing by wind action, is uniform in temperature and is saturated with atmospheric oxygen from top to bottom. Immediately below this is the thermocline, a layer in which there is a rapid fall in temperature with depth. In the thermocline, the concentration of oxygen is relatively constant in lakes poor in plant nutrients (oligotrophic lakes), but it decreases with depth in lakes rich in plant nutrients (eutrophic lakes) (Ruttner, 1963). The hypolimnion is the layer of cold stagnant water practically isolated from the epilimnion, except for solids, both organic and inorganic, that sink through it and accumulate on the mud surface. Bacteria in the surface layer use the oxygen in it to oxidize the organic matter. The oxygenated layer of the mud becomes thinner and thinner and finally disappears. The boundary between the aerobic and anaerobic zones then rises above the mud surface and well into the hypolimnion. When the oxidized layer disappears, phosphate, Fez+,Mn2+,silica, and other soluble substances escape from the mud into the hypolimnion. But when cold weather returns and the layers mix, Fe'+ and Mn?+ are oxidized to Fe(II1) and Mn( IV) oxide hydrates. These precipitates sink to the bottom carrying with them phosphate, silica, and sulfate (Hutchinson, 1957). Thus iron, manganese, phosphate, and silica which were released from the lake mud during the thermal stratification in summer are returned with sulfate to the bottom during the autumn mixing. This cycling of nutrients is less active in oligotrophic lakes, because the muds of these lakes receive less organic matter and therefore remain oxidized. The oxygenated layer in bottom muds regulates the nutrient cycles in lakes. Because of the ecological importance of the oxygenated layer at the surface of lake muds, limnologists have attempted to study the factors that affect its thickness. Mortimer (1942) suggested that the thickness of the layer represented a balance between the diffusion of oxygen into the mud and its consumption, and derived some empirical relationships. Hutchinson (1957) evaluated two equations based on microbial respiration derived by Grote. Recently, Bouldin (1968) proposed six models for the description of diffusion of oxygen across mud surfaces. Two of these are steadystate models similar to those discussed by Hutchinson. The others are transient-state models introducing a new concept-oxygen consumption by mobile and nonmobile reductants, in addition to microbial use. Howeler (1970) and Howeler and Bouldin (1971) found experimentally that about 50% of the total oxygen consumed by the swamp soils they studied was used in oxidizing ( a ) water-soluble iron diffusing upwards and ( b ) reduced iron in the soil matrix. They suggested that oxygen consumption by re-
THE CHEMISTRY OF SUBMERGED SOILS
37
duced muds was best described by models that combine both microbial respiration and chemical oxidation. But Edwards and Rolley (1965) found no relationship between oxygen consumption by river muds and their chemical properties. Because the reduced soil acts as a sink for oxygen, the oxygenated layer at the surface of submerged soils should be quite thin. Howeler (1970) showed that it ranged from 0.2 mm to 6.0 mm in 10 submerged soils, and contained large amounts of freshly precipitated Fe(II1). The presence of Fe(II1) and Mn(1V) (Weijden et al., 1970) oxide hydrates in the surface layer implies that lake and ocean muds can sorb and retain phosphate, silica, manganese, cobalt, nickel, and zinc that are present in the supernatant water or that diffuse to the surface layer from the reduced zone below (Parks, 1967; Jenne, 1968; Hynes and Greib, 1970; Shukla et al., 1971). The oxidized layer thus accumulates phosphorus, silicon, manganese, cobalt, nickel, and zinc. By contrast, permanently reduced ocean muds containing H,S tend to accumulate copper, silver, uranium, molybdenum, and apatite (Goldberg, 1965). Many studies on the absorption capacity of lake muds for nutrients and toxins have been done without recognizing the great difference in the adsorptive properties of oxidized and reduced muds. The presence of oxygen in the soil-water interface profoundly affects the nitrogen economy of paddy soils and lake and ocean bottoms. Ammonium nitrogen broadcast as fertilizer or released from organic matter is converted to nitrate in the oxygenated surface layer. The nitrate diffuses into the anaerobic layer just below it and is denitrified. Denitrification causes substantial losses of ammonium fertilizer broadcast on paddy soils (Mitsui, 1960; Aomine, 1962; Patrick and Mahauatra, 1968) and makes lake and ocean bottoms sinks for nitrate (Kaplan and Rittenberg, 1963; Brezonik and Lee, 1968). Without denitrification, nitrogen deposits in aquatic sediments would deplete the atmosphere of nitrogen in 400 million years (Vaccaro, 1965). Denitrification, however, accounts for only 10% of the nitrogen imbalance in oceans (Martin, 1970). D.
PRESENCE OF MARSHPLANTS
Plants growing in submerged soils have two adaptations that enable the roots to ward off toxic reduction products, accumulate nutrients, and grow in an oxygen-free medium: oxygen transport from the aerial parts and anaerobic respiration. It has been known for quite some time that the rodts of marsh plants receive their oxygen from the aerial parts (shoot, air roots, or stilt roots) through gas spaces connecting these organs (Conway, 1940; Sifton, 1945;
38
F. N. PONNAMPERUMA
Scholander et al., 1955; Ruttner, 1963). Van Raalte (1941) and several other workers cited by me (Ponnamperuma, 1965) showed that rice roots behave similarly. More recently, W. Armstrong (1967, 1970) measured the oxygen flux across the roots of swamp plants, including rice, and found that it is sufficient to meet the oxygen requirements of root cells, to oxidize the rhizosphere, and to ward off the entry of reduced substances. The rhizomes, corms, and leaves of semisubmerged plants apparently can respire anaerobically for long periods of time without injury (Laing, 1940). Anaerobic respiration enables rice to germinate at very low 0, tensions (Erygin, 1936; Aleshin, 1961 ). But how do land plants survive temporary waterlogging? Land plants respond to oxygen stress in the roots by forming intercellular gas spaces in the cortex (Bryant, 1934; McPherson, 1939). Through these spaces, limited amounts of oxygen may be transferred from the shoot to the root cells to enable the plant to survive short periods of soil waterlogging. But land plants may have a permanent system of varying efficiency for oxygen transport. Bartlett (1961 ) found that land plants vary widely in their resistance to waterlogging and that this resistance was linked with the capacity of the root to oxidize the rhizosphere, presumably by oxygen translocation from the shoot. Greenwood (1967) reported that seedlings of the land species he studied contained continuous, nontortuous gas channels in the stems and roots and that the roots grew in oxygen-free media. Apparently, oxygen transport from shoot to root is present in varying degrees even in mesophytes (Greenwood and Goodman, 1971). This enables them to withstand short spells of soil submergence. E.
SOILREDUCTION
The most important chemical difference between a submerged soil and a well-drained soil is that a submerged soil is in a reduced state. Except for the thin, brown, oxidized layer at the surface (and sometimes an oxidized zone in the subsoil), a submerged soil is gray or greenish, has a low oxidation-reduction potential, and contains the reduced counterparts of NO,-, SOqz-,Mn4+,Fe3+, and CO,: NH,', H,S, Mn'+, Fez+,and CH,. Reduction of the soil is a consequence of anaerobic respiration by soil bacteria. During anaerobic respiration organic matter is oxidized and soil components are reduced. 1 . Oxidation-Reduction Potential
Oxidation-reduction is a chemical reaction in which electrons are transferred from a donor to an acceptor. The electron donor loses electrons
THE CHEMISTRY OF SUBMERGED SOILS
39
and increases its oxidation number or is oxidized; the acceptor gains electrons and decreases its oxidation number or is reduced. The source of electrons for biological reductions is organic matter. The driving force of a chemical reaction is the tendency of the free energy of the system to decrease until, at equilibrium, the sum of the free energies of the products equals that of the remaining reactants. In a reversible oxidation-reduction reaction, this force can be measured in calories or in volts. The change in free energy, AG, for the reduction, Ox ne e Red is given by
+
where (Red) and (Ox) are the activities of the reduced and oxidized species and AGO is the free energy change when the activities are unity. Converting calories to volts using the relationship, AG = --nEF, we have
E
=
Eo
RT + -1n nF
(Ox)
__
(Red)
in which E is the voltage of the reaction, Eo is the voltage when (Ox) and (Red) are each unity, and F is the Faraday constant in heat units. If E is measured against the standard hydrogen electrode, it is denoted by Eh. Equation (2) then becomes
Eh
=
Eo
RT + -1nnF
(Ox) (Red)
Eh is a quantitative measure of the tendency of a given system to oxidize or reduce susceptible substances. Eh is positive and high in strongly oxidizing systems; it is negative and low in strongly reducing systems. There is, however, no neutral point, as in pH. Eh, like pH, is an intensity factor. Any chemical reaction which involves the exchange of electrons will be influenced by redox potential (Eh) . If oxidation-reduction reactions are arranged one below the other in descending order of Eo as in Table I, a given system theoretically can oxidize any one below it under standard conditions. Changes in pH and activities of the reactants and resultants can, however, alter the order.
2 . The p E Concept Sillen (1964, 1967) has suggested that it is more logical and more convenient to use pE instead of Eh in the study of redox equilibria. The com-
40
F . N. PONNAMPERUMA
TABLE I Soine Hedos Systeiiis in Surface Media
Systeiii 20 31 14 PO 4 17 3 5
80 06
11
80 33 87 74 12 .' 86 1 64 3 59 - 1 79 -3 ox 0 00
-7
I'sing
31
13 12 7 6
-2 -3 -3
-3 -4
-4 -5 -5 -5
-7 -7
80 66 11 80 67 13 36 65 14 69 16 29 58 00 51
values (Latimer, 1954), unless otherwi3e indicated.
* pEo c.orrt.cteti to pIf
7.0.
Clark (1960). r51~io11 (1966).
mon reagent in redox equilibria is the electron and, according to Sillen, should be treated like other participating species. Just as pH is a measure of proton activity, so is pE, the negative logarithm of the electron activity, a measure of clcctron activity. It can be shown that pE = -log(e) =Eh/2.303RTF-' or pE = Eh/0.0591, and pEo = Eo/0.0591 at 25OC (Sillen, 1964, 1967; Stumm and Morgan, 1970; Ponnamperuma, 1972). Thus for the equilibrium, Ox ne mH* % Red, (Ox) mRT KT E h = EO - 111 ___ __ In H+ (4) (Red) nF nF or RT (Ox) 2.303RTm Ell = Eo 2.303 -- log ___- (5) I)H nF (Red) nF or I (Ox) m pE = pEo - log - - 1111 n (Red) n or
+
+
+
+
+
+
pE
=
1 pEo - - ),(OX) n
~
+ n1 p(Red) - mn pH -
--
(7)
THE CHEMISTRY OF SUBMERGED SOILS
41
where p ( 0 x ) and p(Red) denote the negative logarithms of the activities of the oxidized and reduced species. The pEo can also be expressed in terms of AGO or the equilibrium constant ( K ) of the reaction. Thus pEo
=
1 n
AG0/1.364n = -log K
In strongly oxidizing systems the electron activity is low, so pE is large and positive. In reducing systems, pE is small or negative. Table I lists some redox systems in soils arranged in descending order of pEo corrected to pH 7.0. pE is an intensity factor, for it is a measure of the electron free energy level per mole of electrons. It has nothing to do with capacity.
3. Poise Poise is a useful concept in understanding potential measurements and the behavior of mixed systems. The poise of a redox system is its resistance to changes in potential upon the addition of small amounts of oxidant or reductant. Poise increases with the total concentration of oxidant plus reductant, and for a fixed total concentration it is maximum when the ratio of oxidant to reductant is 1. The analysis of the electrode kinetics of the Fe3+-Fez+system by Stumm and Morgan (1970) leads to a similar conclusion. The poor poise of natural aerated systems is due to the absence of a reversible system in sufficiently high concentration. Poise also has a bearing on the potentials of mixed systems. When two or more systems are mixed, a redistribution of electrons takes place (if energy barriers are absent) which brings the potentials of the systems to a common value. The final potential is determined by the system which is present in excess. 4 . Measurement The state of reduction of a soil can be defined quantitatively by measures of intensity (redox potential) or capacity (total concentration of reduction products). a. Redox Potential. At first sight, Eh or pE appears to be the simplest, quickest, and most meaningful method of characterizing soil reduction. A bright platinum electrode placed in the soil should acquire a potential which is determined by the state of reduction of the soil. This potential can be measured against a reference electrode (usually the saturated calomel electrode) with a high impedance voltmeter such as a pH meter. But in practice, intrinsic and extrinsic errors deprive Eh measurements in most natural media of precise thermodynamic significance. Intrinsic er-
42
F. N. PONNAMPERUMA
rors include electrode malfunctioning, pH effects, absence of true equilibrium, liquid junction potential errors, heterogeneity of the medium, and the discrepancy between the potentials measured in muds and those of their equilibrium solutions. Extrinsic errors are those of sampling muds and extracting the interstitial solutions without altering their composition. Electrode errors can be minimized by preparing the electrodes carefully and cleaning them properly. Many papers during the past fifty years have discussed the choice of metal, size and shape of the electrode, methods of establishing external contact, and ways of cleaning them before use. There is no need to review the merits and demerits of these methods. Suffice it if we refer to two recent accounts (Janz and Ives, 1961; Adams, 1969) and describe a procedure that we have found to be highly reliable. A 2 cm length of B and S gauge 18 bright platinum wire is fused to the end of a 4 mm-bore soft glass tube with 1 cm of the wire outside. A few drops of mercury provide electrical contact with the copper wire connecting it to the voltmeter. Electrodes, new or old, are cleaned by rinsing them successively with a detergent, 1 N HCI, and distilled water. They are checked against a standard redox buffer just before use. A solution of 0.0033 M K,Fe(CN),; and 0.0033 M K,Fe(CN),, in 0.1 M KCI which has an Eh of 0.430 V at 25% (Zobell, 1946) or a suspension of pure quinhydrone in 0.05 M potassium acid phthalate, which has an Eh of 0.463 V at 25OC, can serve as a standard. Good electrodes give potentials which agree to within 1 mV of the standard and to within 0.1 mV among themselves. Although bright platinum electrodes prepared as described above give precise and accurate potentials in buffer solutions and in the solutions of reduced soils, they do not register steady or reproducible potentials in poorly buffered media like aerated soils and natural waters. Morris and Stumm (1967) and Stumm and Morgan (1970) have attributed these defects to the absence of electroactive systems in sufficiently high concentrations in aerobic media and to the presence of mixed systems that are not in equilibrium among themselves. They concluded that even if reproducible potentials are observed in natural systems, the potentials have no precise thermodynamic significance. So they have discouraged the use of redox potential as an environmental parameter. Despite limitations in aerobic media, good platinum electrodes give steady readings within 5 to 10 minutes of insertion in reduced soils and sediments. But the potentials vary widely from spot to spot (Zobell, 1946; Jeffery, 1961; Aomine, 1962; TRRI,' 1966; Yamane and Sato, 1970). The The International Rice Research Institute.
THE CHEMISTRY OF SUBMERGED SOILS
43
low precision of mud potentials, due apparently to heterogeneity of the medium, render mud potentials useless in thermodynamic calculations. Another serious problem is the divergence of potentials measured by electrodes placed in the submerged soil or mud from the potentials of the soil solution drawn out by gravity (Ponnamperuma, 1955; IRRI, 1966), by pressure (Brooks et al., 1968), or by flow under slight hydrostatic pressure into a porous cup embedded in the submerged soil (IRRI, 1970)-a11 under conditions that precluded oxidation. Soil potentials are higher than solution potentials before appreciable soil reduction b.ut several tenths of a volt lower after the soil is reduced (IRRI, 1966, 1970). Published values for potentials of reduced groundwaters (Hem, 1960; Bass-Becking et al., 1960; Back and Barnes, 1965; Balashova, 1969), the interstitial solutions of reduced ocean muds (Brooks et al., 1968; Friedman and Gavish, 1970), reduced lake waters (Mortimer, 1941, 1942; Kjensmo, 1970), and the solutions of reduced soils (Ponnamperuma et al., 1966a) usually range from 0.2 to 0.0 V and rarely drop below -0.05 V. By contrast, reduced soils and lake and ocean muds give potentials of 0.0 to -0.3 V with occasional plunges to values as low as -0.4 V (Bass-Becking et d.,1960). Since the liquid junction potential error accounts for only about 0.025 V, other explanations have been suggested. Ponnamperuma and Castro (1964) noted some similarities between the potentials and dE/dpH values of reduced soils and those of bacterial suspensions. We hypothesized that soil potentials were highly localized bacterial potentials. Later work (IRRI, 1966) suggested that the differences between mud and solution potentials may be due to the presence of oxidant or reductant in the solid phase. Brooks et al. (1968) noted that potentials of marine mud cores were about 0.2 V lower than their interstitial waters. They attributed the low mud potentials to some interaction between the electrode and the mud. But we now have evidence that if a reduced soil is equilibrated anoxically with water and CO, (at the partial pressure of CO, in the mud) and allowed to settle, Eh of the sediment and supernatant solution are nearly identical and this potential is almost that of the soil solution drawn by gravity (IRRI, 1967). In other words, potentials of soil solutions are equilibrium potentials. The variability of soil potentials and their divergence from those of the equilibrium soil solutions explain why several workers who used soil potentials as a measure of reduction (Jeffery, 1961; Ponnamperuma and Castro, 1964; Ponnamperuma, 1965; Bohn, 1968, 1969) failed to obtain the expected theoretical relationships among Eh, pH, and ion activities. Those who measured solution potentials (Hem, 1960; Back and Barnes, 1965; Ponnamperuma et al., 1966a, 1967, 1969b; Skopintsev et al., 1966) were more successful. It is also noteworthy that the Eh, pH, and Fez+concentra-
44
F . N. PONNAMPERUMA
tions in reduced lake waters reported by Pearsall and Mortimer (1939), Mortimer (1941, 1942), and Kjensmo (1970) conform to the equilibrium values for the Fe(OH),-Fe” system. The hydrogen ion concentration affects Eh by direct participation in the oxidation-reduction or by influencing the dissociation of oxidant or reductant (Clark, 1960). So dE/dpH varies with the system. Although most workers use -0.059 V/pH at 25OC as the correction factor, the experimental values range from about -0.06 for aerobic soils to as steep a slope as -0.232 for some reduced soils (Patrick, 1960). This uncertainty makes many potentials corrected to pH 7 unreliable. We have shown theoretically and experimentally that the dE/dpH slope for the solutions of reduced ferruginous soils after the peak of water-soluble Fez+is -0.059 (Ponnamperuma et al., 1967). For most mineral muds, adjustment of Eh for pH is an unnecessary refinement because the pH values of reduced muds are about 7 and mud potentials are in any case imprecise. Although soil or mud potentials have no precise thermodynamic significance, they are semiquantitative measures of soil reduction. They are therefore useful in describing the state of reduction of wet soils and lake and ocean sediments. These potentials are best measured in situ and with minimum disturbance of the mud. Several probes for the simultaneous measurement of Eh and pH have been described (Mortimer, 1942; Starkey and Wight. 1946; Matsuo and Kato, 1956; Whitfield, 1969). If in situ measurement is not possible, the potential should be determined in cores of mud, immediately after collection, without dilution with water or exposure to air. Avoiding direct contact of the calomel reference electrode with the mud minimizes the liquid junction potential error (Jenny et al., 1950; Peech et al., 1953). Devices for extracting undisturbcd mud cores from waterlogged soils and lake and ocean muds have been described by Mortimer (1942), Kawaguchi et al. (1956), Walker (1964), and Mackereth (1969). The potential of the liquid phase of a reduced mud is far more meaningful and reliable than the potential of the mud itself. The liquid phase, that is, the interstitial or pore water or the soil solution, is a homogeneous phase in quasi, dynamic, or near equilibrium with the solid and gas phase of the mud, as the liquid phases of similar natural systems are believed to be (Garrels, 1959; Schuffelen and Koenigs, 1962; Morgan, 1967). The potential and pH of such a solution can be measured precisely with a minimum of the liquid junction potential error. (We have routinely obtained potentials that agree within 0.1 mV at duplicate elcctrodes in the solutions of reduced soils.) The main problem is to extract the solution and transfer it to the electrometric cell without altering its composition. The method of extraction depends on the sample. The solutions of
THE CHEMISTRY OF SUBMERGED SOILS
45
permeable submerged soils in pots can be drawn by gravity into flasks filled with purified nitrogen, transferred into the electrometric cell under slight nitrogen pressure, and the potential and pH measured within a few minutes of sample collection. The interstitial solutions of muds of low permeability and of core samples from paddy fields or lake and ocean bottoms have to be squeezed out by applying gas, mechanical, or centrifugal pressure. Excessive pressure alters the ionic composition of the solution. Manheim (1966) and Presley et al. (1967) used presses for extracting the interstitial water of ocean sediments. Takai et al. (1963) obtained the soil solution by centrifugation. We have found that low-temperature centrifugation gives clear solutions differing little from those drawn by gravity. Suction or contact for more than a few minutes with even an inert gas should be avoided because these treatments lead to loss of CO, from the solution and a consequent increase in pH and a decrease in Eh (Ponnamperuma et al., 1966a; IRRI, 1970). Thus Millipore filtration in an inert atmosphere may lead to loss of CO,. These extrinsic problems do not arise when the measurements are done in situ in natural or waste waters using the probes described by Back and Barnes (1965) and Schmidt (1970). b. Reducing Capacity. Three methods have been proposed for measuring the reducing capacity of muds : titrating with an oxidizing agent (Sturgis, 1936; Starkey and Wight, 1946; Zobell, 1946); determining total reduced Fe(I1) (Ponnamperuma, 1955; Jeffery, 1961; IRRI, 1964); and oxygen consumption by the mud (Howeler and Bouldin, 1971). The titration method is unsatisfactory because the vaIue obtained depends on the conditions of oxidation. If strong, reducing capacity is overestimated because organic matter and Fe(I1) in clay minerals are included; if mild, genuine reduction products are not estimated. Consumption of dichromate under standard conditions as proposed by Sturgis (1936) may, however, provide comparative values of reducing capacity. Total reduced Fe(I1) is not a good measure of reduction capacity because other reduction products are excluded. Oxygen consumption by the mud (Howeler and Bouldin, 1971) may give an ecologically more significant measure of reducing capacity than the chemical methods. The reducing capacity of soil solutions can be found by determining either the biological oxygen demand (BOD) or the chemical oxygen demand (COD) by standard methods (American Public Health Association, 1971). But oxygen consumption by the solution constitutes only a small fraction of the total reduction capacity.
5 . Anaerobic Respiration Submerging a soil cuts off its oxygen supply. The aerobic organisms use up the oxygen present in the soil and become quiescent or die. The faculta-
46
F . N. PONNAMPERUMA
tive and obligate anaerobes then proliferate (Takai et al., 1956; Takeda and Furusaka, 1970) using carbon compounds as substrate and using oxidized soil components and dissimilation products of organic matter as electron acceptors in respiration (Doelle, 1969). The switch from aerobic to anaerobic respiration occurs at the very low oxygen concentration of 3X M (Greenwood, 1961). Aerobic and anaerobic respiration probably follow common paths until the formation of pyruvic acid (Clifton, 1957; Doelle, 1969). The overall reaction may be represented by C',H1206
+ .LNAD++ 2 A T P 4 nCII3COCOOH + 2NADII + 2HHf+ iATP
In aerobic respiration the electrons picked up by nicotinamide adenine dinucleotide are transferred to atmospheric oxygen through the mediation of carriers and the terminal oxidases. Pyruvic acid itself is oxidized through the TCA cycle with oxygen as terminal electron acceptor. The regeneration of NAD' enables the system to operate cyclically and complcte the oxidation of substrate. In the absence of oxygen, facultative and obligate anaerobes use NO,-, Mn( IV), Fe(III), SO,?-, dissimilation products of organic matter, CO:, N2, and even H ions as electron acceptors in their respiration reducing NO,- to N2, Mn(1V) to Mn(II), Fe(II1) to Fe(II), SO,2- to H,S, CO, to CH,, N, to NH?, and H+ to H,. Also, anaerobic respiration produces substances that reduce soil components chemically (Bloomfield, 1951). Thus the switch from aerobic to anaerobic respiration ushers in the reduction of the soil. The requirements for soil reduction are the absence of oxygen, the presence of decomposable organic matter, and anaerobic bacterial activity. The course, rate, and degree of reduction are influenced by the nature and content of organic matter, temperature, the nature and content of electron acceptors, and pH. Also, air-drying a wet soil intensifies reduction after submergence (Aomine, 1962; Yoshizawa, 1966) and N, P, K fertilizers accelerate reduction in soils deficient in these nutrients (Chiang and Yang, 1970).
6. Sequential Reduction Reduction of a submerged soil proceeds roughly in the sequence (Table I ) predicted by thermodynamics (Ponnamperuma, 1955; Ponnamperuma and Castro, 1964; Takai and Kamura, 1966; Turner and Patrick, 1968). The same sequence is observed in the vertical distribution of redox components in a wcll eutrophied lake and in the succession of redox reactions in anaerobic batch digesters (Stumm and Morgan, 1970). The sequence is also reflected in the microbial succession-aerobes, facultative anaer-
THE CHEMISTRY OF SUBMERGED SOILS
47
obes, strict anaerobes-after submerging a soil (Takeda and Furusaka, 1970). Oxygen is the first soil component to be reduced, and it becomes undetectable within a day after submerging a soil (Section 111, A). The next oxidant to be attacked is nitrate, but nitrate reduction begins only after the oxygen concentration has dropped to a very low value (Mortimer, 1941; Skerman and MacRae, 1957; Bremner and Shaw, 1958; Greenwood, 1962; Turner and Patrick, 1968). Just as the presence of oxygen retards nitrate reduction, so does the presence of nitrate retard the reduction of other redox components. Nitrate stabilizes potentials at 0.2 to 0.4 V, and prevents the release of Mn”, Fez+, Sz-, CH,, and H, in lake waters and muds (Pearsall, 1938; Pearsall and Mortimer, 1939; Mortimer, 1941, 1942) and in submerged soils (Ponnamperuma, 1955; Ponnamperuma and Castro, 1964; Yamane and Sato, 1968; Turner and Patrick, 1968). Nitrate also suppresses odor development in effluents and sewage (Sanborn, 1941; Heukelekian, 1943) and methane formation in anaerobic sewage digesters (Brezonik and Lee, 1966). Nitrate prevents the formation of volatile acids (Greenwood and Lees, 1960) but is not as effective an oxidant of organic matter as oxygen (Schroeder and Busch, 1967). Manganese dioxide follows nitrate in the reduction sequence. But its influence is weaker than that of nitrate because it is insoluble in water and is used as an electron acceptor in respiration by only a limited number of bacteria. However, native or added MnO, retards the decrease in Eh of flooded soils and prevents the buildup of high concentrations of Fe2+ and other reduction products (Ponnamperuma and Castro, 1964; Ponnamperuma et al., 1965). The next mineral system in thermodynamic sequence is the Fe( OH) ,-Fez+ system. Because of the low standard potential of this system (Table I), its influence on soil reduction is not as obvious as that of NO3or MnO,. But I noted that soils high in Fe(I1I) showed a slower fall in Eh in the zone -0.05 to -0.2 V (Ponnamperuma, 1965), while Asami and Takai (1970) found that addition of amorphous Fe,03 to paddy soils depressed CH, formation. Although the reduction of a submerged soil proceeds in a stepwise manner, roughly in thermodynamic sequence, attempts to define the potentials at which one system comes into operation and yields to the next have not been successful. This is apparent from the wide range of critical potentials reported by various workers. I have previously listed these potentials and discussed the reasons for their variability (Ponnamperuma, 1972). But the following critical potentials reported by Patrick ( 1964), Connell and
48
F . N. PONNAMPERUMA
Patrick (1968), and Turner and Patrick (1968) for stirred soil suspensions may provide a rough guide to the progress of reduction:
IV.
Obseri-atiori
Ei (volt)
Osygen (undertahlr) Kitrate (undetectahle) Jfanga nese (detectable) Iron idetertahle) Sulfate (undetectable)
0 53 0 22 O PO n 12 -0 1 5
Electrochemical Changes in Submerged Soils
Submerging a soil brings about a variety of electrochemical changes. These include ( a ) a decrease in redox potential, ( b ) an increase in pH of acid soils and a decrease in pH of alkaline soils, (c) changes in specific conductance and ionic strength, ( d ) drastic shifts in mineral equilibria, (e) cation and anion exchange reactions, and ( f ) sorption and desorption of ions. I discuss changes in pH, Eh, and specific conductance in this section and mineral equilibria in Section VI. The information on ion exchange rcactions and sorption and desorption of ions in submerged soils is too meager and unreliable for review. A.
REDOXPOTENTIAL
The single electrochemical property that serves to distinguish a submerged soil from a well-drained soil is its redox potential. The low potentials (0.2 to -0.4 V ) of submerged soils and sediments reflect this reduced state, the high potentials (0.8 to 0 . 3 V ) of aerobic media, their oxidized condition.
1. Submerged Soils and Muds When an aerobic soil is submerged, its Eh decreases during the first few days and reaches a minimum; then it increases, attains a maximum, and decreases again asymptotically to a value characteristic of the soil, after 8-12 weeks of submergence (Ponnamperuma, 1955, 1965; Motomura, 1962; Yamane and Sato, 1968). The course, rate, and magnithde of the Eh decrease depend on the kind and amount of organic matter,
THE CHEMISTRY OF SUBMERGED SOILS
49
the nature, and content of electron acceptors, temperature,2 and the duration of submergence. The presence of native or added organic matter sharpens and hastens the first minimum, nitrate abolishes it (Ponnamperuma, 1955; Yamane and Sato, 1968). The rapid initial decrease of Eh is apparently due to the release of reducing substances accompanying oxygen depletion before Mn(1V) and Fe(II1) oxide hydrates can mobilize their buffer capacity. According to Yamane and Sato (1968), the first minimum potential can be as low as -0.42 V and can be accompanied by the evolution of hydrogen. Nitrate stabilizes potentials for some time at an E, value of about 0.2 V; Mn(1V) and Fe(II1) oxides, at lower values (Ponnamperuma and Castro, 1964; Ponnamperuma, 1965). The influence of soil factors on Eh changes have been summarized as follows (Ponnamperuma and Castro, 1964; Ponnamperuma, 1965) : (a) soils high in nitrate (more than 275 ppm NO,-) have positive potentials for several weeks after submergence; ( b ) soils low in organic matter (less than 1.5%) or high in manganese (more than 0.2%) maintain positive potentials even 6 months after submergence; (c) soils low in active manganese and iron (sandy soils) with more than 3% organic matter attain Eh values of -0.2 to -0.3 V within 2 weeks of submergence; and ( d ) the fairly stable potentials reached after several weeks of submergence lie between 0.2 and -0.3 V. Temperatures above and below 25OC retard the decrease in Eh but the degree of retardation varies with the soil (IRRI, 1967, 1969; Cho and Ponnamperuma, 1971 ) . The retardation of reduction is most pronounced in acid soils and hardly noticeable in neutral soils high in organic matter. The fairly stable potentials attained after about 12 weeks of submergence are practically independent of temperature in the range 15 to 45OC. The surfaces of submerged soils and oxygenated lake and ocean muds have potentials (0.3 to 0.5 V) which differ little from those of the overlying water. But below the oxygenated layer which is a few millimeters thick, the potential drops sharply and may be strongly negative (Section 111, B). The oxidized state of the surface layer is not permanent: during stagnation the surface may undergo reduction. According to Mortimer (1941, 1942), the critical potential for reduction of the soil-water interface is 0.2 V. Lake and ocean muds have potentials of 0.3 to -0.3 V, with occasional plunges to -0.4 V (Bass-Becking et al., 1960). Unless otherwise indicated, the temperature of the studies of the chemical and electrochemical kinetics described in this chapter was 25-32°C. All laboratory equilibrations were done at 25°C.
50
F. N. PONNAMPERUMA
2 . Natural Waters The redox potentials of lake and sea waters and their muds reflect their state of oxidation-reduction. Waters in contact with the atmosphere have potentials of 0.5 to 0.3 V (Hutchinson, 1957; Bass-Becking et af., 1960; Kaplan and Rittenberg, 1963). Anoxic waters have potentials of 0.2 to 0.0 V (Mortimer, 1941, 1942; Hutchinson, 1957; Kjensmo, 1970), although potentials as low as -0.15 V have been reported for stagnant sea water (Richards, 1965). The potentials of interstitial waters of marine sediments lie between 0.45 and -0.11 V (Friedman and Gavish, 1970). The fairly stable potentials attained by the solutions of flooded soils after several weeks of submergence range from 0.2 to 0.0 V with an occasional dip to -0.05 V. The vertical changes in potentials in natural waters depend on the degree of mixing and the nutrient content of the water. If thermal or mechanical mixing is active, the surface potential is maintained to great depths. But in eutrophic lakes that undergo thermal stratification (Section 111, C), the potential drops sharply in the thermocline and may have, in the hypolimnion, values as low as those of the reduced sediment (Mortimer, 1941, 1942). 3. Practical Significance
Despite theoretical and practical problems, the redox potential of a soil or sediment provides a quick, useful, semiquantitative measure of its oxidation-reduction status. Since the oxidation-reduction sratus of soils affects the growth of plants and that of sediments influences aquatic life and the capacity of sediments to absorb certain terrestrial wastes, Eh can be a useful environmental parameter. Several workers have confirmed this. Bradfield et al. (1934) successfully used Eh of the soil in early spring to demarcate low-yielding orchard sites associated with impeded drainage where measurements of groundwater alone were of little avail. Pearsall and Mortimer (1939) noted an association between Eh and the distribution of plant species in marshes and stressed the ecological significance of the oxidation-reduction state of a soil. Aomine (1962) reported that a mosaic of high and low redox spots in flooded fields benefits rice. Starkey and Wight (1946) found that anaerobic corrosion of iron pipes was severe below 0.1 V; above 0.4 V it was absent. Mortimer (1941, 1942) found that when the E7 of the mud-water interface dropped below 0.2 V, the mud underwent reduction, lost its absorptive capacity, and released manganese, iron, phosphate, and silica into the overlying water. Whitfield (1969) admitted the shortcomings of Eh measurements but found them a valuable guide in mapping the distribution of estuarine sediments. BassBecking et a/. (1960) examined 6200 pairs of Eh-pH readings in natural
THE CHEMISTRY OF SUBMERGED SOILS
51
media and established Eh-pH limits for different natural environments and bacterial ecosystems. Pohland and Mancy ( 1969) discussed the theoretical difficulties in the use of Eh for characterizing heterogeneous biochemical systems but concluded that Eh-pH can be used as an operational parameter in anaerobic digestion of waste organic materials. The optimum range was -0.275 to -0.285 V (Pohland, 1969). Weijden et al. (1970) found that the manganese content of deep-sea sediments increased sharply in the Eh range 0.32-0.42 V. Borchert (1965) defined zones of deposition of limonite, siderite, and pyrites in ocean sediments in terms of pH and Eh. Garrels and Christ (1965) have used Eh and pH to describe mineral associations in nature, and Hem (1960) and others, in the study of groundwaters. Although Eh reveals whether a soil is aerobic or anaerobic, it is unsatisfactory as a measure of oxygen concentration in soils (Ponnamperuma, 1972). Also, for the reasons discussed by me (Ponnamperuma, 1965), it is of little diagnostic value in rice culture. But the redox potentials of reduced lake and ocean waters, the interstitial waters of reduced soils and muds, and groundwaters are thermodynamically meaningful and have been used successfully in the quantitative study of redox equilibria (Section VI) . B.
pH
1 . The p H Values of Submerged Soils and Sediments When an aerobic soil is submerged, its pH decreases during the first few days (Motomura, 1962; Ponnamperuma, 1965), reaches a minimum, and then increases asymptotically to a fairly stable value of 6.7-7.2 a few weeks later. The overall effect of submergence is to increase the pH of acid soils and to depress the pH of sodic and calcareous soils. Thus submergence makes the pH values of acid soils (except those low in iron) and alkaline soils converge to 7 (Fig. 1 ) . The following figures, gleaned from several sources (Pearsall, 1938; Bass-Becking et al., 1960; Ponnamperuma et al., 1966a; Friedman and Gavish, 1970; Weijden et al., 1970), show that the pH of submerged soils and sediments and their interstitial solutions is about 7: Submerged soils Solutions of submerged soils Fresh water sediments Sea sediments Interstitial waters of sea sediments Marsh soils (flat bogs)
6.7-7.2 6.5-7.0 6.0-7.0 7.0-7.8 6.2-7.7 5.0-7.0
The low lower pH limit of marsh soils may be due to humic acids (Ruttner, 1963).
52
F . N. PONNAMPERUMA
7
94
99 28 35 6
PH
40
5
4
3
T
I
I
I
I
I
0
2
4
6
8
10
I 12
I 14
I 16
Weeks submerged
FIG. 1. Kinetics
of the pH values of some submerged soils.
Soil so.
Texture
28
Clay Clay Clay Clay loaiii Clay Clay loam
35 10 37
94 99
pH 1 3 3 8 6 7
9 4 8 7 7 7
0.31. % 2 6 7 2 2 4
9 6 2 2 6 8
Fe % 4 70
2 1 0 0 1
60 50
63 96 55
M n o/c 0 0 0 0 0 0
08 01 00 07 09 08
Draining and exposure to air reverse the pH changes in paddy soils (Dennett, 1932), in mangrove swamps (Hesse and Jeffery, 1963), in lake muds and bogs (Pearsall, 1938; Misra, 1938), and in anaerobic soils (Starkey and Wight, 1946; IRRI, 1965). The pH values of submerged soils measured in air-free aqueous suspensions are slightly higher than those of the corresponding soil solutions (Ponnamperuma et al., 1966a). This may be due to the inversion of the suspension effect by the divalent cations Ca'+, Fe", and Mn'+ (Raupach, 1954) and to dilution and loss of CO, during measurement of the pH of the soil suspension (Ponnamperuma et al., 1966a). Because the soil solution is the thermodynamically meaningful phase and the pH of a solution can be measured with the minimum of the liquid junction potential
THE CHEMISTRY OF SUBMERGED SOILS
53
and CO, errors, I use pH values of the soil solution in the description of pH changes in flooded soils and their quantitative interpretation. Although the pH values of acid soils increase after submergence and those of sodic and calcareous soils decrease, soil properties (and temperature) markedly influence the pattern of changes (Fig. 1). Soils high in organic matter and in reducible iron attain a pH of about 6.5 within a few weeks of submergence. Acid soils low in organic matter or in active iron slowly attain pH values which are less than 6.5. In fact, acid sulfate soils low in iron may not attain a pH of more than 5 even after months of submergence. Organic matter magnifies the decrease in pH of sodic and calcareous soils (IRRI, 1966). Low temperature (IRRI, 1968; Cho and Ponnamperuma, 1971) or the presence of nitrate (Yamane, 1958; IRRI, 1965) retards the increase in pH. 2 . Interpretation of pH Changes The decrease in pH shortly after submergence is probably due to the accumulation of CO, produced by respiration of aerobic bacteria, because CO, depresses the pH even of acid soils (Nicol and Turner, 1957). The subsequent increase in pH of acid soils is due to soil reduction (Ponnamperuma et al., 1966a). The pH values of submerged calcareous and sodic soils are lower than those of the aerobic soils because of the accumulation of CO,, for Whitney and Gardner (1943), Yaalon (1957), and Ponnamperuma et al. (1966a) have shown that the pH of alkaline soils is highly sensitive to changes in the partial pressure of CO, (Pco,). Table I shows that all the important reduction reactions that occur in nature involve the consumption of H+ ions. This means a decrease in acidity or an increase in net OH- ion concentration. The increase in pH is not determined by the absolute number of H+ ions consumed or OH- ions produced but by the ratio, R = H+ consumed/e consumed, as Bostrom (1967) has pointed out. This ratio is merely the coefficient of H+aqin the equations in Table I. It is highest for the reduction of Fe(OH),. The overall value of R for this reaction, however, is 1 because after the peak of water-soluble Fez+,the equilibrium
comes into operation (Ponnamperuma et al., 1967). Since most soils contain more Fe(II1) oxide hydrates than any other oxidant, the increase in pH of acid soils is largely due to the reduction of iron. Thus the pH of reduced ferruginous soils can be related to Eh and Fez+ activity by Eh
=
1.06 - 0.059 log Fez+ - 0.177pH
(10)
54
F . N. PONNAMPERUMA
before the peak of water-soluble iron and afterward by the equations (Ponnamperuma ef al., 1967)
Eh El1
= 1.37 - 0.0885 = 0.43
log Fez+ - 0.236pI-I
(11) (1%
- 0.059pH
The pH values of soils high in sodium sulfate may increase after submergence because Na,SO, is reduced to HIS and NaHCO, forms (Section
v, E l . The pH values of sodic soils can be related to the Na,CO,-H,O-CO, equilibrium and those of calcareous soils to the CaC0,-H,O-CO, equilibrium (Section VI, B). Although the increase in pH of acid soils is brought about by soil reduction, the fairly stable pH attained after a few weeks of submergence is regulated by Pco,. For reduced ferruginous soils the empirical relationship is pH
= 6.1 - 0.58 log
Pco,
(13)
This relationship is almost identical with that for an aqueous suspension nHLO equilibrated with COI (Ponnamperuma et al., 1969a). of Fe 10,. The pH values (and therefore Eh) of submerged soils, whether acid or alkaline, are highly sensitive to loss of CO, (Fig. 2 ) . This must be borne in mind during the sampling of reduced soils and the extraction and handling of their interstitial solutions (Section 111, E, 4, a ) . The pH of most reduced soils equilibrated with CO, at 1 atm is 6.1 (Ponnamperuma et al., 1969a). Sillen (1961 has proposed that the reaction of sea water, which is buffered around pH 8.1, is regulated by an equilibrium of the type SA12Si20;(0H)r
+ G i O ? + 2KC + ?Ca2+ + 9HgO F? ?KC~AI,S~,O,F,(H,O)+ BH+ 6
with log K
=
6 log (H+) - 2 log (K-) - 2 log (Ca2+)
(14)
The carbonate system, CaCO,-H,O-COI, according to Sillen is only an indicator of pH, not the mechanism of the buffer action. Garrels (1965) has suggested that dissolved silica and metallic cations recombine with degraded silicates and release H+ ions. The H+ions produced by this reversal of weathering prevent an increase in the alkalinity of sea water. Presumably on the basis of these observations, Bohn (1969) surmised that silicate equilibria rather than the redox equilibria control the pH of submerged soils. Martin (1970) and Stumm and Morgan (1970) have briefly reviewed the role of silicates vis-5-vis carbonates and have proposed two mechanisms buffering the pH of sea water: the carbonate system, a short-term
THE CHEMISTRY OF SUBMERGED SOILS
55
Brown cloy, pH 4.6
I.o
-0.5I
0
I
I
20
I
I
I
40 Minutes of N2 bubbling
I
I
60
0
I
I
20
I
I
40
I
I
60
16.5
Minutes of N, bubbling
FIG.2. Influence of the loss of CO? (caused by bubbling N t ) on pH and Eh of the solutions of two soils, 10 weeks after submergence.
mechanism; and the silicate system, a long-term mechanism operating perhaps over thousands of years. In waterlogged soils, in paddy soils, and in recent lake and ocean sediments, redox and carbonate systems control the pH.
3. p H Eflects The pH value profoundly influences hydroxide, carbonate, sulfide, phosphate, and silicate equilibria in submerged soils. These equilibria regulate the precipitation and dissolution of solids, the sorption and desorption of ions, and the concentrations of such nutritionally significant ions or substances as A13+,Fez+,H,S, H2C03,and undissociated organic acids. Some of these mineral equilibria are discussed in Section VI. Since excess watersoluble aluminum and iron are the toxic factors in acid sulfate rice soils (Nhung and Ponnamperuma, 1966) and iron deficiency limits the growth of rice on sodic and calcareous soils (Ponnamperuma, 1965), the influence of pH on the solubility of aluminum and iron is of special interest. The concentration of water-soluble aluminum in a soil is related to pH (Raupach, 1963) by pAl,
=
2pH - 4.41
(15)
Thus at a pH of 3.5 (a common pH value for acid sulfate soils), the concentration of water-soluble aluminum is 69 ppm. This concentration is
56
F . N. PONNAMPERUMA
much above the toxic limit for rice. But if the pH is raised to 4.4by liming or if the soil is kept submerged for a few weeks before planting, the aluminum concentration should drop to 1 ppm and aluminum toxicity be averted. Nhung and Ponnampcruma (1966) have confirmed this experimentally. The concentration of water-soluble Fe2+,like that of Al, is highly sensitive to pH changes as Eq. (16) (Ponnamperuma, 1955) indicates:
If solid-phase Fe,(OH), is present, the activities of water-soluble FeZf (concentrations would be higher) are 3.5 ppm at pH 7.5, 35 ppm at pH 7, 350 ppm at pH 6.5, and 3500 ppm at pH 6 . The detection of 6600 ppm Fc:+ in the solution of a flooded acid sulfate soil when its pH was 5.67 (Nhung and Ponnamperuma, 1966) is therefore not surprising. A pH change of 0.5 unit above or below 7 can spell the difference between iron deficiency and toxicity of rice on flooded soils (Ponnamperuma, 1965). The increase in pH of acid soils is one of the benefits of flooding rice soils because it eliminates aluminum toxicity, minimizes iron toxicity, and increases the availability of phosphorus.
C.
SPECIFICCONDUCTANCE I . Kinetics
The specific conductance of the solutions of most soils increases after submergence, attains a maximum, and declines to a fairly stable value, which varies with the soil. The changes in conductance reflect the balance between reactions that produce ions and those that inactivate them or replace them with slower moving ions. The increase in conductance during the first few weeks of flooding is due to the release of Fez+ and Mn2+ from the insoluble Fe(II1) and Mn(1V) oxide hydrates, the accumulation of NH,', HC0,-, and RCOO-, and (in calcareous soils) the dissolution of CaCO, by CO, and organic acids. An additional factor is the displacement of ions, especially cations, from soil colloids by reactions of the following type
57
THE CHEMISTRY OF SUBMERGED SOILS
This is evident from the similarity of the curves for the kinetics of (Fez+ Mn2+),other cations, and specific conductance (Fig. 3 ) . Conductance increases in spite of the replacement of NO,- and SO,*- by the less mobile HC0,- ion formed during denitrification and sulfate reduction and in spite of the neutralization (in acid soils) of the highly mobile H+ ions. The decline after the maximum is due mainly to the precipitation of Fez+ as Fe,Oi.nH,O and Mn*+ as MnCO, (Section V, C, 1 ) . The decrease in conductance of calcareous soils is caused by the fall in partial pressure of C 0 2 and the decomposition of organic acids (Section V, A, 3 ) . The kinetics of specific conductance varies widely with the soil. Neutral and alkaline soils starting with high conductances attain values exceeding 2 mmhos/cm and show a slow decline. Strongly acid soils have low initial conductances. They show steep increases to 2 4 mmhos/cm during the first 4 weeks of flooding, and then decline sharply, in striking similarity to the kinetics of water-soluble iron and manganese (Fig. 3 ) . Among nonsaline soils, the highest specific conductances (4-5 mmhos/cm) are observed in soils that have a low cation exchange capacity and are high in organic matter. A specific conductance exceeding 4 mmhos/cm indicates the presence of too much salt for the healthy growth of rice (IRRI, 1967). Values considerably in excess of 4 mmhos/cm are possible in submerged soils that have a low cation exchange capacity and are high in organic matter (especially if they are fertilized), and in acid sulfate soils.
+
25
20
-E
;7
._ -
15
\
U
10
3.0 5
2.0
5 .c E
-
1.0 E 0
2
4
I
I
I
I
I
I
6
8
10
12
14
16
0
Weeks submerged
FIG. 3. Kinetics of specific conductance and cation concentrations in a submerged ferrallitic soil. 0 Total alkalinity (rneqll); 0 CaZ+ Mg2++ NHa+ Na' + K (rneq/l); v Fez+ MnZ+(rneq/l); Specific conductance (rnrnhos/cm at 25°C).
+
+
+
58
F . N. PONNAMPERUMA
2. Specific Conductance and Ionic Strength The specific conductance of an aqueous solution, at a fixed temperature, depends on the kind and concentration of ions present. Since the kind and concentrations of ions determine to a large extent the ionic strength ( I = ~ ~ X C C , where ~ , ' , I is ionic strength, c i the concentration in moles per liter and i , the valence), there should be a quantitative relationship between specific conductance and ionic strength. When we tested this prediction experimentally (Ponnamperuma et al., 1966b), we found that, in spite of wide variations in the ionic composition of the solutions of reduced soils, the ionic strength in moles per liter was numerically equal to 16 times the specific conductance ( K ) in mhos/cm at 25OC, up to ionic strengths of 0.05. The ionic strength of a solution is chemically and, perhaps, ecologically an important property of a soil solution because it determines the activity Coefficients of the ions present. A knowledge of the activity coefficients of ions is necessary for the thermodynamic study of mineral equilibria in flooded soils. The activity coefficient ( y ) of an ion can be derived from the Debye-Huckel equation if the concentrations of all the ions present are known. Use of I = 1 6 K eliminates the need for chemical analysis and enormously simplifies the calculation of ionic strength. Substituting 1 6 for ~ I , we can rewritc the Debye-Huckel equation and its simpler form as follows
- log 7 = 4A22K1'2
(18)
And for dilute solutions the Henderson-Hasselbalch equation becomes pH
=
7.84
+ log [IICO,-]
- log
PCO,- 2~'''
(19)
where [HCO,-] is the concentration of bicarbonate in moles per Iiter (Ponnamperuma et al., 1966b).
V.
Chemical
Transformations in Submerged Soils
The chemical properties of a soil undergo a drastic transformation on submergence. The oxidized constituents, Fe( III), Mn(IV), NO,-, and SO,2-, that characterize a well drained soil, virtually disappear and are replaced by their reduced counterparts, Fe( 11), Mn(II), NH,', and Ss-, and the course of organic matter decomposition is diverted from CO, production to the formation of an array of unstable organic substances, fol-
THE CHEMISTRY OF SUBMERGED SOILS
59
lowed by the evolution of CO, and CH,. These changes have important implications for geochemistry, limnology, rice culture, and pollution control. A.
CARBON
The two main transformations of carbon in nature are photosynthesis and respiration. On the balance between these two processes depend ( a ) the amount of organic matter that accumulates in soils.and sediments, and ( b ) the quality of streams, lakes, and estuaries. In submerged soils, respiration (decomposition of organic matter) is the main transformation. 1. Decomposition of Organic Matter The decomposition of organic matter in a submerged soil differs from that in a well drained soil in two respects: it is slower; and the end products are different. In a well drained soil, decomposition of plant residues is accomplished by a large group of microorganisms assisted by the soil fauna. Owing to the high energy release associated with the aerobic respiration of these organisms, decomposition of substrate and synthesis of cell substance proceed rapidly. The bulk of freshly added organic matter disappears as CO,, leaving a residue of resistant material, chiefly altered lignin. Also, there is a heavy demand on nutritional elements, especially nitrogen. In submerged soils, the decomposition of organic matter is almost entirely the work of facultative and obligate anaerobes. Since anaerobic bacteria operate at a much lower energy level than aerobic organisms, both decomposition and assimilation are much slower in submerged soils than in aerobic soils. The accumulation of plant residues in marshes and in underwater sediments (Degens, 1965) illustrates this point. The most striking difference between anaerobic and aerobic decomposition lies in the nature of the end products. In a normal well drained soil the main end products are CO,, nitrate, sulfate, and resistant residues (humus); in submerged soils, they are CO,, hydrogen, methane, ammonia, amines, mercaptans, hydrogen sulfide, and partially humified residues. 2 . Pyruvic Acid Metabolism Carbohydrate metabolism is probably the same in both aerobic and anaerobic soils until the formation of the key metabolite, pyruvic acid. For example, cellulose is hydrolyzed to soluble sugars by extracellular enzymes; the sugars enter the microbial cell and are hydrolyzed further to glucose, which is then oxidized to pyruvate by the EMP, HMP, ED, or the phos-
60
F. N. PONNAMPERUMA
phoketolase pathway (Doelle, 1969). The overall oxidation of glucose to pyruvic acid may be represented as follows Cp,H1?Os
+ 2hTP + 2XAD+ F? PCH3COCOOIT + 4hTP + 2LK.iDH + 211’
When oxygen is absent, the reduced nicotinamide adenine dinucleotide (NADH) formed during this process transfers its H ions and electrons to oxidized soil components or to organic metabolites (Section 111, E, 5 ) . If the electron acceptor is inorganic, the process is called anaerobic respiration; if they are organic substances, fermentation (Doelle, 1969). Fermentations are largely the reactions of pyruvic acid and its alteration products. Pyruvic acid undergoes a variety of changes determined by pH, redox potential, osmotic pressure, the available electron acceptors, and the microorganisms present (Werkman and Schlenk, 1951). Since most soils contain a wide variety of microorganisms, the physical and chemical environment determine the fate of pyruvic acid. The main anaerobic transformations of pyruvic acid gleaned from Werkman and Schlenk (1951), Wood (1961), Wilkinson and Rose (1963), and Doelle (1969) include: ( a ) reduction to lactic acid, (b) decarboxylation to CO, and acetaldehyde, (c) dissimilation to lactic and acetic acids and CO:, ( d ) cleavage to acetic and formic acids, H2, and CO,, ( e ) carboxylation to oxaloacetic acid, (f) condensation with itself or acetaldehyde to give acetylmethylcarbinol, and (g) dissimilation to butyric and acetic acids. The reaction products of pyruvic acid may undergo further biochemical transformations (Barker, 1956; Clifton, 1957; Wilkinson and Rose, 1963; Doelle, 1969). Among these reactions are: ( a ) reduction of acetaldehyde to ethanol, ( b ) reaction of ethanol and acetic acid yielding butyric and caproic acids. (c) decomposition of ethanol to CH, and CO1, ( d ) dissimilation of lactic acid to propionic and acetic acids, (e) decomposition of formic acid to CO, and H,, (f) reduction of oxaloacetic acid to succinic acid, (g) reduction of acetylmethylcarbinol to 2,3-butanediol, and (h) reduction of butyric acid to butanol and acetone to 2-propanol. Thus the transformations of pyruvic acid and its reaction products produce an array of substances that include: ethanol, butanol, 2-propanol, glycerol, 2,3-butanediol, and acetylmethylcarbinol; acetone and acetaldehyde; formic, acetic, butyric, valeric, caproic, lactic, oxaloacetic, malonic, fumaric, and succinic acids; and CO,, H,, CH, and C,H,. Almost all these products and others have been identified collectively in anaerobic soils and cultures containing soils (Acharya, 1935; Roberts, 1947; Takijima, 1964; IRRI, 1965; Wang and Chuang, 1967; Yamane and Sato, 1967; Cotoh and Onikura. 1967, 1971; Smith and Scott Russell, 1969; Gotoh, 1970) and in sewage and polluted waters (Pohland and Bloodgood, 1963; Tezuka et al., 1963; Mahr, 1965; Smith and Mah, 1966). The main
THE CHEMISTRY OF SUBMERGED SOILS
61
products of the anaerobic decomposition of carbohydrates, however, are CO,, the lower fatty acids, and CH,. Their formation sequence corresponds roughly to the three main stages of the dissimilation of carbohydrate following soil submergence: (a) respiration by aerobes and facultative anaerobes, (b) acid production, and (c) decomposition of acids and reduction of CO, to CH, by strict anaerobes.
3. Kinetics of CO, One to three tons of CO, are produced in the plowed layer of 1 hectare of a soil during the first few weeks of submergence (IRRI, 1964). Being chemically active, it forms carbonic acid, bicarbonates, and insoluble carbonates. The excess accumulates as gas. The partial pressure of CO,, which is a good measure of CO, accumulation, can be calculated from pH, HC0,- concentration, and specific conductance of the solutions of flooded soils (Ponnamperuma et al., 1966b) or from pH and total CO, determined by gas chromatography (IRRI, 1967). The partial pressure of CO, in a soil increases after submergence, reaches a peak of 0.2-0.8 atm 1-3 weeks later, and declines to a fairly stable value of 0.05-0.2 atm (IRRI, 1964). The pattern of Pco, kinetics depends on soil properties and temperature (Cho and Ponnamperuma, 1971). Acid soils high in organic matter but low in iron and manganese show a rapid increase in Pcoz to about 0.5 atm within 1-2 weeks of flooding followed by a slow decline to about 0.3 atm. Acid soils high in both organic matter and iron build up Pcoz values as high as 0.8 atm, but the peaks are followed by a rapid decline to a fairly constant value of about 0.1 atm. Sandy soils tend to give higher mean Pcoz values than clay soils of comparable organic matter content. The decline in Pcoz after 1 - 4 weeks of submergence is due to escape, leaching, removal as insoluble carbonates, dilution by CH, produced during the decomposition of organic acids, and bacterial reduction of CO, to CH,. Since low temperature retards methane production, Pco, tends to be high when the temperature is low (Cho and Ponnamperuma, 1971). The Pco, values reported above are considerably lower than those suggested by the composition of the gases of anaerobic soiIs reported by Takai et al. (1963). The degassing method used by these workers probably displaced varying amounts of CO, from HC0,-, thus inflating the concentration of CO,. The practical implications for rice culture are that CO, injury may occur in acid soils low in iron, that organic matter or low temperature may aggravate this injury, that high Pcoz values are short-lived in ferruginous soils, and that planting should be delayed at least 2 weeks after flooding to avoid CO, injury.
62
F. N. PONNAMPERUMA
4 . Kinetics of Volatile Organic Acids The main organic acids found in anaerobic soils and sewage are formic, acetic, propionic, and butyric acids. Of these, acetic acid is by far the most abundant (Tezuka et al., 1963; Painter, 1971; Gotoh and Onikura, 1971). When a soil is submerged, the concentration in the soil solution of volatile organic acids increases, reaches a peak value of 10-40 mmoles/liter in 1-2 weeks and then declines to less than 1 mmole/liter a few weeks later. Soils high in native or added organic matter produce high concentrations of acids (Motomura, 1962). Low temperature retards acid formation slightly, but acid destruction markedly (Yamane and Sato, 1967; Cho and Ponnamperuma, 1971). Thus organic acids persist longer in cold soils than in warm soils. Ammonium sulfate appears to increase acetic acid formation but suppresses the formation of propionic and butyric acids (IRRI, 1971). The volatile organic acids are ecologically important because they are intermediates in the degradation of organic matter to CH, in rice soils, in sewage, polluted and anoxic waters, and lake and ocean sediments. These acids have been shown to be toxic to rice in culture solutions at pH values below 6.0 (Takijima, 1964; Tanaka and Navasero, 1967). But because of their transitory existence, organic acid injury to rice is unlikely except in peaty soils (Takijima, 1963), cold acid soils (Cho and Ponnamperuma, 1971), and soils to which green manure or straw has been added (IRRI, 1971) . Accumulation of volatile organic acids in anaerobic sewage digesters indicates digester failure.
5 . Methane Fermentation Methane is the typical end product of the anaerobic decomposition of organic matter. The gas escapes in large amounts from flooded soils, marshes, lake muds, anoxic lake and ocean waters, sewage disposal units, and from the stomachs of ruminants, accompanied usually by smaller amounts of carbon dioxide and hydrogen. Some of the methane is oxidized bacterially at the surface of paddy soils (Harrison and Aiyer, 1913, 1915) and in the oxygenated strata of lakes (Hutchinson, 1957). Methane formation is ecologically important because it helps the disposal of large amounts of organic matter sedimented in lakes or accumulated in cities. Methane is produced by a small group of obligate anaerobes found in reduced muds, in anaerobic sewage digesters, and in rumens. Methane bacteria function best at temperatures above 3OoC, but Methansarcina methanica, the species which is most abundant in natural anaerobic waters, produces methane even at 5OC (Ruttner, 1963). Methane bacteria are highly substrate specific and can metabolize only a small number of simple organic and inorganic substances, usually the products of fermentation.
THE CHEMISTRY OF SUBMERGED SOILS
63
These include: formic, acetic, propionic, n-butyric, n-valeric, and n-caproic acids; methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and n-pentanol; and hydrogen, carbon monoxide, and carbon dioxide (Barker, 1956). These substances may be derived from the breakdown of carbohydrates, proteins, or fats. Some typical reactions of methane bacteria listed by Barker (1956) and Stadtman (1967) include: (a) oxidation of ethanol to acetic acid coupled to the reduction of CO, to CH,; (b) P-oxidation of propionic acid to acetic acids (Motomura, 1962). Low temperature retards acid formation slightly, to CH, and CO,; (d) decomposition of formic acid to CH, and C 0 2 ; and (e) reduction of CO, to CH,. The end result of these reactions is the almost complete degradation of the lower fatty acids and some alcohols to carbon dioxide and methane (Stadtman, 1967). Thus soluble carbon compounds produced by anaerobic degradation of insoluble carbohydrates, proteins, and fats are gassified and released to the atmosphere as CO, and CH,. Almost all natural organic substances and many synthetic compounds can be fermented to CO, and CH, (Loehr, 1968). Pfeffer (1966) and Loehr (1968) have discussed the advantages of anaerobic digestion over aerobic digestion for the disposal of sewage and industrial wastes and Loehr ( 1968) has listed the conditions for efficient anaerobic digestion. Cessation or retardation of methane formation, the accumulation of acids, and bad odors signify digester failure. The primary cause of digester failure is probably the accumulation of substrates for which the appropriate species of bacteria are not present in sufficient numbers (Gaudy and Gaudy, 1966). The gasification of the greater part of the organic matter in sewage and industrial wastes relieves streams and lakes of a heavy oxygen demand, and prevents their pollution. Toerien and Hattingh (1969) and KotzC et al. (1969) have reviewed the microbiology and mechanisms of anaerobic digestion in wastewater treatment.
6. Reducing Substances The solutions of submerged soils contain substances that reduce KMnO, under mild conditions. These substances compete with rice roots for oxygen, and if present in excess, may deprive them of the oxygen vital for respiration and nutrient uptake. Since water-soluble Fez+ (the main inorganic reductant) accounts for less than 25% of the reducing capacity, the bulk of the reducing substances must be organic. But aldehydes, ketones, alcohols, reducing sugars, aliphatic hydroxy and unsaturated acids, and mercaptans and organic sulfides are present only in traces (IRRI, 1965).
64
F. N. PONNAMPERUMA
Two phenolic substances-ferulic acid ( 3-methoxy-4-hydroxycinnamic acid) and sinapic acid (3,5-dimethoxy-4-hydroxycinnamicacid) -have been dctected in the solutions of reduced soils (IRRI, 1970), but the nature of the bulk of the reducing substances is unknown. They are probably complex molecules containing phenolic and olefinic groups (Fotiyev, 1966; Christman and Ghassemi, 1966; Lamar and Goerlitz, 1964).
7 . Detergents and Peyticidey In recent years, pollution of drinking water, streams and lakes, and even the sea by detergents and pesticides has received wide attention, and methods of minimizing it have been investigated. Physical, chemical, and microbiological factors determine the fate of these substances in soils and waters. For pollution control, microbiological decomposability is a desirable characteristic of detergents and pesticides. And data on biodegradability of these pollutants in aerobic media are available. But the fate of these substances may be different in anaerobic media. Branched-chain anionic detergents apparently resist biodegradation both in aerobic and anaerobic media, but linear-chain anionic detergents are broken down to a much lesser degree anaerobically than aerobically (Rismondo and Zilio-Grandi, 1968). The herbicides atrazine ( 2-chloro-4-ethylamino-6-isopropylamino-s-tnazine ) and t rifluraline (~,~,~-trifluoro-2,6-dinitro-N,N-dipropyl-~-toluidine ) disappear more rapidly under anaerobic conditions than under aerobic conditions (Kearney et a/., 1967). Anaerobic biodegradation of the insecticide parathion (0,O-diethyl-Op-nitrophenyl phosphorothioate) stops at the aminoparathion stage; aerobically it is biodegraded further (Graetz et a/., 1970). Aerobically, diazinon [O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate] is first hydrolyzed to 2-isopropyl-6-methyl-4-hydroxypyrimidine, and the pyrimidine ring is then oxidized to CO, by soil microflora; anaerobically, the hydrolysis product accumulates (Sethunathan and Yoshida, 1969). The insecticide, Y-BHC (./-isomer of 1,2,3,4,5,6-hexachlorocyclohexane) persists for several years in aerobic soils, but is biodegraded partly to COJ in submerged soils (MacRae et al., 1967b). A high content of organic matter hastens the biodegradation; addition of nitrate or manganese dioxide retards it (Yoshida and Castro, 1970). DDT (dichlorodiphenyltrichloroethane) is fairly stable in aerobic soils but is degraded to DDD in submerged soils (Castro and Yoshida, 1971). Alternate oxidation and reduction may provide an environment more favorable to the degradation of mixed detergents and pesticides than oxi-
THE CHEMISTRY OF SUBMERGED SOILS
65
dation or reduction alone. If so, these substances are less likely to persist in paddy soils (Section 11, C) and in eutrophic lakes than in aerobic soils and oligotrophic lakes (Section 111, E) .
B. NITROGEN Nitrogen occurs in soils and sediments chiefly as complex organic substances, ammonia, molecular nitrogen, nitrite, and nitrate. The transformations that they undergo are largely microbiological interconversions regulated by the physical and chemical environment. The main interconversions may be depicted as follows Nz Proteins F! amino acids
Nz
T + NH4+ F? NOzF! NOS1
The equilibrium point of this system in a soil depends on the nature and content of organic matter, oxidation-reduction potential, pH, and temperature. In submerged soils, the main transformations are the accumulation of ammonia, denitrification, and nitrogen fixation. These transformations have an important bearing on the nutrition of rice and aquatic plants, the pollution of streams, lakes, and estuaries, and the nitrogen balance in nature.
I . Accumulation of Ammonia The mineralization of organic nitrogen in submerged soils stops at the ammonia3 stage because of the lack of oxygen to carry the process via nitrite to nitrate. So ammonia accumulates in anaerobic soils, anoxic waters, and in anaerobic sewage digesters, Ammonia is derived from anaerobic deamination of amino acids, degradation of purines, and hydrolysis of urea. Less than 1 % comes from nitrate reduction (Woldendorp, 1965). Some examples of deamination reactions taken from Clifton (1957), Barker (1961), and Doelle (1969) include: (a) oxidation of alanine to pyruvic acid; (b) reduction of aspartic acid to succinic acid; (c) oxidation of alanine to pyruvic acid coupled to the reduction of glycine (the Stickland Reaction); (d) oxidation of alanine to acetic acid coupled to the reduction of alanine; (e) desaturation of aspartic acid to fumaric acid; (f) dissimilation of tryptophan to indole and pyruvic acid; (g) dissimilation of glutamic acid to acetic and propionic acids, CO,, and H,; (h) dissimilation of arginine to ornithine; (i) dissimilation of cysteine to H,S; and ( j ) dissim ilation of purines to acetic acid and CO,. The end products of these reaca
Ammonia stands for (NHz
+ NH,OH + NH++).
66
F. N. PONNAMPERUMA
tions are ammonia, CO,, and volatile fatty acids. The fatty acids are subsequently fermented to methane. Bacteria that effect these changes have been isolated from soils, muds, decomposing organic matter, sewage, and rumen fluids. Greenwood and Lees (1960) found that during the anaerobic decomposition of amino acids added to soil volatile fatty acids were formed, and, in 10 days, 80% of the N in the amino acids was released as ammonia. Anaerobic deamination was much slower than the aerobic process. Nitrate suppressed volatile acid formation but not ammonia production. Some amino acids decomposed much more slowly than others both aerobically and anaerobically. Although aerobic deamination may be more rapid than the anaerobic process, inorganic nitrogen is released in larger quantities and faster in anaerobic soils than in aerobic soils (Joachim, 1931; Waring and Bremner, 1964; Broadbent and Reyes, 1971 ) because less immobilization of nitrogen occurs in anaerobic media. Ammonification in an aerobic soil is accomplished by a wide group of microorganisms of which fungi are normally the most vigorous. The aerobic metabolism of fungi with its high energy release enables them to carry on vigorous decomposition of organic matter coupled to high synthetic activity. Thus decomposition is rapid but no nitrogen is released unless the nitrogen content of the substrate exceeds 1-2% (Bartholomew, 1965). On submergence, fungi are replaced by anaerobic bacteria. These bacteria, operating at a lower energy level, synthesize much less cell material. Thus the nitrogen factor (the number of grams of additional nitrogen required to decompose 100 g of material) is much less anaerobically than aerobically (Acharya, 1935). But Broadbent and Nakashima (1970) found that the nitrogen factor varied with quantity of straw added, the nitrogen content of straw, the nature of soluble nitrogen added, the degree of anaerobiosis, and the soil, but in no case was it inconsiderable. Williams et al. (1968) observed an increase in the yield of rice on flooded fields when the nitrogen content of the straw that was plowed in exceeded 0.6%, and a decrease in yield when the nitrogen content was below 0.5%. Thus in spite of the lower nitrogen factor in flooded soils, materials with wide C : N ratios such as straw and weeds, instead of supplying nitrogen may depress its availability to rice, especially in soils low in organic matter (IRRI, 1964), while green manures like Sesbania sesban release the greater part of their nitrogen within 2-3 weeks of incorporation in flooded soils (Joachim, 1931 ;IRRI, 1964). Temperature has a marked effect on ammonia release in flooded soils. Mitsui (1960) reported a virtual doubling of ammonia production when the temperature of anaerobic incubation was raised from 26 to 4OOC. Kawa-
THE CHEMISTRY OF SUBMERGED SOILS
67
guchi and Kyuma (1969) and Cho and Ponnamperuma (1971) have reported similar increases in the range 15-40°C. Ammonia production in submerged soils follows a roughly asymptotic course and the kinetics of ammonia release can be described by log ( A - y)
=
log A - ct
(20)
where A is the mean maximum NH,+-N concentration, y is the actual concentration t days after submergence, and c is a parameter depending on the soil (Ponnamperuma, 1965). A is a characteristic for a soil under a given temperature regime and was found to be highly correlated, in 31 tropical mineral paddy soils, with the organic matter content of the soil. Waring and Bremner (1964) proposed ammonia released after 2 weeks anaerobic incubation at 30°C as a measure of nitrogen available to dryland plants. Kawaguchi and Kyuma (1969) used ammonia produced after 2 weeks’ anaerobic incubation at 40°C as an index of available nitrogen in paddy soils. Almost all the mineralizable nitrogen in a soil is converted to ammonia within 2 weeks of submergence if the temperature is favorable, the soil is not strongly acid or very deficient in available phosphorus. The amount of ammonia produced during the first 2 weeks of submergence may range from 50 to 350 ppm N on the basis of the dry soil. The soil solution may contain 2 to 100 ppm N depending on texture and organic matter content of the soil (IRRI, 1964). The A values of nitrogen in flooded soils may be as high as 30% of the total N (Broadbent and Reyes, 1971). I have previously discussed the implications of the kinetics of ammonia release for rice culture (Ponnamperuma, 1965). 2. Denitrification Nitrate undergoes two transformations in submerged soils : assimilation or reduction of nitrate with incorporation of the products into cell substance; and dissimilation or nitrate respiration in which nitrate functions as an alternative to oxygen as an electron acceptor. A substantial proportion of added nitrate may, in some submerged soils, be assimilated, and enter the pool of soil organic matter (MacRae et al., 1967a), but the bulk of native or added nitrate disappears within a few days in most soils as a result of nitrate respiration. Nicholas (1963) defines denitrification as a special case of nitrate respiration in which nitrate, nitrite, or some intermediate is converted to nitrogen or its oxides. Denitrification has been extensively reviewed during the past decade with emphasis on varying aspects : microbiology (Alexander, 1961) ; environmental factors (Broadbent and Clark, 1965; Woldendorp, 1968) ; biochemistry (Nicholas, 1963; Campbell and Lees, 1967) ; paddy soils
68
F . N. PONNAMPERUMA
(Patrick and Mahapatra, 1968); sewage (Painter, 1971); and the sea (Martin, 1970). The main fe.atures are critically reviewed below. Denitrification is brought about by a large number of bacteria and fungi which include heterotrophic and autotrophic species (Painter, 1971 ) . These facultative organisms transform nitrate to nitrogen and its oxides only at very low oxygen concentrations (Skerman and MacRae, 1957; Bremner and Shaw, 1958; Greenwood, 1962; Turner and Patrick, 1968). I have shown theoretically that nitrate will become undetectable ( M) in water only at an infinitesimal partial pressure of oxygen (Ponnamperuma, 1972). If denitrification occurs in aerobic soils, it is due to the presence of anaerobic pockets (Russell, 1961 ; Greenwood, 1962). Nitrite, however, is denitrified by bacteria even in the presence of oxygen (Kefauver and Allison, 1957; Skerman et al., 1958; Mechsner and Wuhrmann, 1963). This observation agrees with the thermodynamic prediction that nitrite is highly unstable in aqueous systems in equilibrium with air (Ponnamperuma, 1972). Because nitrite is an intermediate both in the reduction of nitrate and the oxidation of ammonia to nitrate, aerobic denitrification via nitrite may be more substantial and widespread than realized, especially in soils that are alternately wet and dry. Denitrifying organisms need a source of H+ ions and electrons to reduce nitrate and a carbon source and ammonia for cell synthesis. In soils, organic matter is the source of all these ingredients. Thus denitrification is absent or slow in soils low in organic matter and is enhanced in such soils by adding organic matter (Bremner and Shaw, 1958; McGarity, 1961; MacRae et al., 1967a). The limiting factor in denitrification by marine bacteria, according to Lloyd (1939), is the supply of organic matter. Most reviewers state that the rate of denitrification increases with temperature up to 6OOC. While this is generally true €or experiments lasting a few hours or days, over a period of 2 weeks the overall rate is essentially the same in the range 15-40GC,as the figures of Bremner and Shaw (1958) and the findings of Yamane and Sat0 (1961) and Cho and Ponnamperuma ( 197 1 ) indicate. Although Cooper and Smith (1963) found that a decrease of temperature from 2 5 O to 2OoC halved the rate of nitrate disappearance in the first few hours, at the end of 60 hours there was no nitrate left at 30°, 25", or 2OOC. Only temperatures near freezing can delay denitrification over a period of weeks. Thus in submerged soils and sediments, and anoxic lake and ocean waters, where changes are measured over weeks, months, or years, temperature may be irrelevant to denitrification losses. Patrick (1960) and Broadbent and Clark (1965), impressed by the approximate linearity of the nitrate/time curves, reported that the rate of denitrification was independent of the nitrate concentration. But Patrick's
THE CHEMISTRY OF SUBMERGED SOILS
69
curves and those of Cooper and Smith (1963) cited by Broadbent and Clark reveal a roughly exponential trend. So do the nitrate curves of Turner and Patrick (1968). Mathematical formulation of the kinetics of nitrate in the solutions of flooded soils showed that the disappearance of nitrate follows first-order kinetics (--dc/dt = kc) with high velocity constants for nearly neutral soils and low ones for acid soils (IRRI, 1965). This means that the rate of denitrification depends on the nitrate concentration, and it is slower in acid soils than in neutral soils, as has been reported by De and Sarkar (1936), Ponnamperuma (1955), Bremner and Shaw (1958), and several others. Some reviewers discount redox potential as a factor in denitrification. But it is, at least theoretically, the best quantitative measure of the tendency toward denitrification because it is the quintessence of the factors that bring about a milieu favorable or unfavorable for denitrification. Several workers have confirmed this (Section 111, E, 6 ) . It may, however, be repeated that over a wide range of soils under natural vegetation Pearsall (1938) found that whenever the potential was higher than 0.35 V, nitrate was present and whenever the potential was less than 0.32 V at pH 5.0, nitrate was absent. Patrick (1960) noted that the potential at pH 5.1 at which nitrate in soil suspensions became unstable was 338 mV. The partial pressure of oxygen at this potential and pH is 10-31.seatm. During denitrification, nitrogen is the ultimate product of nitrate reduction and CO,, the oxidation product of carbohydrate; ammonia is produced in only very small amounts; and nitrite and nitrous oxides are intermediates (Broadbent and Clark, 1965). Nitric oxide is an unlikely intermediate (Ponnamperuma, 1972), but may arise in soils by chemical reactions. Hydroxylamine is believed to be an intermediate both in nitrate reduction and ammonia oxidation (Doelle, 1969), but its presence in submerged soils has not been established. Jordan et al. (1967) found that of 59 microorganisms isolated from a flooded soil only 22 reduced NO,- to NO,-, but not all these reduced NO,-. Martin (1970) reports that about half of the marine bacterial species reduce NO,- to NO,- but less than 5% can reduce NO,- or NO,to nitrogen. In spite of this, nitrite does not accumulate in anaerobic media. Concentrations exceeding 3 ppm are rare and transitory in flooded soils (Ponnamperuma, 1955). Almost all the nitrate present in a soil disappears within a few days of submergence, the bulk of it being lost as N,. After submergence, ammonia diffusing up to the oxidized surface layer or broadcast on the surface undergoes nitrification; the nitrate formed moves down by diffusion and mass flow into the reduced soil below and is denitrified (Section 111, B). Alternate wetting and drying increases the denitrification loss (Patrick and
70
F . N. PONNAMPERUMA
Wyatt, 1964); continuous submergence minimizes it and even leads to a substantial accumulation of nitrogen (IRRI, 1969). Denitrification in cultivated soils is undesirable because it causes the loss of a valuable plant nutrient, so good farmers try to minimize this loss. But in efflue'nts from sewage works, in groundwaters, streams, lakes, and estuaries, loss of nitrogen by denitrification is highly desirable because it helps to prevent the contamination of drinking water by nitrate and the pollution of natural bodies of water by excessive growth of aquatic plants. 3 . Nitrogen Fixation
Biological nitrogen fixation is the reduction of nitrogen gas to ammonia (Mortenson, 1962). This reaction needs reductants at a very high electron activity or a very low pE (Table I ) . Photosynthesis and anaerobic respiration are the two major natural sources of these reductants. Submerged soils with blue-green algae (and sometimes, nitrogen-fixing photosynthetic bacteria) at the surface and nitrogen-fixing bacteria in the bulk of the soil are thus favorably placed for nitrogen fixation. Harada (1954), Mitsui (1960), and Singh (1961) have reviewed the role of blue-green algae in the nitrogen economy of rice fields. From their reviews the following conclusions may be drawn: ( a ) members of the genera, Nostoc, A nabaena, Ocillatoria, Tolypothrix, Calothrix, Phormidium, Aulosira, and several others can fix nitrogen; ( b ) some of these algae are present in most paddy fields; ( c ) some species may fix as much as 22 kg/ha of nitrogen in a season; (d) a slightly alkaline reaction and available phosphorus favor fixation and nitrogen fertilizers retard it; (e) fixation is apparently increased by the presence of the rice plant; and ( f ) the greater part of the nitrogen fixed is not available for the current needs of the crop. Nitrogen fixation by free-living bacteria are perhaps equally important because the environment of a flooded soil is suited to both aerobic and anaerobic nitrogen fixers. Aerobic bacteria can thrive in the oxygenated surface layer and in the oxygenated rhizosphere of rice, sustained by ethanol, acetate, and other soluble substrates diffusing from the anaerobic soil matrix. The low oxygen concentration in their habitat would favor nitrogen fixation (Jensen, 1965). The anaerobic bulk of the soil would be an ideal medium for such anaerobic nitrogen fixers as Clostridium, especially if organic matter is present. In this connection the finding of Rice et al. (1967) that a combination of aerobic and anaerobic conditions dramatically increased nitrogen fixation in a thin layer of soil amended with straw is significant. But under field conditions, except at the soilwater interface, the nitrogen supply may be insufficient because the gas can reach the interior of the soil only by the extremely slow process of
THE CHEMISTRY OF SUBMERGED SOILS
71
molecular diffusion in the interstitial water. The presence of rice plants alters the situation: nitrogen is transported along with oxygen from the shoot to the root and presumably diffuses out with oxygen (Section 111, D). This ensures a supply of nitrogen for the aerobic fixers in the rhizosphere and perhaps for the anaerobes just outside the oxygenated rhizosphere. No data are available on the amount of nitrogen diffusing out of rice roots in flooded fields but the following observations are significant: (a) more nitrogen is fixed in the presence of rice plants than in their absence (Green, 1953; Singh, 1961; IRRI, 1970); (b) nitrogen is fixed in the root zone of rice (Yoshida and Ancajas, 1971); and (c) aerobic nitrogen fixing bacteria are present on rice roots (IRRI, 1970).
C.
IRON
The most important chemical change that takes place when a soil is submerged is the reduction of iron and the accompanying increase in its solubility. Rice benefits from the increase in availability of iron but may suffer, in acid soils, from an excess. The presence of iron in natural waters is undesirable because it imparts a color and makes purification of water for domestic and industrial use difficult. Besides, it is an indication of natural or man-made organic pollution. The reduction of iron present at the soil-water interface in lake bottoms during thermal stratification of lakes has even greater implications : the mud loses its absorptive capacity and releases nutrients into the water (Section 111, C), shifting the photosynthesis-respiration balance of the lake toward the accumulation of organic matter. The reduction of iron has important chemical consequences : (a) the concentration of water-soluble iron increases; (b) pH increases; (c) cations are displaced from exchange sites; (d) the solubility of phosphorus and silica increases (Section V, F and G ) ; and (e) new minerals are formed. The reduction of iron is a consequence of the anaerobic metabolism of bacteria and appears to be chiefly a chemical reduction by bacterial metabolites (Bloomfield, 1951; Motomura, 1961) , although direct reduction coupled with respiration may be involved (Kamura et al., 1963; Ottow and Glathe, 1971). The kinetics of iron(I1) follows a roughly asymptotic course (Takai et al., 1963; IRRI, 1964). Five to 50% of the free iron oxides present in a soil may be reduced within a few weeks of submergence depending on the temperature, the organic matter content, and the crystallinity of the oxides. The lower the degree of crystallinity, the higher is the reduction percentage (Asami, 1970). Soil properties influence the kinetics of water-soluble Fe2+more drastically than that of total Fe( 11). Acid soils high in organic matter and iron
72
F. N. PONNAMPERUMA
build up concentrations as high as 600 ppm within 1-3 weeks of submergence and show a steep roughly exponential decrease to levels of 50-100 ppm which persist for several months. Low temperature retards the peak and broadens the area under it (Cho and Ponnamperuma, 1971). Soils high in organic matter but low in iron give high concentrations that persist for several months. In neutral and calcareous soils the concentration of water-soluble iron rarely exceeds 20 ppm. The increase in concentration of water-soluble iron can, in most soils, be related to the potential and pH of the Fe(OH) <-Fez+system (Ponnamperuma et d.,1967) Eh = 1.058 - 0 059 log Fez' - 0.177pH
(21)
The subsequent decrease is due probably to precipitation of Fe,(OH)s or Fe,iO,-nH,O brought about by the increase in pH following a decline in Pro, (Section V, A, 3 ) . The final stable concentration appears to be regulated by the Fe,(OH), - Fez+equilibrium with Eh
= 1.373 - 0.0885 log
FeZb- 0.Q36pH
(2%)
In ferruginous soils, the concentration of water-soluble Fez+after the peak conforms closely to Eq. (23) (Ponnamperuma, 1972) pH
+
15
log Fez+ = 5.4
(23)
For other soils the value of the constant may be as low as 4.9 (IRRI, 1967), reflecting the variability of the composition of Fe,O,.nH,O that Sillen (1966) refers to. The bulk of the water-soluble iron is present as bicarbonate and salts of the lower fatty acids, for the kinetics of iron (and manganese) parallels that of total alkalinity (HC0,- RCOO-) (Fig. 3 ) . In strongly colored solutions of reduced soils, Fe2+may also be present as complexes of humic acid (Oldham and Gloyna, 1969). The dynamics of the concentration of Fez+(and Mn*+) affects the concentrations of other cations. This is clear from the similarity of the kinetics of (Ca'+ $. Mg?+ Na+ NH,+ K+) to that of (Fez+ Mn2+) (Fig. 3 ) . But values for exchangeable and water-soluble Fe2+must be viewed cautiously because they are highly dependent on the pH of the extractant, as can be seen from Eq. (23 ) and the following equilibrium:
+
+
+
+
+
Fe8Or.nH~O Fez+F1 Fez+ (clay)
The cations displaced may be lost by leaching while the supply of Fez+ is maintained by the dissociation of Fe,O,-nH,O. On drying and oxidation, the soil is acidified (Brinkman, 1970). Water-soluble Fez+diffusing to the oxygenated soil-water interface and
THE CHEMISTRY OF SUBMERGED SOILS
73
moving by mass flow and diffusion from the surface of rice roots and to the oxidized zone below the plow sole is deposited as mottles, tubules, and nodules, respectively. The grayish color of submerged soils is attributed to iron sulfide, but I have cited evidence (Ponnamperuma, 1972) to show that the bulk of the reduced iron in most paddy soils is probably hydrated magnetite [Fe,O, nH,O or Fe, (OH) along with some hydrotroilite (FeS * nH,O) . If anaerobic conditions persist, these precipitates may age, producing the typical minerals of reduced sediments, magnetite (Fe,O,), and pyrite (FeS,). Also, Fe( 11) silicates (greenalite and chamosite), siderite (FeCO,) and vivianite [Fe, (PO,) 2 . 8H20] may form (Rankama and Sahama, 1950; Rode, 1962; Rosenquist, 1970).
D. MANGANESE The main transformations of manganese in submerged soils are the reduction of Mn(IV) oxides to Mn(II), an increase in the concentration of water-soluble Mn2+,precipitation of manganous carbonate, and reoxidation of Mn2+diffusing or moving by mass flow to oxygenated interfaces in the soil. Within 1-3 weeks of flooding, almost all the EDTA-dithionate extractable manganese present in soils, except those low in organic matter, is reduced (IRRI, 1964). The reduction is both chemical and biological and precedes the reduction of iron. The kinetics of water-soluble MnZ+reflect the influence of soil properties on the transformations of manganese (IRRI, 1964). Acid soils high in manganese and in organic matter build up watersoluble Mn2+concentrations as high as 90 ppm within a week or two of submergence then show an equally rapid decline to a fairly stable level of about 10 ppm. Soils high in manganese but low in organic matter also give high peaks but they are late and broad like those of Fez+at low temperatures (Cho and Ponnamperuma, 1971). Alkaline soils and soils low in manganese rarely contain more than 10 ppm water-soluble Mn2+ at any stage of submergence. Manganese is present in anoxic soil solutions as Mn2+,MnHCO,', and as organic complexes. Its concentration represents a balance between the release of Mn2+by reduction and removal by cation exchange reactions, precipitation, and formation of insoluble complexes. After the peak, the concentration of Mn2+(Ponnamperuma, 1972) conforms to pH
+ >& log Mn2+ + )k
log Pco, = 4.4
(94)
Patrick and Turner (1968) noted that as the content of reducible Mn of an anaerobic silty clay decreased, the content of exchangeable Mn in-
74
F. N. PONNAMPERUMA
creased. But binding at cation exchange sites does not explain the steep decline in water-soluble Mn’+ that occurs in acid soils high in Mn and organic matter. We have suggested that the steep fall in concentration after the peak is due to precipitation of MnCO, (Ponnamperuma et al., 1969b) (Section VI, B ) . MnCO, is appreciably soluble in ammonium acetate. That may explain the high values Patrick and Turner (1968) got for exchangeable Mn2+. Water-soluble Mn2+,like Fez+,diffuses from reduced soils and sediments to the oxygenated interface and is sorbed by Fe(II1) and Mn(IV) oxide hydrates and oxidized to produce Mn-rich nodules. Penetrating the oxidized zone below the plow sole of paddy soils, it is oxidized below the B , y horizon to give a B,,, horizon (Koenigs, 1950; Kanno, 1957). When a reduced soil is drained, air enters the soil and reoxidizes it. Iron(I1) being more easily oxidized than Mn(I1) is rapidly converted to Fe(II1) oxide hydrates. These precipitates may sorb Mn2+or form coprecipitates to give stains, nodules, and concretions whose composition apparently is determined by the milieu in which they are formed (Ponnamperuma el al., 1969b). Most flooded soils contain sufficient water-soluble Mn for the growth of rice, and manganese toxicity is not known to occur in flooded soils. But native or added M n 0 2 retards soil reduction and counteracts the adverse effects of excess Fez+and other reduction products (Nhung and Ponnamperuma, 1966; Yuan and Ponnamperuma, 1966). Hutchinson (1957) and Tanaka (1964) have described the oxidation and reduction of manganese in lakes. Little is known about the mineralogy of manganese in submerged soils. A search for the following precipitates may be fruitful: MnCO,, (Ca,Fe,Mn)CO,, MnS, Mn,O,, (Mn,O,,Fe,O,), and Mn,(PO,),.
E. SULFUR Sulfur is one of the most abundant elements on earth, and its transformations are of great geochemical, pedological, biological, and ecological interest. In aerated soils and waters the main transformations are ( a ) the oxidation of elemental sulfur, sulfides, and organic sulfur compounds to sulfate, and ( b ) the reduction of SO,?- and incorporation of sulfur into plant and microbial tissues. In anaerobic media, the main changes are the reduction of SO,*- to sulfide and the dissimilation of the amino acids, cysteine, cystine, and methionine (derived from the hydrolysis of proteins) to H,S, thiols, ammonia, and fatty acids (Barker, 1961; Freney, 1967). Methyl thiol has been found in submerged soils (Takai and Asami, 1962; IRRI,
THE CHEMISTRY OF SUBMERGED SOILS
75
1965), and the bad odor of putrefying blue-green algae in a reservoir has been attributed to dimethyl sulfide and methyl, butyl, and isobutyl thiols (Jenkins et al., 1967). The main product of the anaerobic transformations of sulfur is H,S and it is derived largely from SO,,- reduction (Timar, 1964; Postgate, 1965). The H,S formed may react with heavy metals to give insoluble sulfides, it may function as hydrogen donor to photosynthetic green and purple sulfur bacteria, and it may be oxidized chemically and bacterially at aerobic/anaerobic boundaries. The reduction of sulfate is brought about by a small group of obligate anaerobic bacteria of the genus Desulfovibrio, which use as the terminal electron acceptor in respiration. These bacteria use a variety of fermentation products and molecular hydrogen to reduce SO,,-, they tolerate high concentrations of salt and H,S, and function best in the pH range 5.5-9.0 (Starkey, 1966). Nitrate (Vamos, 1958) or low temperature (Cho and Ponnamperuma, 1971 ) retards the reduction. The kinetics of water-soluble sulfate (which is a measure of sulfate reduction) in anaerobic soils depends on soil properties (IRRI, 1965). In may neutral and alkaline soils concentrations as high as 1500 ppm be reduced to zero within 6 weeks of submergence. Acid soils first show an increase in water-soluble then a slow decrease spread over several months. The initial increase in SO,,- concentration is due to the release (following increase in pH) of SO,,- which according to Harward and Reisenauer (1966) is strongly sorbed at low pH by clay and hydrous oxides of iron and aluminum. Sulfate reduction follows first-order reaction kinetics with velocity constants which are several hundred times higher for alkaline soils than for acid soils (IRRI, 1965). Sulfate reduction proceeds slowly in submerged acid sulfate soils, despite their strong acidity; lime accelerates reduction considerably (Nhung and Ponnamperuma, 1966). Although large amounts of H,S are produced in submerged soils, lake and ocean muds, and anoxic waters, the concentration of water-soluble H2S may be so small that it is chemically almost undetectable (Misra, 1938; Mortimer, 1941; Hutchinson, 1957; IRRI, 1965). This is due to its removal as insoluble sulfides, chiefly FeS. Thus the solutions of submerged soils rarely contain more than 0.1 ppm H,S. But enormous amounts of H,S can accumulate in the stagnant waters of inland seas, salt lakes, and in suffer-containing waste waters (Hutchinson, 1957). When such waters mix with overlying inhabited strata, there can be a catastrophic kill of fish (Ruttner, 1963; Richards, 1965). At the pH values of most anaerobic soils and sediments the bulk of the H,S in the interstitial water is present as undissociated H,S and HS-. But the concentration of S2- is high enough to precipitate FeS. FeS may also be formed by the action of H,S on FeP04 (Sperber, 1958) and on
76
F. N. PONNAMPERUMA
crystalline Fe(II1) oxides (Berner, 1964). Finely precipitated iron sulfide is probably black hydrotroilite (FeS .nH,O) disseminated throughout the solid phase (Berner, 1964 ) . FeS nH,O is present occasionally in colloidal solution (Shioiri and Tanada, 1954). Hydrotroilite gradually changes to mackinawite and later to pyrites (FeS,) (Berner, 1964). The requirements for pyrite accumulation in a sediment are absence of oxygen, the presence of sulfate-reducing bacteria, a supply of SO,2- and fresh organic matter, and sufficient iron to immobilize the H,S produced. These conditions are found in ideal combination in deltas and estuaries in the tropics. When these sediments or “mud clays” (which are neutral to alkaline in reaction) are drained and exposed to air, the pyrites are oxidized to basic ferric sulfate and H,SO, giving acid sulfate soils or “catclays” with pH values as low as 2 (Ponnamperuma, 1972). Sulfate reduction can lead to the formation of a diametrically opposite type of soil-an alkali soil. Sodium sulfate present in arid soils may be converted to H,S and sodium bicarbonate during periods of waterlogging. This reaction decreases salinity (Ogata and Bower, 1965) and increases alkalinity (Timar, 1964; Janitzky and Whittig, 1964). Reduced lake and ocean muds accumulate insoluble sulfides and function as sinks for silver, copper, lead, cadmium, and iron (Hutchinson, 1957; Goldberg, 1965). The reduction of sulfate in submerged soils has three implications for rice culture: the sulfur supply may become insufficient, zinc and copper may be immobilized, and H,S toxicity may arise in soils low in iron. F. PHOSPHORUS
Phosphorus, like nitrogen, exists in valence states from +5 to -3. Although, thermodynamically, phosphite, hypophosphite, and phosphine can form from phosphate in anaerobic media, and their presence has indeed been demonstrated (Hutchinson, 1957; Tsubota, 1959), the main transformation of phosphorus in anaerobic media are the movements of the orthophosphate ion. Phosphorus is present in soils, sediments, and natural waters are soluble phosphates, both organic and inorganic, and as slightly soluble solids. The solids include (a) iron( 111) and aluminum phosphates, (b) phosphates adsorbed or coprecipitated with Fe( 111) and Mn( IV) hydrous oxides, (c) phosphates held by anion exchange on clay and hydrous oxides, (d) calcium phosphates, and (e) organic phosphates. Phosphates associated with iron( 111) and aluminum predominate in acid soils and sediments; calcium phosphates predominate in neutral and alkaline soils (Jackson, 1964; Stumm and Morgan, 1970). Iron( 111) and aluminum phosphates release
THE CHEMISTRY OF SUBMERGED SOILS
77
phosphate as pH increases while calcium phosphates liberate phosphate as pH decreases (Stumm and Morgan, 1970). When a soil is submerged or when a lake undergoes thermal stratification, the oxygen supply is cut off and reduction sets in (Section 111, E). A striking consequence of reduction is the increase in the concentration of water-soluble and available P. Mortimer (1941) observed a hundredfold increase in soluble P in the hypolimnion of a lake after reduction of the mud-water interface. Kaplan and Rittenberg (1963) reported that the interstitial waters of reduced sediments contain 50 times as much P as sea water and that stagnant lake and sea waters containing H,S have high P concentrations. The increase in concentration of water-soluble P on soil submergence, though appreciable, is not as pronounced as that in stagnant lake waters.
4.0
3.0
-E a
a
2.0
I .o
0
0
2
4
6
8
1 0 1 2
14
Weeks submerged
FIG.4. Kinetics of water-soluble P in some submerged soils. Soil No. 1 14 25 26 27
Texture
pH
O.M. %
Fe %
Sandyloam Clay Sandy loam Clay loam Clay
7.6 4.6
2.9
0.18
2.8
a, 18
4.8
4.4 1.5
0.18
2.0
1.60
7.6 6.6
0.50
78
F. N. PONNAMPERUMA
Besides, it is markedly affected by soil properties (Fig. 4 ) . The increases on flooding and the peak values are highest in the sandy calcareous soil low in iron, moderate in the acid sandy soil low in iron, small in the nearly neutral clay, and least in the acid ferrallitic clay. The availability of native and added P whether judged by solubility in soil-test extractants or uptake by the rice plant increases on submerging a soil (Shapiro, 1958; Mitsui, 1960; Davide, 1961; Broeshart et al., 1965; Mahapatra and Patrick, 1969). In noncalcareous soils the increase in solubility of P is associated with a decrease in Eh or an increase in Fe(I1) (Ponnamperuma, 1955; Patrick, 1964; Savant and Ellis, 1964) suggesting a role for Fe(I1I) bonded phosphates. But neither soil test data (Chang, 1965) nor P fractionation studies with submerged soils throw much light. Fractionation of P in reduced soils by the method of Chang and Jackson (1957) is unreliable unless precautions are taken to avoid the oxidation of Fe(I1) and unless a distinction is drawn between calcium bonded P and P that may, in some soils, be bonded by Fe(I1) and Mn(I1). The increase in concentration of water-soluble P when acid soils are submerged result from ( a ) hydrolysis of Fe(II1) and A1 phosphates, ( b ) release of P held by anion exchange on clay and hydrous oxides of Fe(II1) and Al, and (c) reduction of Ee(II1) to Fe(I1) with liberation of sorbed and chemically bonded P. The first two reactions are due to the pH increase brought about by soil reduction. In alkaline soils the increase in solubility of P is a consequence of the decrease in pH of these soils on flooding, for the solubility of hydroxylapatite increases as pH decreases (Stumm and Morgan, 1970). The phosphate ions released by these reactions and from the decomposition of organic matter may be resorbed by clay and hydrous oxides of A1 in the anaerobic zone (Gasser and Bloomfield, 1955; Bromfield, 1960) or they map diffuse to oxidized zones and be reprecipitated (Hynes and Greib, 1970). Noncalcareous sediments absorb and retain more P than calcareous lake sediments (Williams et al., 1970; Shukla et af., 1971). That explains why the increase in concentration of water-soluble P is small and transitory in acid clay soils compared to calcareous soils (Fig. 4 ) . Quantitative studies of phosphate equilibria in submerged soils face the following difficulties: (a) up to 60% of water-soluble P may be organic; ( b ) P may be present as soluble complexes of Ca, Mg, Al, and Fe, and as colloids; (c) the identity of the solids, which may range from adsorption products to chemical compounds, is not known; and (d) sorption may be more important than precipitation in regulating the concentration of soluble P. But the solubility of P in alkali and calcareous soils appears to be regulated by the solubility of hydroxylapatite [Ca,,( OH) (PO,,)3] and
THE CHEMISTRY OF SUBMERGED SOILS
79
in reduced acid soils by adsorption on kaolinite, montmorillonite, and hydrous oxides of Al. Vivianite [Fe, (PO,) * 8H,O] may not be involved. If vivianite with an activity product constant of 10-29.9 is in equilibrium with dissolved Fez+at pH 7.0, the total P concentration should be about M . The actual concentrations in ferruginous soils are much less (Fig. 4 ) . That explains why vivianite is found only in old sediments (Rosenquist, 1970). Phosphate is one of the main pollutants of streams and lakes, so stringent efforts are being made to minimize its escape from waste-water treatment plants. Also, much attention is focused on the role of lake sediments as sinks for phosphate. Hutchinson (1957) has reviewed the phosphorus cycle in lakes, and Martin (1970) that in the sea. Stumm and Morgan (1970) have discussed the chemistry of phosphates in surface waters and the role of hydrous oxides in phosphate equilibria. Leckie and Stumm (1970) have reviewed precipitation reactions. Ponnamperuma ( 1965) and Patrick and Mahapatra (1968) have reviewed P transformations in paddy soils.
G. SILICON Silicon occurs in soils as crystalline and amorphous silica, as silicates, of Si(OH), in equilibrium with amorphous silica at 25OC is 120-140 ppm and Mn(IV), and as silica dissolved in the soil solution. Dissolved silica is present as monomeric Si( OH) 4. The concentration of Si(OH), in equilibrium with amorphous silica at 25OC is 120-140 ppm as SiO, and is independent of pH in the range 2-9. The concentrations of silica in natural waters are much less than 120 ppm (Stumm and Morgan, 1970; Martin, 1970), and in soil solutions decrease with increase in pH up to about pH 8 (Jones and Handreck, 1967). The low concentration of silica in natural waters has been attributed to sorption of silica by hydrous oxides of Fe(II1) and A1 (Jones and Handreck, 1967) and to recombination of silica with aluminum silicates (Mackenzie et al., 1967). But the concentration of dissolved silica in interstitial solutions of river, lake, and ocean sediments are higher than those of the overlying waters (Kaplan and Rittenberg, 1963; Harris, 1967). And when lake muds undergo reduction, large amounts of soluble silica are released into the hypolimnion (Hutchinson, 1957). The concentration of silica in the solutions of submerged soils increases slightly after flooding and then decreases gradually, and after several months of submergence the concentration may be lower than at the start (IRRI, 1964). The increase in concentration after flooding may be due to the release of silica following (a) reduction
80
F. N. PONNAMPERUMA
of hydrous oxides of Fe(II1) sorbing silica, and ( b ) action of CO, on aluminosilicates (Bricker and Godfrey, 1967). The subsequent decrease may be the result of recombination with aluminosilicates, following the decrease in Pco,. H.
TRACE ELEMENTS
Although the forms of boron, cobalt, copper, molybdenum, and zinc present in soils are probably not involved in oxidation-reduction reactions, their mobility may be affected by some of the consequences of soil submergence. Thus the reduction of the hydrous oxides of Fe(II1) and Mn (IV) and the production of organic complexing agents should increase the solubility of Co, Cu, and Zn. The increase in pH of acid soils and the formation of sulfides should lower their solubility. The net result of soil submergence is to increase the availability of Co and Cu (Mitchell, 1964; Adams and Honeysett, 1964) and of Mo (Jenne, 1968) and to depress that of zinc (IRRI, 1970). Elements mobilized in reduced sediments may diffuse upward and accumulate in the oxidized surface layer (Hynes and Greib, 1970). VI.
Mineral Equilibria in Submerged Soils
In recent years, the application of equilibrium thermodynamics to the quantitative study of chemical transformations in nature has assumed increasing importance. Despite many theoretical and practical difficulties, equilibrium thermodynamics has been useful in understanding mineral associations in nature (Garrels and Christ, 1965), the composition of sea water (Sillen, 1961) and other natural waters (Stumm and Morgan, 1970), the chemical changes in submerged soils (Ponnamperuma et al., 1965, 1966a, 1967, 1969a,b), and biological oxidation-reduction reactions (Ponnamperuma, 1972). Stumm and Morgan (1970) have listed the difficulties in applying equilibrium thermodynamic concepts to the nonequilibrium conditions encountered in nature. Ponnamperuma et al. (1966a, 1967) have discussed the difficulties relevant to oxidation-reduction reactions, These difficulties are minimal in submerged soils, because submerged soils are more or less closed, isobaric, isothermal systems in which many reactions are catalyzed by bacteria. A.
REDOXSYSTEMS
Reduction of the soil is the most important chemical change brmght about by submergence. According to Table I, the reduction of the main
THE CHEMISTRY OF SUBMERGED SOILS
81
components proceeds in the following sequence: 0,, NO3-, Mn(IV), Fe(III), SO4,-, CO,, N,, and H+. Since the oxidized and reduced forms of each component coexist along with H+ ions and electrons, they may be assumed to be in equilibrium if energy barriers are absent and reaction rates are not too slow. 1 . The 0,-H,O System The 0,-H,O system dominates the redox picture in all environments exposed to atmospheric oxygen. Oxygen is such a powerful oxidant that at a Po, of 0.21 atm it should, theoretically, convert carbon, hydrogen, nitrogen, sulfur, iron, and manganese to CO,, H,O, NO3-, SO,,-, Fe,03, and MnO,. These are the stable forms of the elements in the surface oxygenated soil-water interface of submerged soils and sediments. If the system, O2 4H+ 4e = 2Hz0, dominates aerobic media, the potential (Eh) and pE (-log e ) values should conform to
+
+
Eh = 1.229
+ 0.015 log Po, - 0.059pH
(25)
pE = 20.80
- MpO) - pH
(96)
where PO, is the negative logarithm of Po,. Aqueous systems in equilibrium with air should, therefore, have a dE/dpH of -0.059 and an Eh value of 0.82 V. Experimental values of dE/dpH for aerobic soils and natural waters are of the right order, but their Eh values at pH 7.0 are considerably lower: they range from 0.35 to 0.56 V and depend on pretreatment of the platinum electrode. For this reason and also because of poor poise and low sensitivity (-1 5 mV or %pE unit for a 10-fold decrease in 0, concentration), Eh or pE is not a satisfactory measure of soil aeration. The high pE of the 0,-H,O system and its small decrease with Po, mean that very small concentrations of 0, are sufficient to maintain an environment in the oxidized state. According to Turner and Patrick (1968), the E, at which oxygen becomes undetectable is 0.33 V or a pE of 5.63. 2. The Nitrogen Systems Nitrogen and its oxidation and reduction products are abundant and widespread, .they are soluble in water, and they are in constant contact with atmospheric oxygen or electrons liberated from organic matter by microorganisms. Besides, the three main transformation of N-nitrification, denitrification, and nitrogen fixation-are oxidation-reduction reactions catalyzed by enzymes. So they are best studied from the standpoint of electron activity.
82
F. N. PONNAMPEKUMA
a. The NO,--N, System. In anaerobic media the NO,--N, which pE = 21.06
- 'ip?rTO3- + 'iopN2 - ~
~ ~ ) I I
system for (27)
should be in equilibrium with the 0,-H,O system for which pE is 13.63 at a Po, of 0.2 atm and pH 7.0. Substituting 13.63 for pE and 0.78 atm for PA,, we have 10' as the activity of NO,-. This means that nitrate is very stable in aerobic media. Equation (27) also reveals that the pE M ) in an aqueous system at pH at which NO,- is just detectable ( 7.0 in equilibrium with atmospheric N, is 11.27. At this pE, Po, is lo-'".', which explains the stability of NO,- at very low 0, concentrations even in the presence of denitrifying bacteria (Bremner and Shaw, 1958; Skerman and MacRae, 1957). But at a pE of 5.63 at pH 7.0 (the potential at which 0, becomes undetectable), the Ps2 in equilibrium with lo-, M NO,- is loG6"?. This means that NO,- is highly unstable in anaerobic media, as we have seen in Section V, B, 2. b. The iV02--N2 Svstem. The equations for this system are
+ %H+., + e = >/6Nzg+ pE = 95.69 - '$pNOz- + !'fpNv?- %pH
?$H201
(28) (99)
In aerated media the system should be in equilibrium with the 0,-H,O system and have a common pE. Combining Eq. (29) with Eq. (26) and substituting 0.78 for Ps2 and 0.21 for Po? we have p m ? - = 14.1 - pH
(30)
This equation reveals that the concentration of NO,- in equilibrium with air and water at pH 7.0 is lo-' M . Thus NO,- occurs in the merest traces or not at all in aerobic soils and waters (Hutchinson, 1957; Campbell and Lees, 1967 ) and, unlike NO,-, is denitrified even in the presence of oxygen (Skerman et al., 1958). In anaerobic media it is even more unstable. c. The NO,--NH,+ and NO,--NH,+ Systems. The equations for these systems are
+ 3$H+,, + e = l$SHqfsq + XH?01 PI? = 11.91 16pSOj- + 'QpNII1+ - 9;pH liSO1-es + .?jH+,, + e = I$SHq+Aq+ 1$H?01 PF, = 15.17 - 'dpS01- + '(pSHI+ - $$pH lgS08-,,
-
(31) (32) (33)
(34)
These equations show that in aerated media NO,-/NH,+ is i6 and NO,-/NH,- is :', dramatizing the tremendous driving force of the oxidation of ammonia to nitrite and nitrate. But at the potential of flooded soils (pE = -1 to 3 ) the concentrations of NO,- or NOJ- that can be
THE CHEMISTRY OF SUBMERGED SOILS
83
in equilibrium with NH,+ is infinitesimal. Measurements in lake waters, soils, and sediments support this prediction (Pearsall, 1938; Hutchinson, 1957; Patrick, 1960). Thermodynamically, the reduction of NO3- and NO,- to NH,+ is possible in anaerobic media. But not more than a few percent of NO,- goes to NH,+ apparently because of the tremendous driving force of the competing denitrification reactions. Plants and bacteria reduce nitrate to ammonia with the help of the powerful reductants, NADH, NADPH, and ferredoxin, generated by light or by anaerobic respiration. d . The N,-NH4+ System. The following equations reveal that a high electron activity is necessary for the reduction of N, to NH,+ %Nz, pE = 4.64
+ ?4HfaP-te
= %NH4+,,
- j4pNz + >gpNHf
- %pH
(35) (36)
At a pH 7.0 and a PNz 0.78 atm, a detectable amount of NH,+ (lo-* M ) will be present only at a pE of -2.35. The source of electrons at high activity are NADH, NADPH, and ferredoxin (Arnon, 1965; Rabinowitch and Govindjee, 1969). Thermodynamically, algae and anaerobic bacteria present in submerged soils can fix N,. Further even aerobic N, fixers should function more efficiently at a low 0, concentration as Jensen (1965) has reported.
3. The Manganese Systems Manganese is present in surface media in at least 36 oxide minerals with the metal in the valence states $2, +3, +4, and mixtures of these (Jenne, 1968). The complexity of the oxides of manganese and their variability make quantitative studies of the manganese systems difficult. Reduced soils have a pE of -1 to 3, a pH of 6.5 to 7.0 and a Pcoz of 0.1 to 0.5 atm. Under these conditions, pELpH diagrams show that the stable solid phases are Mn,O, and MnC03 (Ponnamperuma et al., 1969b). The equations for these systems are j@fn&
+ sCOng + H+,, + e = %MnCOa, + j@Mh pE = 18.57 - %pCOz - pH
(37)
(38)
The observed relationship for three reduced soils high in manganese equilibrated with different Pcoz values (Ponnamperuma, 1972) was pE
=
10.86
- I.PlpC02 - pH
(39)
The coefficients of pC0, and pH are of the right order for the Mn,04-MnCO, system, but pEo is considerably less than that for hausmannite (Mn,O,) . This discrepancy suggests that the Mn,Or present
84
F . N . PONNAMPERUMA
in these soils was far less reactive than the ideal oxide. But the value of % log Mn?+ % log Pco, was fairly constant and the expression pH equal to 4.4 suggesting that the concentration of dissolved Mn2+in reduced soils is apparently determined by the solubility of MnCO,.
+
+
4 . The Iron Systemy
We have previously reviewed the chemistry of iron and the thermodynamics of its transformations in submerged soils (Ponnamperuma et al., 1967; Ponnamperuma, 1972). In this section, I review the main equilibria. a. The Fe( OH):<-Fe?+System. The equilibrium between “ferric hydroxide” and Fe’* is given by
+ 3€If,, + e = Fez+,, -i-3H201 pE = 17.87 + pFe2+ - 3pH
Fe(OH)a,
(40) (41)
The pEo values calculated from Eh, pH, and Fez+activities of the solutions of 32 submerged soils ranged from 17.46 to 18.13 (Ponnamperuma, 1972). They were closest to the theoretical value of 17.87 for the ferruginous soils. The solubility of iron in submerged paddy soils is controlled by the F e ( 0 H ) 3-Fe2+ equilibrium. In this connection, the report of Kawaguchi and Kyuma (1969) that no crystalline oxides of Fe(II1) were present in the A horizon of Southeast Asian rice soils is noteworthy. In other soils the concentration of soluble Fez+ may be governed by minerals intermediate in properties between ferric hydroxide and hematite (Barnes and Back, 1964). b. The Fe,(OH),-Fe2+ System. The gray-green color of reduced soils, often attributed to FeS, may be due to the presence of hydrated magnetite, Fe,O,.nH,O. If this solid is present after the peak of water-soluble Fez+ in submerged soils, as solubility criteria suggest, then
The pEo values calculated from Eh, pH, and Fe activities of the solutions of 32 submerged soils after the peak of water-soluble Fez+ ranged from 21.8 to 23.5. The mean values for the three ferruginous soils in the group was 23.31. This is almost identical with the theoretical value for the Fe, (OH) \--Fez+ system. These findings suggest that the concentration of water-soluble iron after its peak is controlled by the solubility of Fe,,O,.nH,O whose thermochemical properties vary with the soil.
THE CHEMISTRY OF SUBMERGED SOILS
85
c. The Fe( O H ),-Fe3 ( O H ) System. After the formation of Fe, (OH) the following equations are relevant SFe(OH)a,
+ H+=, + e = Fes(OH)e, + HzOl
(44)
- pH
(45)
pE = 7.26
Thus the dpE/dpH slopes for the solutions of reduced ferruginous soils is -1 and pE can be derived from pH if the pEo characteristic of the soil is known (IRRI, 1966).
5. The Sulfur Systems The main sulfur systems can be described by
+ XH+., + e = JQHzS,, + > ~ H z O I pE = 5.12 - > ~ P S O + ~ ~>QpHzS - XpH + H+aQ+ e = J$HZS,,
>QSOP.,
>@rh
pE = 9.40
+
+ >$pHzS - pH
>@04z-., %H+.,
pE = 6.04
+ e = 36% + ~ $ H z O I
- >6pSOhz- - $$pH
(46) (47)
(48) (49)
(50) (51)
These equations show that SO4,- is highly stable in air, that a chemically M SO,,- solution M) will appear in a detectable amount of H,S ( at pH 7.0 only at the low pE of -3.26, and that H,S and S are readily oxidized in air to SO,". The oxidation of S and pyrites in mines and acid sulfate soils produce strong acidity.
B. CARBONATE SYSTEMS Submerged soils have all the ingredients required for carbonate equilibria: (a) high concentrations of CO,; (b) the presence of the divalent cations, Fez+,Mn2+,Caz+,and Mg2+,in most soils, CaCO, in calcareous soils, and NaHCO, in sodic soils; (c) intimate contact between solid, solution, and gas phases; and (d) virtual isolation of the system from the surroundings. Thus sodic soils behave like NaHCO,, calcareous soils like CaCO,, ferruginous soils like Fe,O,. nH,O, and manganiferrous soils like MnC03 when submerged and equilibrated with CO, (Ponnamperuma et al., 1969a).
I . The Na,CO,-H,O-CO,
System
The partial pressure of CO, and the concentration of Na+ associated with HC03-, CO,,-, and OH- determine the pH of this system according
F . N. PONNAMPERUMA
86
to the equation (Ponnamperuma, 1967) pH
= 7.85
+ log [Xa']
- log Pco? - 0.5111'2
(52)
The pH values of soils containing NaHCO, equilibrated with CO, at different Pco, values conformed closely to Eq. ( 5 2 ) (Ponnamperuma et al., 1966a, 1969a). Nakayama (1970) has derived equations for the CaC0,-NaHC0,-H,O-CO, system and proved them experimentally. 2. The CaC0,-H,O-CO, System The theoretical equations for this system (neglecting the effect of complexes) are pH = 5.99
pH
- 25
log Pco-
+ '3/
(log YIlros- - log yca2*)
+ 15 log Ca2+ + 15 log Pco? = 4.92
(53) (54)
Equation (53) can be simplified to pH = 6.03 -
?$ log Pco,
(55)
Calcareous soils, both aerobic and anaerobic, conformed closely to Eq.
(55) (Ponnamperuma et al., 1966a, 1969a; IRRI, 1966), but the slightly higher values obtained for the constant (6.1) indicated that the CaCO, in these soils was slightly more soluble than calcite. Olsen and Watanabe (1960) reported that calcareous material in soils is apparently more soluble than calcite. 3. The MnC0,-H,O-CO,System
When a manganiferrous soil is submerged, the concentration of watersoluble Mn increases rapidly, reaches a peak, and declines rapidly (Section V, D). The decrease has been attributed to the precipitation of MnCO, (IRRI, 1964, 1965; Ponnamperuma et al., 1969b). If MnCO, is present, the following equations should apply (Ponnamperuma, 1967)
3.5log Pco, + 1.5 (log y E C O 3 - - log yyn2+) + '$ log MII*++ ?5 log Pco, = 4.06
pH = 5.4% pH
(56)
(57)
When we plotted the pH values of the solutions of reduced manganiferrous soils against log Pco,, we obtained straight lines with slopes of 0.6-0.7 but intercepts of about 5.9 (IRRI, 1965). An equilibrium study in the laboratory with three reduced soils containing more than 0.3% reducible Mn yielded slopes of 0.64-0.67 and intercepts of 6.08-6.11. A plot of
THE CHEMISTRY OF SUBMERGED SOILS
87
+
pH 1/2 log Mn2+against log Pco,, in the same study, gave slopes of 0.46 to 0.53 and intercepts of 4.37 to 4.56. These data indicate that the MnCO, present in submerged soils (probably as a fine precipitate coating soil particles) is more soluble than rhodocrosite. 4 . The FeC0,-H,O-CO, System
Conditions in submerged soils seem to be ideal for the precipitation of FeCO,, and indeed siderite is found in marshes. But apparently it does not precipitate during short periods of submergence, for the solubility of iron in the solutions of submerged soils is almost 100 times that permitted by the solubility product of FeCO, (10-10.24) given by Singer and Stumm (1970). There is no satisfactory explanation of this anomaly (Ponnamperuma, 1972). Reduced ferruginous soils do not satisfy the equations for the FeC0,-H20-C02 equilibrium, but when such soils are equilibrated with CO, at different Poo, values they give a pH-log Pcoz relationship almost identical with that for Fe,O,.nH,O (Ponnamperuma et al., 1969a) namely pH = 6.1
VII.
- 0.58 log Pco,
(58)
Perspectives
Two recent developments have stimulated interest in the chemistry of submerged soils: the breeding of lowland rice varieties with a high yield potential; and the pollution of streams, lakes, and seas by domestic, agricultural, and industrial wastes. The chemistry of submerged soils is valuable (a) in understanding soil problems limiting the performance of highyielding rice varieties, and (b) in assessing the role of lake, estuarine, and ocean sediments as reservoirs of nutrients for aquatic plants and as sinks for terrestrial wastes. The chemical and electrochemical changes in paddy soils have been described adequately, but information on the dynamics of cold, cumulative, underwater lake and ocean muds is meager. In spite of the availability of a large body of infohation on the chemistry of submerged soils, the following observations have not been satisfactorily explained : (a) the difference in oxidation-reduction potential between submerged soil or sediment and the interstitial solution; (b) the release of water-soluble phosphate when a soil or sediment undergoes reduction; ( c ) the dynamics of water-soluble silica in submerged soils; (d) the
88
F . N. PONNAMPERUMA
decrease in the solubility of zinc brought about by soil submergence; (e) the supersaturation of solutions of submerged soils with respect to siderite; and ( f ) the much lower reactivity of the manganese oxides occurring in paddy soils compared to that of the pure oxides. Quantitative studies of redox, carbonate, and silicate equilibria in submerged soils are likely to yield valuable information on the dynamics of ecologically important elements. A prerequisite to these studies is mineralogical and thermodynamic research on the metastable precipitates, the complex oxides and coprecipitates, the solid solutions, and the surface-active solids present in soils that undergo alternate oxidation and reduction. The chemistry of hydrated magnetite and its role in mineral equilibria in submerged soils and lake muds require further investigation. The redox potential of the interstitial solutions of muds is an ecologically and thermodynamically meaningful parameter, so its use should be encouraged. The role of alternate oxidation and reduction in the biodegradation of detergents and pesticides merits study. A submerged soil appears to be an ideal medium for both aerobic and anaerobic nitrogen fixation, especially in the presence of rice plants. But quantitative data on the amounts fixed and methods of increasing fixation are meager. Augmenting nitrogen fixation in paddy fields can be a boon to the farmers of developing countries.
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PHYSIOLOGICAL GENETICS OF CROP YIELD D. H. Wallace, J. L. Ozbun, and H. M. Munger Departments of Plant Breeding and Biometry, and Vegetable Crops, Cornell University, Ithaca, N e w Yark
1. Introduction ................................................... 11. Identification of Genetic Variation . . . . . . . . . . . . . . . . . A. Growth Analjrsis ............................ B. Light Interception and Utilization . . . . . . . . . . . . . . . . ........ C. Net C02 Exchange and Components . . . . . . . . . . . D. Translocation and Partitioning ................................ E. Dark and Photorespiration . . . . . . . . . . . . . . . . . . . .. 111. Genetics and Heritability ................................... A. Introduction and Defi .............................. B. Yield ................................ ................. C. Relative Growth Rate and Net Assimilati .............. D. Leaf Area ........................... .............. E. Leaf Angle and Light Interception ............................ F. Net CO, Exchange Rate . . . . . . . . . . . . . . . . . . . .... ...... G. Stornatal Number and Resistance .............................. H. Enzyme Activity ............................................ I. Dark and Photorespiration . . . . ................... J. Conclusions Relative to Heritabi IV. Relative Importance of Physiological Components .............. V. Using Genetic Differentiation for Elucidation of Physiological and Biochemical Pathways . . . . . . . . . VI. Summary and Applications in Plant Breeding . . ................ References .....................................................
I.
97 105 119 123 124 125 126 127 127 129 129 130
132 138 142
Introduction
Spectacular gains in economic yield of certain crop varieties have been achieved by exploiting major genes whose presence could be identified by morphology or disease reaction. Less spectacular but larger in number have been yield improvements resulting from empirical recombination of individually unrecognized genes. Recent studies confirm the expectation that varieties differ extensively in the physiological processes determining yield. 97
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Identification of these physiological components of yield and their genetic controls should make it possible to plan crosses to maximize segregation of genotypes possessing the physiological complementation and balance required for high yield, thereby leading to more rapid and predictable yield improvement. Efficient development of high-yielding varieties requires cooperative effort of many disciplines. Benefits from cooperation between plant breeders and plant pathologists have long been recognized. Disease control by chemicals or preferably by genetic resistance is essential before genes conditioning optimum physiological activity for high yield can be expressed. The same holds for insect control. Statistical genetics discerns gene action as additive and dominance effects without identifying the physiological activities controlled by the genes. Variation in physiological activity and its genetic control can be ascertained by cooperative effort involving plant physiologists, plant geneticists, statistical geneticists and plant breeders. This paper will review reports of genetic variations in physiological components of yield which have been recognized for only about 30 years, and genetic and inheritance studies dating back only about 10 years. Research with dry beans, Phaseolus vulgaris L., at Cornell will serve as a central example illustrating the objectives, problems, and progress. The work was begun in 1954, using varieties differing in yield by as much as 30% (Table I ) . Marrow, yelloweye, and pea classes of dry bean were each represented by two varieties with respective yield differences of 21, 20 and 9%. Other varieties were sometimes studied. The goal was to determine physiological bases for yield differences and how these physiologicalgenetic variations could be used in breeding higher yielding varieties. Understanding of the physiological bases of yield with any crop is, with modifications, applicable to all crops. T.-WLE T Relative Seed Yields of Seven Dry Bean Varieties as Determined over 6 Years from 18-20 Yield Trials
Class of dry bean Marrow
Variety PERRY M A R R O W
CORNELL
7-16
Yellow eye
YELLOWEYE
Pea
MICHELITE
STEUBEN
MONROE
Red kidney
XED KIDNEY
Relative yield 100 141 9s 111 111 1?1 108
Harvest indes
Yield advantage
%
%
GO St GO
41
Gr?
20
55 57 57
9
PHYSIOLOGICAL GENETICS OF CROP YIELD
II.
99
Identification of Genetic Variation
A. GROWTH ANALYSIS Watson ( 1952) reviewed growth analysis procedures. More recently, Radford ( 1967) reviewed the mathematics, assumptions, and acceptable usage, and Eagles ( 1971a) used additional mathematical procedures. With growth analysis, mean total accumulated plant weights (W; also called biological yield), mean leaf areas (L) , and mean dry weights of the different plant organs including weights of economically important organs (economic yield) are obtained at the beginning and end of a time period of plant growth. These are used to calculate relative growth rate (RGR; dry weight accumulated per unit of plant dry weight per unit of time), net assimilation rate (NAR; dry weight accumulated per unit of leaf area per unit of time), leaf area ratio (LAR; leaf area per unit of plant dry weight), leaf area index (LAI; leaf area per unit of land area), leaf area duration (LAD; leaf area integrated over time), harvest index (HI; economic yield divided by total plant dry weight X loo), crop growth rate (CGR; dry weight accumulated per unit of land area per unit of time), and other measures of capacity and efficiency of growth and yield. Growth analysis usually ignores weights and role of roots since accurate data are difficult to obtain. That variation in root systems constitutes an important physiological-genetic component of yield is, however, suggested by findings that roots of high-yielding semidwarf winter wheats (Triticum vulgare) are heavier, longer, and extend to lower soil depths than roots of older varieties (Lupton and Bingham, 1970). Khan and Tsunoda ( 1 970a) found cultivated and spring wheats to have larger root systems than wild (Triticurn and Aegilops species) and winter wheats, respectively. Watson (1952) reviewed particularly contributions of leaf area ( L ) and net assimilation rate (NAR) to biological yield (W; symbolized as A W when it refers to W accumulated over a period of time). From wheat, sugar beet (Beta vulgaris), and potato (SoEanum tuberosum) plantings over several years he concluded that varietal, fertilizer, and seasonal effects on economic yield were highly correlated with variation in L but not with NAR. Similarly, for both marrow and yelloweye beans the higher yielding variety usually had about 10% more L than the lower-yielding one (Fig. 1 ) . Such data suggest that varietal differences in biological and economic yield are mediated by variation in L , particularly since NAR differences were inconsistent and generally nonsignificant. That L of pea bean seedlings was about 60% of that of marrow bean seedlings, and was as much as 80% larger (Wallace and Munger, 1965) during late growth indicates, however, that not all variations in biological and economic yield were accounted for by correlation with L. These conclusions are typical of at-
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OZBUN, AND H. M. MUNGER
0.8
0.701 30
40
50
60
70
80
DAYS AFTER PLANTING
FIG. 1. Logarithm of leaf area in square decimeters at 5 sampling dates for 2 varieties of each of 3 dry bean types: -, line connecting midpoints of marrow type; s, PERRY MARROW; e, CORNELL 7-16. ---, Line connecting midpoints of pea type; *, MICHELITE; e , MONROE. ...., Line connecting midpoints of Yelloweye type; YELLOWEYE; B,STEUBEN. (From Wallace and Munger, 1965.)
a,
tempts to relate L to varietal differences in yield (Rhodes, 1971). Change of the pea beans during growth from smallest to largest L, with simultaneous reversal from smallest to largest W (Fig. 2) is related to findings (Elmore et al., 1967) that interspecies differences in U' were related to relative leaf growth rate (RLGR). Across the bean varieties, neither L nor RLGR was highly correlated with economic yield. The higher RLGR of the pea beans is closely related to their much higher leaf area ratio (LAR; Fig. 3 ) . The LAR = L / W . LAR influences
101
PHYSIOLOGICAL GENETICS OF CROP YIELD
RLGR because a AW for a variety with high LAR is accompanied by a larger addition to L than the same AW for a variety with low LAR. The LAR also indicates the relative amount of L supporting a unit of W, i.e., photosynthetic capacity per unit of respiring and growing tissue. The function of LAR is further indicated in that LAR and NAR are physiological components of RGR (RGR = NAR x LAR). Absolute differences in LAR between bean varieties were almost constant throughout growth, but the LAR of all varieties declined steadily, so LAR of the pea beans as compared with the other bean classes was about 15% higher for seedlings but 50% higher during late growth (Fig. 3 ) . Thus, the higher RGR of the pea bean varieties (Fig. 2) resulted in part from their higher LAR.
30
40 50 60 DAYS AFTER PLANTING
70
80
FIG.2. Logarithm, at 5 times after Rlanting, of the total dry weight in grams of the aerial shoot for 2 varieties of each of 3 dry bean types. Symbols as for Fig. 1. (From Wallace and Munger, 1965.)
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D. H . WALLACE, J. L . OZBUN, AND H. M. MUNGER
30
40
50
60
70
80
DAYS AFTER PLANTING
FIG. 3. Leaf area ratios at 5 times after planting €or 2 varieties of each of 3 dry bean types. Symbols as for Fig. 1 . (From Wallace and Munger, 1965.)
Blackman (1950) found that differences in RGR among three strains of bluebell were accounted for primarily by variation in LAR. Similarly, Khan and Tsunoda (1970b) found differences in RGR among wheats and related species to correlate with LAR. On the other hand, Wilson and Cooper (1970a) found LAR and NAR of ryegrass (Loliurn perenne and L. muitiflorum) selections to be negatively correlated, minimizing effectiveness of selection for either factor. In contrast to above examples where LAR was the main physiological component correlated with genetic differences in RGR, Wilson and Cooper (1969a) found differences in NAR to account for variation in RGR among ryegrass populations. Similarly, Eagles ( 1967) reported that genetic control of NAR accounted for a difference in RGR between two populations of orchardgrass (Dactylis gfomerala). Eagles (1971 a,b) later demon-
PHYSIOLOGICAL GENETICS OF CROP YIELD
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strated that varied temperature and light intensity, which caused population X temperature and population X light intensity interactions for RGR, respectively, conditioned RGR primarily by changes of LAR and NAR. He also showed (Eagles, 1969, 1971a,b) that relative contributions of LAR and NAR to RGR varied with physiological stage of plant development, giving population X sampling date interactions. Jones (1971) found changes in leaf thickness in beans (see Section 11, C), caused by temperature or other environmental factors, to result in similar variety and sampling date interactions. Loach (1970) found NAR during late, but not early, growth to be correlated with yield of sugar beet varieties. Such population x sampling date interactions, plus usual field variability and need for many replicates, and possible stage of plant development x environment interactions were probably why consistent differences in NAR were not found among the bean varieties studied (Fig. 4). The pea beans clearly had the higher RGR (Fig. 2). Their high LAR would contribute to the higher RGR, but that leaves of the pea bean varieties have the highest net CO, exchange rate (Section 11, C) implies that a difference in NAR should also have been detected. The pea beans did have the highest, but not significantly so, NAR as seedlings (Fig. 4 ) . A statisti-
90
I
* 1
LSD
26- 39
39 -'52
52:66
DAYS AFTER PLANTING
FIG. 4. Net assimilation rates, milligrams of dry weight accumulated per square decimeter of leaf area per day, at 3 times after planting for 2 varieties of each of 3 dry bean types. Symbols as for Fig. 1. (From Wallace and Munger, 1965.)
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D. H. WALLACE, J . L . OZBUN, AND H. M . MUNGER
cally significant difference was only observed for the last sampling period, when the pea beans had the lowest rather than highest NAR. During this growth period the pea beans had the largest L, and NAR is often negatively correlated with L (Watson, 1958), presumably because mutual shading increases as L increases. The examples indicate that genetic differentiation in RGR occurs, and that it arises because of genetic differences for either or both of its major physiological components, LAR and NAR. Both components, and consequently RGR, are influenced by environment and change relative to each other with stage of plant development (time). Therefore, varietal differences in NAR are difficult to identify. Watson's (1952) conclusion that variation in N AR is inconsequential relative to variation in L undoubtedly resulted from greater difficulty in measuring NAR than L, and in accounting for changes of NAR with plant development and environmental influence. This also accounts for a greater number of examples of genetic variation in L than NAR. The absolute increment of W ( h W ) for any time period is h W = NAR X L , and RGR = NAR X LAR [ = N A R X ( L / W ) ] . All are essential and important physiological factors, and all have genetic variability. In growth analysis literature, L is frequently expressed as leaf area index (LAI), i.e., as the number of units of leaf area above one unit of land area, since economic interest rests in productivity per unit land area. Similarly biological yield is expressed on a land area (and time) basis as crop growth rate (CGR) . There are numerous reports that biological yield is more closely related to leaf area duration (LAD) than L (Welbank er al., 1966). The LAD integrates L over time, or, simply stated, it equals active L x time. The time span of active photosynthesis by leaves, represented by the LAD, is an obvious component of biological yield (AH'). It integrates genetic control of early vs late maturity (Takahashi and Yasuda, 1971; Murfet, 1971). It also integrates responses to environmental factors such as photoperiod (Chang et al., 1969) or temperature, which affect rate and duration of photosynthesis directly, or indirectly by triggering or preventing flowering, bulbing, tuber formation, or simply photosynthate partitioning among the different vegetative organs of the plant. Genetic variation for response to photoperiod, temperature, vernalization, nutrition, etc., are all components of the physiological genetics of yield. Donald (1962), Wallace and Munger (1966), and Singh and Stoskopf (197 1) present evidence that genetic improvement in economic yield of several crops derives in part from higher percentages of biological yield being partitioned to the plant organs constituting economic yield. The ratio of economic yields to biological yield is called the harvest index (HI).
PHYSIOLOGICAL GENETICS OF CROP YIELD
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In the early 1900’s, for example, HI of wheat varieties was 32% (Van Dobben, 1962); for current high-yielding dwarf wheats it is 49% (Thorne et al., 1969). HI of wheat is positively correlated with economic yield but negatively correlated with biological yield (Singh and Stoskopf, 1971 ). Improved HI represents increased physiological capacity (often termed sink power or sink capacity) to mobilize photosynthate and translocate it to organs having economic value (see Section 11, D ) . The bean varieties have large differences in H I (Table I ) (Wallace and Munger, 1966), ranging from 55 to 64%. Variety Charlottetown has a still higher HI of 67%, but its relative yield of 56% (compare Table I) is lowest because early maturity results in low leaf area duration (LAD).
B. LIGHTINTERCEPTION AND UTILIZATION A major weakness of growth analysis as outlined (Section 11, A) is that the role of light energy is not evaluated. It was concluded that variations in biological and economic yield result from variation in L and LAR, and to a lesser extent from variation in NAR, but most growth analysis studies have not considered relationships of these physiological yield components to interception and utilization of radiant energy. This omission is serious since energy input is essential to photosynthesis and constitutes an extremely variable environmental factor. Effects of light intensity and quality on photosynthesis of single leaves and in theoretical considerations of photosynthesis in the field have been extensively considered, but data on energy interception, absorption, and utilization by field grown plants are scarce, particularly with varietal comparisons. This arises because of scarcity and cost of equipment for measuring radiant energy, and because equipment for field studies is not readily available which fully separates ultraviolet and infrared radiation from photosynthetically active (visible) radiation. Emecz (1962) suggested that growth analysis data be expressed on an accumulated light energy rather than on a time basis. Hayashi (1966, 1967, 1968, 1969) presents the most complete comparison of energy use by different varieties. He combines conventional growth analysis of rice (Oryza sativa) with measurements of radiant energy incident above and transmitted through the leaf canopy. The average energy (measured in calories and accumulated over several days, weeks, or the entire growing season) reaching a unit land area beneath the rice leaf canopy was subtracted from that incident above the canopy to give the calories intercepted by the leaf canopy. The percentage of incident energy intercepted [ ( 100 X intercepted calories) /(total incident calories)] was called the efficiency of interception ( E i ) . Efficiency of utilization (E,,) of intercepted energy by each variety was obtained by converting A W for
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the same time period and land area to its calorie equivalent (aW x 4000 = calories of energy stored in the biochemical products of photosynthesis; see Lieth, 1968) and dividing this by the number of intercepted calories. Varietal variation in E , reflects primarily differences in LAI, but integrates effects of leaf size and vertical vs horizontal leaf orientation on light interception. E,, is partially equivalent to NAR, expressing photosynthetic rate per unit of intercepted light while NAR expresses it per unit of leaf area E , x E , gives the efficiency of crop growth ( E c ) , which mathematically also equals 100 X AW expressed in calories and divided by total calories of incident radiant energy. E , is roughly equivalent to both CGR (crop growth rate) and RCiR of growth analysis, but the latter are expressed, respectively, on a per unit land area and per unit W basis (in addition to per unit time) while E, is expressed per unit of total incident radiant energy. Figure 5 , which summarizes Hayashi's varietal comparisons of light energy use, is complex and difficult to interpret, but careful study of it and Fig. 6 will greatly assist in comprehending the scope and complexity of yield physiology. E , is plotted against E , for thin, medium, and dense stands of each of five varieties. Since E , X E,, = E,, E , , values of 0.7, 0.8, 0.9 . . . 1.4% are each identified by diagonal dashed lines. Any point on each dashed line represents a specific pair of E , and E,, values whose product is the given E , value. Each E, value also represents a given biological yield (total dry weight), as indicated in the figure, since E , = A W x 4000/total incident radiant energy. Varietal differences in light energy use are illustrated by comparing
FIG.5. Total dry matter production in relation to E , , E,, and E , of NORIN 8 (N8), 22 ( N 2 2 ) , NORIN 29 (N29), KINMAZE (Kin), and KUSABUE (Ku). 0, Thin; A , medium; 0, dense planting. (From Hayashi, 1966.)
NORIN
PHYSIOLOGICAL GENETICS OF CROP YIELD
46
-
321 30
107
-
2kO 300 $50 LSD 15%)
800
tc-----(
I
J
1000 1200 1400 Total dry weight (gm-*)
FIG.6 . Ear dry weight in relation to total dry weight and harvest index of NORIN 8 (N8), NORIN 22 (N22), NORIN 29 (N29), KINMAZE (Kin), and KUSABUE (KU). 0, Thin; A, medium; 0, dense planting. (From Hayashi, 1966.)
varieties N22 and Ku with variety Kin. At all three planting densities (thin, medium, and dense) Ku has higher E i than N22, but N22 usually has a much higher E,, so their E, values and corresponding W differ little. At medium and dense plantings, variety Kin has a much higher AW (biological yield) (which results from both high E i and high E , ) than either N22 or Ku. Biological yields of these varieties are clearly shown to result from different inputs of Ei (efficiency of energy interception) and E , (efficiency of utilization of intercepted energy), two physiological components of net photosynthesis. It will be shown that the varietal differences in E i and E, depend in turn on morphological and biochemical differences conferred by genetic differentiation. Figure 6 shows that economic yield (ear dry weight) of the rice varieties was dependent upon both biological yield and harvest index (see Sections 11, A and XI, D) . At medium and dense populations the AW (Fig. 5 ) of N29 and Ku were much lower, especially at high density, than that of Kin. However, because of higher harvest index N29 produced about the same economic yield. Thus, variations in light interception and utilization, and in harvest index, accounted for variation in economic yield among the five rice varieties. Hayashi attributed the high E , of Kin, particularly with dense planting to its better arrangement of leaves. Kin had more erect leaves which resulted in a smaller light extinction coefficient, i.e., in less complete light interception by the uppermost leaf area and more interception of incident radiant energy by the lower leaf canopy. Tsunoda (1959) was among the first to indicate an advantage for erect leaves. He found that rice varieties with good economic yield response to fertilizer had shorter, more erect and thicker leaves than nonresponding varieties. Murata ( 1961) then related
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D. H. WALLACE, J. L . OZBUN, AND H. M. MUNGER
this plant habit to a lower light extinction coefficient, as compared to varieties with taller and more horizontal leaves, and indicated that the reduced light interception by the uppermost leaf surface resulted in more equal distribution of incident light energy over total leaf area and therefore in high NAR. These relationships were verified at the International Rice Research Institute (IRRI), discussed in its first and second annual reports (IRRI, 1963, 1964), and further elucidated in technical reports (Tanaka et nl., 1964; Tanaka et al., 1966). IRRI rice breeders selected short, erect, thick, dark-green leaves and short slender stems (IRRI, 1964). This resulted in IR8 (IRRI, 1967) and other high-yielding tropical rice varieties (IRRI, 1970); it is probably the best available example of utilizing yield physiology in the breeding of higher yielding varieties. These varieties can, with adequate fertilization and environment, give 3 - 4 times the yield of older tropical varieties. Although the plant habit described is controlled primarily by a single recessive gene (Section 111, E) , it increases yield via several physiological pathways. First, as indicated above, erectness of leaves results in more uniform distribution of incident radiant energy over total leaf area and, therefore, in higher NAR. In turn, the better light distribution permits a very high LA1 without a decrease in crop growth rate as occurs in other varieties (IRRI, 1969). One way of achieving high LA1 is to fertilize; a second pathway is therefore that these varieties are very responsive to nitrogen fertilization. Closely related is that the shortness, erectness, and thickness of leaves largely eliminates lodging caused by fertilizer additions to older and taller varieties, and that mean net CO, uptake per unit leaf area is generally correlated with leaf thickness (Hayashi, 1968; Takano and Tsunoda, 1971) and with nitrogen content per unit leaf area (Takano and Tsunoda, 1971). A third pathway is mobilization and partitioning of a larger proportion of total photosynthate to the seed as indicated by a higher harvest index. It should be noted that in mixed populations of tall and short plants, the tall plants intercept the most light and give the highest yields; the short plants are poor competitors, and it is only in pure stands that their advantageous yield physiology is expressed (Jennings and Aquino, 1968). Seniidwarf habit has also been used to breed wheat varieties giving high yield in many countries (Reitz and Salmon, 1968; Syme, 1970). AS with rice, responsiveness to nitrogen, freedom from lodging, and high harvest index are physiological aspects contributing to the high yield (Thorne et al., 1969; Thorne and Blacklock, 1971; Syme, 1970; Khan and Tsunoda, 1970b). It can be assumed that light distribution over the leaves is also more uniform (Tanner et al., 1966). Recent studies implicate high photosynthetic rates of single leaves (Khan and Tsunoda, 1970c,d,e) and parti-
PHYSIOLOGICAL GENETICS OF CROP YIELD
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tioning of more photosynthate to the roots (Lupton and Bingham, 1970; Khan and Tsunoda, 1970a; see Sections I and 11, D) as additional contributing physiological factors. Photoperiod insensitivity has been bred into both the wheat (Syme, 1970) and rice varieties (IRRI, 1967), and is a physiological-genetic component of yield, since by controlling flowering it controls both harvest index and time required to achieve maturity, i.e., leaf area duration. Thorne and Blacklock (1971) found as much variation among as between semidwarf and tall varieties and emphasize that, while semidwarfness may contribute certain advantages, the older and taller wheat varieties also differed in harvest index, lodging, light interception, etc. Further, genetic capability for yield has improved over the years and some tall varieties yield as well as the new semidwarfs. Berdahl, Rasmusson, and Moss (1972) studied the effect of leaf size in barley using F5 selections from the second backcross of crosses between parents with large and small leaves. NCE was similar in the laboratory so the larger leaves had higher total photosynthesis. Similarly, for field canopies, there was no consistent difference in NCE of large and small leaves, but canopies with large leaves had the larger LA1 and hence the higher total photosynthesis. Photosynthesis was limited by light at all observed incident radiation intensities. Consideration of leaf angle has given similar, but also contradictory results in corn (Zea mays). A backcross-derived isogenic single cross hybrid carrying the liguleless gene for erect leaf produced 40% more grain than its counterpart with more horizontal leaves (Pendleton et al., 1968). Later studies by Sinclair (1971) have failed to show this effect. Pendleton et al. also found that tying the leaves of a commercial corn hybrid to support them in a more vertical position gave increased yield. In a reverse test of this phenomenon Tanaka et al. (1969) found that horizontal positioning of the leaves of upright-leaf rice varieties, achieved by attaching weights to the tips of the leaves, decreased yields. In comparison with the Gramineae species discussed above, dicotyledonous plants have more horizontal leaves with consequent higher light extinction coefficients (Newton and Blackman, 1970; Verhagen et al., 1963). Tsunoda (1959) and Watson and Witts (1959) were among the first to compare light interception of varieties of dicotyledonous species. Watson and Witts found that sugar beet varieties with upright leaves had higher NAR than varieties with prostrate leaves. Tsunoda found that sweet potato (Zpornea batatas) and soybean (Glycine m u ) varieties with good economic yield response to high fertilizer rates had relatively thick, small leaves. Further, the leaves were positioned near the central axis of the plant so that the canopy did not completely fill the space between rows, and vertically oriented leaves were most responsive. Varieties with large, thin hori-
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D. H. WALLACE, J. L. OZBUN, AND H. M. MUNGER
zontal leaves and leaves distant from the central axis had poor response. Thus, responsive varieties have thick leaves with a leaf arrangement permitting good penetration of light into the leaf canopy and a consequent low light extinction coefficient. Vertical leaf orientation permits LA1 to be large without causing excessive mutual shading of leaves. Singh et al. (1968) found light interception by the soybean variety WAYNE to be greater than that of HAROSOY 63 and attributed it to larger leaves and higher LAI. Hicks et ai. (1969) found with near isogenic soybean lines that light penetrated farther into canopies with narrow than wide leaflets, but this was not correlated with economic yield. Another mechanism to explain varietal differences in light interception of dicotyledonous plants has been observed in soybeans (Kawashima, 1969) and in beans (Dubetz, 1969). Kawashima found that soybean leaves move in response to direction and intensity of the brightest light source. Upper leaves respond most, but all leaves are somewhat reactive. Individual leaflets bend at their pulvinus (point of attachment to the petiole) upward, downward, and/or sideways. Upper leaves orient at about 60° to horizontal on bright days so that only about 50% of the radiant energy that a leaf could intercept is intercepted. The remainder enters the canopy and can be intercepted by lower leaves. The leaves, especially the upper more responsive ones, tend to follow the sun. This maximizes distribution of available radiant energy over total leaf area, and efficiency of light interception over time. On overcast days when the brightest light source is not so distinct, the leaves assume a less vertical orientation. Wien and Wallace (1971 ) have found bean varieties to differ in responsiveness to this phenomenon. Looking down on the leaf canopy of Red Kidney, the leaves are oriented so that only 2-3 layers of leaves can be seen. There are additional layers, but they are obscured from view, and from direct sunlight, by the mostly horizontal upper leaves. In contrast, many layers of leaves can be seen within the canopy of responsive lines because the leaves, more so at the top of the canopy and decreasing with depth, are vertically oriented as described by Kawashima. Comparison of responsive and nonresponsive varieties is most striking when leaf canopies are viewed from the direction of the sun. It is possible that high responsiveness to this phenomenon will confer some of the same physiological advantages to dicotyledonous species that is conferred by short erect leaves in rice, wheat, and other monocotyledonous species. Incident radiant energy will be more uniformly distributed over total leaf area and the light extinction coefficient will be low with a resultant increase in NAR. With highly responsive varieties, it may be possible to plant at higher densities or to get better response from fertilizer, neither of which gives significant economic yield increases with current dry bean varieties. The lower light extinction coefficient may permit crop
PHYSIOLOGICAL GENETICS OF CROP YIELD
111
growth rates to remain high even at the high LA1 brought about by high fertilizer rates and dense populations. Wien and Wallace (1971) found the pulvinus to be the photoreceptor for this phenomenon. Leaves turn up when the pulvinus is illuminated on top and down when it is illuminated from below. Light energy intercepted by the leaf blade has little effect. Wien (1971) used the methods of Hayashi, except that he sampled light interception weekly rather than continuously, to compare efficiency of light interception (Ei)and light utilization E, of bean varieties RED KIDNEY and BLACK TURTLE SOUP at Cornell, and RED KIDNEY and GREAT NORTHERN in the Philippines. Varietal differences in E , were not large and, because of differences in angle of leaf orientation, did not correlate precisely with LAI. In correlation with changes in LAI, there was a time x variety interaction, with RED KIDNEY intercepting slightly more light during early growth and GREAT NORTHERN and BLACK TURTLE SOUP intercepting the most during later growth. E, varied inconsistently from one sampling date to another, but was highest for RED KIDNEY when averaged over time at any point in the growing season. The data indicate that meaningful results require, because leaf angle varies with light intensity as discussed above and perhaps with moisture stress, continuous measurement of light interception rather than sampling at intervals of time. C. NET CO, EXCHANGE AND COMPONENTS Growth analysis of beans (Section 11, A ) indicated that MICHELITE had both higher RGR and LAR than other varieties. Since RGR = LAR X NAR, the difference in RGR could all be accounted for by the difference in LAR. But it seemed likely that a difference in NAR also contributed since, although not statistically significant, the NAR of MICHELITE tended to be higher during early growth than NAR of the other varieties (Fig. 4). Higher NAR could in turn be caused in whole or in part by a difference in net C 0 2 exchange rate (NCE) of individual leaves, or by leaf orientation and consequent light distribution over leaf area as discussed in Section 11, B. Possible contribution of NCE was evaluated using infrared CO, analysis, and it was found (Izhar and Wallace, 1967) that MICHELITE-62 (M-62), a mosaic-resistant MICHELITE, had an NCE 9 % to 3 1% higher than four other varieties. Compared to RED KIDNEY, the NCE of M I C H E L I T E - ~was ~ about 25% higher at all light intensities (Fig. 7). Since 1967 there are additional reports of varietal differences in NCE of single attached leaves. Irvine (1967) measured 14C0, uptake and reported a 2.5-fold range among 10 sugarcane (Saccharurn oficinarurn) varieties. Ojima et al. (1970) reported differences of about 20% among
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D. H. WALLACE, J. L. OZBUN, AND H. M. MUNGER
5.3
16.0
26.7
37.4
48.0
Kilolur
FIG.7. Net co, exchange at 5 different light intensities for and MICHEI.ITE-62 (I@-@). (From Izhar and Wallace, 1967.)
RED KIDNEY
(.-a)
soybeans. Heichel and Musgrave ( 1969a) reported 3-fold differences among 27 corn inbreds and varieties, and Dreger et al. (1969) reported that three soybean varieties had a common mean NCE 4% to 23% higher than 6 other varieties. Dornhoff and Shibles (1970) reported that the highest NCE among 20 soybean varieties exceeded the lowest by 48%. Khan and Tsunoda (1970e,e) found in two experiments that the semidwarf wheat variety MEXIPAK took up CO, at rates 26% and 36% higher than variety C273; four other wheat varieties had intermediate rates. This work and that of Evans and Dunstone (1970) indicate that in the evolution of wheat NCE has decreased as leaf area has increased, the increase in leaf area being sufficient that total photosynthesis and economic yield have also increased (Khan and Tsunoda, 1970b,c). The high NCE of MEXIPAK reverses this trend for reduced NCE. Apel and Lehmann (1969) compared 1 15 barley (Hordeum vulgare) varieties from many geographical origins, using detached leaves. They found seven modern high-yielding varieties to have high NCE rates. There are more reports of varietal differences in NCE, some of which will be cited elsewhere, but sufficient have been given to suggest that genetic variability in NCE exists in all species. Except for the study of barley by Apel and Lehmann (1969), all above rcports of varietal differences in NCE were for single, intact, attached leaves, using either infrared CO, analysis or **CO, uptake measurements. The difference in NCE between M-62 and RED KIDNEY has been observed many times but results are erratic when detached leaves or leaf disks are used. Contrariwise, Wilson et al. (1969) found NCE of attached and detached leaves of ryegrass to be highly correlated, although absolute values for detached leaves were 60% lower immediately after excision and 20%
PHYSIOLOGICAL GENETICS OF CROP YIELD
113
lower after a recovery period. They concluded that infrared gas analysis, 14C0, measurement, and manometric measurements with a Warburg apparatus which can use only leaf segments, all measure the same phenomenon. Different responses of bean and ryegrass leaf segments probably indicate different causal mechanisms for the varietal differences in NCE. This review indicates that there are many physiological and biochemical pathways by which genetically controlled differentiation of NCE can arise. It is therefore expected that each specific pair or set of varieties, for comparisons both within and between species, will interact differently with environmental factors and experimental methods. Other investigators have compared NCE of varieties, using whole-leaf canopies enclosed in. gas-tight air-conditioned chambers rather than single attached leaves or leaf segments. Using infrared CO, analysis, Norcio (1970) showed that the leaf canopy of rice variety IR8, which has short erect leaves, had higher NCE at medium and high light intensities than a tall variety with drooping leaves (Fig. 8). Further, IR8 required a higher light intensity for light saturation. Similarly, Rosario ( 1967) found varietal differences among sugar cane varieties. It is instructive to compare interpretations of varietal differences in NCE obtained with single attached leaves and with leaf canopies. Environmental factors can be precisely defined and duplicated time afer time when single attached leaves are accurately positioned in small leaf chambers. With leaf
E 16
4
8
12
16
20
24
LIGHT ENERGY (gm-col/rq c m / m i n )
FIG. 8 . Photosynthetic rates of varieties with light saturation rates at 15 g-caI/cm' per minute. 0-0,C4-63, Y = 1.9484X - 0.0621X2, Ra = 0.90; 0-0, Peta, Y = 1.6857X - O.O539P, R2 = 0.97; A-A, BPI-76, Y = 1.2270X - 0.0306X2, R2 = 0.47. (From Norcio, 1970.)
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D. H . WALLACE, J . L. OZBUN, AND H. M. MUNGER
canopies when using an artificial light source, each leaf will receive an amount of radiant energy dependent upon its distance from and angle with respect to the light source. If sunlight is used as the source, effects of varied leaf angle and mutual shading remain. In addition, incident energy changes with time of day and fluctuates wildly with changes of cloud cover. Therefore many readings followed by regression analysis, using light intensity as the independent variable, are required to get valid means. Measurements with single attached leaves are easier to get, to repeat, and to analyze. Mean NCE rates of single leaves of different varieties, obtained with the leaves at a fixed angle (usually 90°) and distance relative to the light source, indicate the relative genetically determined maximum NCE obtainable with all leaves uniformly exposed to light. That is, they indicate relative genetic potentials for photosynthetic rates when differences among varieties in mean leaf angle and uniformity of light distribution over total leaf area, as actually occur in the field, are eliminated by experimental technique. On the other hand, these effects of leaf angle and light distribution are fully integrated into the NCE obtained from leaf canopies. The relative varietal NCE of leaf canopies is therefore more comparable to NAR values obtained by growth analysis (Section 11, A ) and should approximate photosynthesis rates occurring in the field. These NCE values must, however, because of unwanted environmental changes caused by the chamber enclosing the leaf canopy, be for instantaneous time, or for short periods of a minute, hour, or day. On the contrary, NAR and E , , E,,, and E, are obtained from unaltered field canopies and must be the mean rate of a relatively long time period, i.e., a day, week, or as long as 2 weeks for NAR and the entire growing season for E , , E,,, and E, . Since genotypic differences in NCE have been observed, it is timely to ask what physiological factors (components of NCE) cause them. One cause could be stomata frequency. When stornatal number of M-62 and RED KIDNEY were compared it was found, however, that the lower epidermis of RED KIDNEY leaves had 40% more stomata per square millimeter than M-62 leaves (Izhar and Wallace, 1967). When the variety REDKOTE, a disease-resistant RED KIDNEY, was substituted, M-62 again had fewer stomata but higher NCE (Martin, 1970). This agrees with the finding of Heichel (1971b) that a corn inbred with high NCE had fewer stomata than an inbred with lower NCE, and with data of Miskin et al. (1971; Miskin and Rasmusson, 1970) showing no correlation between stornatal frequency and NCE of five barley populations. It contrasts with the finding of Moss (1971 ) that NCE of other barley lines is correlated with stomata number. It is the resistance imposed by stomata to COL diffusion from air into intercellular space within the leaf which actually controls NCE (Jackson
PHYSIOLOGICAL GENETICS OF CROP YIELD
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and Volk, 1970; Heath, 1969). This resistance depends on number and also on extent and size of stomatal openings. Clark et al. (1941) found stomata of some high-yielding corn varieties to open earlier in the morning and stay open later in the afternoon than stomata of lower-yielding varieties. Additional evidence that stomate opening and closing is genetically controlled is presented by Tal ( 1966) ; rapid wilting was caused by a different recessive gene for each of three tomato mutants. All three genes prevented closure of stomata. A similar potato mutant was identified by Waggoner and Simmonds ( 1966). Bjorkman et al. (1971) showed that NCE differences among Atriplex roseu, A . patula, and their F, hybrid were not correlated with stomatal resistances. Further, 0, concentration altered NCE rates differentially without affecting stomatal resistances. In contrast, Holmgrem ( 1968) found the higher NCE of a hybrid clone of SoEidago virgaurea L., when grown at high irradiance, to be associated with lower stomatal resistance of hybrid than parental leaves. All these comparisons, except the latter one, were determined by measuring water vapor losses (transpiration) through the stomata rather than CO, exchange. Resistance to CO, diffusion is difficult to measure directly since CO, influx (photosynthesis) and efflux (respiration) proceed simultaneously and CO, concentration at the chloroplast cannot be measured accurately. It is of interest to compare C, and C, species (see Sections 11, E and V). C, species consistently have higher NCE in spite of higher stomatal resistances (Downes, 1970) as indicated by transpiration data. A favorable consequence for C, species is higher dry weight accumulation per unit of water transpired. Stomata are but one of numerous anatomical, morphological, physiological, and biochemical characteristics of a leaf which control rate of CO, movement from air to the chloroplast within the leaf and thereby influence NCE. Resistances to CO, flow beyond the stomata are frequently lumped into one category called rnesophyEZ resistance, although varietal differences in some components of mesophyll resistance are sometimes compared. After moving past the resistance to diffusion presented by stomata, CO, must first diffuse through intercellular space within the leaf, then through the air-water interface presented by the mesophyll cell walls, then through the cytoplasm of mesophyll cells, and finally move into the chloroplasts. Initial and continuing rates of CO, fixation by the chloroplasts will also depend on levels of enzymes essential to photosynthesis, their specific activities and their affinity for CO,. The continuing rate will also depend on the rate at which photosynthate is moved out of the chloroplast and translocated to active sinks. Relative contributions to control of NCE by each of the components of mesophyll resistance is difficult to ascertain.
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D. H. WALLACE, J. L. OZBUN, AND H. M. MUNGER
Quantity and specific activity of chloroplast enzymes are sometimes determined as are relative rates of translocation, but this is usually done without concurrent assays of other components of total mesophyll resistance to CO, fixation. Instances where genotypic comparisons, usually qualitative rather than quantitative have been made for one or more of these component resistances will be cited below. Most studies of mesophyll resistance have considered only one genotype or made comparisons between rather than within species. However, Holmgren (1968) showed that the difference in NCE of clones of Sotidago virgaurea L. from exposed and shaded habitats, when both were preconditioned to high irradiance, was correlated with differences in both mesophyll and stornatal resistance. Treharne and Eagles ( 1970) found differences in mesophyfl resistance to be partially responsible for NCE differences between races of orchardgrass, and Heichel (1971 b) drew the same conclusion for corn inbreds. An aspect of mesophyll resistance, which has not been quantitized in terms of actual resistance, is positive correlation of NCE with leaf thickness. For example Pearce et al. (1969) found NCE rates of 13 alfalfa (Medicago sativa) clones to be correlated with specific leaf weight (leaf dry weight per unit leaf area) which largely reflects leaf thickness. The correlation held when specific leaf weight was altered by light intensity. Tsunoda (1959) and Murata (1961) earlier reported a similar relationship in rice. Tsunoda (1959) and Takano and Tsunoda (1971) emphasized positive correlation between leaf thickness and plant response to nitrogen fertilization in rice, and wheat (Khan and Tsunoda, 1970d,e, 1971). Hayashi (1968) found that the thicker a rice leaf, the larger the increase in NAR with an increase in light intensity, and decrease with decrease in light intensity. Varieties with thick leaves had the highest light saturation points and accompanying highest NAR. M-62 and the other varieties studied (Table I) provide an exception to this general relationship. MICHEL I T E - ~has ~ lower specific leaf weight (Wallace, 1958) but higher NCE (Izhar and Wallace, 1967). Wilson and Cooper ( 1969b,c) also point out that specific leaf weight cannot be used reliably to estimate relative NCE of different genotypes. They found certain ryegrass genotypes with high NCE to have thinner leaves; these leaves had a larger number of smaller mesophyll cells. Number and size of mesophyll cells is closely related to leaf thickness and resistance to CO, movement toward the chloroplast and thereby on NCE. In comparisons among rather than within species, El-Sharkawy et ai. (1965) found the ratio of internal exposed cell surface to cell volume to be correlated with NCE. Using both inter- and intraspecies comparison of Brassica, Sasahara ( 1971) showed that NCE was correlated with total
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PHYSIOLOGICAL GENETICS OF CROP YIELD
cell surface per unit leaf area. High total mesophyll cell surface and high NCE were, in general, associated with small mesophyll cell size. Wilson and Cooper ( 1970a) reported similar correlations between mesophyll cell size, NCE and seedling growth of ryegrass populations. Ryegrass is crosspollinated and therefore heterogeneous, and the same relationships were found among genotypes within populations. Further, selection for small mesophyll cells generally resulted in progenies with smaller cells, greater dry weight accumulation, and higher NAR and NCE than selection for large cells. We previously indicated that genotypes with thick leaves have higher yield, higher NCE, and greater response to nitrogen fertilization. Murata (1961) and Osada and Murata (1965) have observed that rice genotypes with high nitrogen content per unit of leaf area have high NCE. Khan and Tsunoda (1970d,e, 1971) showed the same relationship among wheat
-
d:
3
0
I4
0
0
0.2
17
0.4
0.6
Nitrogen content per leaf area (mg/cm2)
FIG.9. Relationship between specific leaf weight (dry weight: leaf area) and leaf areal nitrogen content (nitrogen content: leaf area). 0 , November 27-December 18, r = +0.952; 0, December 18-January 8, r = +0.897. Both significant at 1%. (From Khan and Tsunoda, 197Oa.)
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13. H. WALLACE, J. L. OZBUN, AND H. M. MUNGER
genotypes and demonstrated (Fig. 9) that specific leaf weight (leaf thickness) and nitrogen content per unit leaf area are highly correlated. Osada ( 1965) found NCE of some rice varieties to be increased by nitrogen, while other varieties did not respond. Since enzymes are proteins, and proteins contain much of the plant nitrogen, leaf nitrogen content may be indicative of overall enzyme levels. Either quantity or specific activity of any enzyme essential to component processes of photosynthesis and photosynthate utilization may be limiting. If limiting, the enzyme will control photosynthetic rates. Ribulose- 1,Sdiphosphate carboxylase ( RUDPase) , located in chloroplasts, is a key enzyme in the Calvin-Benson (C,) photosynthetic pathway and accounts for about half of total leaf protein. It catalyzes attachment of CO, to 1,5-ribulose diphosphate. After showing RUDPase activity to be higher in sun than shade-adapted species, Bjorkman (1968) found the same relative difference in activity for ecotypes within Solidago virguurea. Evolutionary response to environment has given clones from exposed habitats a genetically determined, higher capacity to synthesize RUPase than clones from shaded habitats. Similarly, Eagles and Treharne (1969; z
-
%
RudP CARBOXYLASE ACTIVITY (CPM x I O - ~ )
FIG. 10. Ribulose- 1,Sdiphosphate carboxylase activity and relative areas under sedimentation peaks from several different tomato genotypes. Each point on the graph represents a separate assay from a different tomato genotype. Most of the genotypes included here represent mutant phenotypes used as genetic marker stocks. The solid line represents normal specific enzyme activity. The dashed lines represent differential enzyme activities found in 4 mutant forms. For further details, see text. (From Andersen el a / . , 1970.)
PHYSIOLOGICAL GENETICS OF CROP YIELD
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Treharne and Eagles, 1970) found with populations of orchardgrass from Portugal and Norway that differences in NCE, caused by both genotype and light intensity, were accompanied by corresponding levels of RUDPase activity. Huffaker et al. (1970) found RUDPase activity of a temperaturesensitive alfalfa mutant to be limiting in leaves grown at 10°C. Above 18OC, there was a large increase in RUDPase activity. A single recessive tetrasomic gene permitted synthesis in leaves grown at high temperatures but prevented it at 10°C. In contrast to these examples of genetic control over quantity of RUDPase synthesized, Andersen et al. (1970) found four of sixty mutant tomato genotypes to differ, not in quantity but in specific activity per unit of RUDPase protein. Two had enzymes with high specific activity and catalyzed more CO, fixation per unit protein than normal, and two catalyzed less (Fig. 10). Altered specific activity suggests change in molecular structure of the RUDPase, a possibility supported by different electrophoretic mobilities of the enzymes with high and low specific activities. Closely related to these examples of within-species differences in quantity and quality of RUDPase, is the finding of Treharne and Cooper (1969) that RUDPase of corn, a C, species, has a temperature optimum about 10°C higher than RUDPase of temperate gramineae (C,) species, and a later report that RUDPase of the strain of orchardgrass from Norway is more active at low temperature than that of the strain from Portugal (Treharne and Eagles, 1970).
D. TRANSLOCATION AND PARTITIONING Neales and Incoll (1968) reviewed evidence for and against control of NCE by relative accumulation or translocation of photosynthate from the leaf. There are indications that NCE is controlled by feedback signals generated by photosynthate accumulation, and therefore depends on number and capacity of metabolically active sinks, i.e., the capability for using photosynthate as energy for growth, as structural components, or partitioning it to storage in molecular forms that do not limit NCE by feedback signals. Additional evidence that rate of translocation influences NCE is the finding of Hofstra and Nelson (1969) that photosynthate is translocated more rapidly and completely from leaves of C, than C, species. Similarly, Liu (1970) found that photosynthate was translocated more rapidly and completely from M-62 than RED KIDNEY bean leaves (Fig. 1 1) . Thus, for both inter- and intraspecies comparisons, NCE rates were correlated with speed and extent of translocation of photosynthate from the leaf. In Section 11, A evidence was reviewed that RGR is sometimes con-
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D. H. WALLACE, J. L. OZBUN, AND H. M. MUNGER 0
M-62 o RK
0
1
2
3
4
5
6
7
8
TIME [ h r l
FIG. 11. Percent "C remaining in leaf at different times after labeling.
0,RED KIDNEY. (From Liu, 1970.)
0, M-62;
trolled by relative leaf growth rate (RLGR) and leaf area ratio (LAR). Varietal variation in RLGR and LAR represent genetically controlled differences in partitioning of photosynthate. Some varieties have superior capacity to mobilize photosynthate and translocate it to the plant organs where it will most effectively further growth (Slatyer, 1970). An important aspect of differential partitioning of photosynthate is genetic control of harvest index (HI) discussed in Section 11, A. High HI represents capacity (often termed sink power or sink capacity) to mobilize photosynthate and translocate it to the plant organs having economic value. Genetic variation in partitioning of the photosynthate produced after flowering or initiation of growth of economically important organs may be a more exact indicator of sink capacity. Almost nothing is known of the hormonal or other physiological mechanisms through which genetic control of partitioning is mediated. Such data are needed to explain variation in HI, RLGR, dark and photorespiration, etc. Determining the physiological basis of sink capacity is a current frontier of the physiological genetics of yield (Watson, 1968). Lupton (1969) used 'TO, to measure both NCE of wheat and rates of translocation to the ears. The combined data gave estimates of varietal yields in reasonable agreement with measured economic yields. Evans and Dunstone ( 1970) found modern wheats to have seeds as puch as 24 times larger than species considered to be progenitors of wheat. This increased
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sink size was accompanied by more rapid translocation of photosynthate to the seed and by longer duration of translocation and grain growth. Cross-sectional phloem area of these wheats and relatives is correlated with rates of translocation, although it is also affected by environmental factors such as vernalization (Evans et al., 1970). Khan and Tsunoda (1971) found NCE to be negatively correlated with distances between vascular bundles of leaves of wheat varieties and related species. Takeda and Fukuyama (1971 ) found the same relationship among other Gramineae species. Allison (1971) found that corn hybrids translocated a higher fraction of the photosynthate produced after flowering to the ears than the inbred parents. High-yielding sugar beet varieties translocate a larger proportion of their photosynthate to roots than low-yielding ones (Loach, 1970).
E. DARKAND
PHOTORESPIRATION
It is because respiration and photosynthesis proceed simultaneously in plant leaves that photosynthetic rates have been discussed as net rates, i.e., net assimilation rate (NAR) or net C 0 2 exchange (NCE). Early investigators assumed that respiration in the light and dark were equivalent. Recent studies, reviewed by Goldsworthy (1970) and Jackson and Volk ( 1970), divide the plant kingdom into apparent-photorespiring and nonphotorespiring species, and show that at least part of photorespiration proceeds via biochemical pathways activated by light and not functioning during respiration in the dark. Dark respiration in this review refers to CO, release through stomata in the dark and photorespiration (Jackson and Volk, 1970) to CO, release through stomata in the light, regardless of the biochemical pathways by which CO, is released and 0, consumed. Nonphotorespiring species (monocotyledonous species Zea mays, Sorghum bicolor, and certain other tropical grasses, and dicotyledonous species of the genus Amaranthus) all fix CO, via a metabolic pathway in which 4rather than 3-carbon compounds are the first product of carboxylation. The C, species have higher NCE rates, especially at light saturation, than C, species. Assuming that lack of photorespiration represents the ability to partition photosynthate to leaf organelles where it will best further growth and thereby confer these high NCE rates, several investigators have searched for within-species variation in photorespiration. It has also been assumed that the near-zero CO, compensation point (0-5 ppm) of C, species is one measure of photorespiration. Low compensation point presumably reflects ability of C, species to reduce CO, concentration at the carboxylation cite (chloroplast) to 0-S ppm, in contrast to a limit of about 60 ppm indi-
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cated by the higher compensation points of C, species. This would give differentials in C 0 2 concentration in air outside the leaf and at the chloroplast of about 315 ppm for species with low compensation points and about 260 ppm for high compensation point species. This difference of about 20% in CO, gradient could account for higher NCE rates of low compensation (C, species), even when stomata1 resistances are higher (see Section 11, C ) . Heichel and Musgrave (1969b), who found differences among corn genotypes, present the only report of significant within-species variation in compensation point. Moss and co-workers (Moss, 1970) have tested many genotypes within many species, including Zea mays, and failed to find lines with CO, compensation points intermediate between those cited for C, and C, species. Similarly, Dvorak and Natr (1971) found no differences among wheat varieties. Zelitch and Day (1968) reported differences in photorespiration rates of two tobacco genotypes, as measured by "CO, release of leaf disks to a C0,-free atmosphere. Low '*CO, release (low photorespiration) was correlated with high NCE. Similarly, the higher NCE of M-62 bean leaves, using attached leaves rather than leaf disks, is correlated with a lower 14C0, release than RED KIDNEY leaves (Marlin et al., 1972). Carlson et al. (1971) used the difference between the highest rate of CO, release, measured immediately after turning off the light, and the steady state release rate in the dark to show that two clones of orchardgrass differed in estimated photorespiration rates. Dark respiration, unlike photorespiration, is not a component of NCE; the definition given above for photorespiration excludes it. It is a negative component of NAR. Dark respiration is frequently correlated with NCE, whether variation in NCE is caused by environmental factors or genetic control (Thomas and Hill, 1949; Gaastra, 1959; McCree and Troughton, 1966). As an example of genetic control, dark respiration of M-62 is higher than that of RED KIDNEY and REDKOTE (Izhar and Wallace, 1967; Martin, 1970). In contrast, Heichel ( 1 9 7 1 ~ )compared two corn inbreds and concluded that low dark respiration accounted for the faster dry weight accumulation of one. This inbred partitioned a larger fraction of its phorosynthate to structural components of growth, and less to dark respiration. Wu (1971 ) found that dark respiration rate did not differ for another pair of corn inbreds that differed in NCE. Martin (1970) found that the correlation between dark respiration and NCE rates of 122 F, plants from the cross M-62 x REDKOTE was low ( r = 0.21) and nonsignificant. Plant means of both NCE and dark respiration differed significantly, suggesting independent genetic control of NCE and dark respiration rates. On the other hand, Khan and Tsunoda (1970d) found NCE and dark respiration rate of 20 varieties of Triticum and Aegilops to be significantly correlated (r = 0.58, p. 3 12). As discussed in Section 111, I, positive
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vs negative benefit from partitioning photosynthate to dark respiration is dependent on the extent of coupling to energy transfer and growth.
Ill.
A.
Genetics and Heritability
INTRODUCTION AND DEFINITIONS
Until now, this review has concentrated on evidence that genotypes within species exhibit variation in the many physiological components of yield. The evidence indicates that genetic variation exists for most if not all components. To the extent that data are available, we will now review genetic mechanisms controlling these variations and their heritabilities. Understanding genetic mechanisms and heritabilities will assist with intelligent use of physiological components in plant breeding. Before proceeding it is necessary to deiine genetic mechanism and heritability. For genetic mechanism the intent is to indicate whether observed variation is controlled by one, two, or more recognized genes, or by numerous unrecognized genes as for quantitative inheritance. For both few (qualitative) and many (quantitative) genes we will, when data are available, indicate if gene action is overdominant (the F, exhibits a phenotype more extreme than that of one of the parents; i.e., positive or negative heterosis is expressed), dominant (the F, exhibits the phenotype of one of the parents), additive (the F, exhibits a phenotype intermediate between the parents), or intermediate between dominant and additive. Heritability is divided into broad and narrow sense heritabilities (Allard, 1960) and is normally expressed as a percentage. Broad sense heritability (BSH) indicates the percentage of the total variation among plants of a population (usually among F, plants or F, progenies because genetic segregation, i.e., variation, is maximized in these early filial generations) that is caused by genetic influence. The remaining variation is of environmental origin. In statistical-genetics terms, using the F, generation as an example, P, +F,)]/V,,, where V,, is the total variance BSH = [V,, - V(P, among plants of the heterogeneous (many genotypes represented) F, population and V(P, P, F,) is the pooled among-plant variance of the three homogeneous (all plants of the same genotype) populations, the parents P, and P,, and their F,. High and low BSH, respectively, indicate much and little genotypic variation within the population. Narrow sense heritability is derived, directly or through variance component analysis, from regression of F, progeny on F, parent or F, progeny on F, parent, etc. It estimates effectiveness of the mean of a plant or progeny (parent,
+
+ +
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I). H.
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L. OZBUN, AND H. M. MUNGER
F2, F,, etc.) in predicting its progeny mean. Low NSH indicates that performance of selections will be relatively ineffective in indicating progeny performance and that large populations with progeny tests and extensive statistical procedures are required to ultimately identify superior genotypes. High NSH indicates that superior genotypes can be identified from among relatively few progenies (if BSH is also high) and that the progenies will perform like their parents. BSH and NSH indicate completely different aspects of heritability and tend to be negatively correlated (Kempthorne, 1957). BSH indicates the proportion of genetic as contrasted with environmental determination of variation in phenotype and thereby the probability that progenies of selected plants will differ from each other. In contrast, NSH indicates the accuracy with which the mean of a selected plant (or progeny or population) predicts the mean performance of its progeny. Overall effectiveness of selection depends upon both potential for selected progenies to differ (BSH) and ability to predict the progeny performances (NSH). See Section 111, J for further conclusions regarding heritability. B.
YIELD
Yield has long been classified as a character controlled by quantitative genetics i.e., one influenced by many genes with the effects of indivdual genes normally unidentified (von der Pahlen and Goldberg, 1971). This review also demonstrates that yield is a complex character; its expression depends on functioning and interaction of many physiological component processes, especially the limiting components which vary with variety. A simplest possible description of the genetics of yield is to assume that each physiological component is controlled by a single gene. The minimum estimate of gene number controlling yield is then the number of physiological components. However, as shown, particularly in Section 11, C where many components of NCE were discussed and, as will be seen in this section (111), identified physiological components (complex components) can often be further subdivided into subcomponents, i.e., components of components. These are also physiological components of yield. A maximum estimate of gene number affecting yield is to assume that all genes of a plant affect yield. This maximum estimate is probably closer to reality than the minimum estimate. Almost any gene which affects photosynthesis or partitions photosynthate by diverting it from one metabolic pathway to another, which includes almost all genes, influences yield to some extent. These concepts agree with conclusions of Grafius (1959), Williams and Gilbert (1969), and Malborn (1969) that genes do not exist for yield
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per se. Genetic control is indirect, through control of the physiological components which interact to give economic yield (Adams and Grafius, 1971; Thomas et al., 1971).
c.
RELATIVE GROWTHRATEAND NET ASSIMILATION RATE
Relative growth rate (RGR) is a complex physiological component of yield. Varietal differences in RGR must therefore be explained in terms of interactions of its less complex components ( L , W , and NAR; RGR = L / W X NAR), and their subcomponents. High biological yield must (unless initial W ,i.e., seed or embryo weight is larger) result from high RGR. Studies of the physiological basis of heterosis provide most of the available information on inheritance of RGR (Whaley, 1952; Allison, 1971 ) . Heterosis arises via numerous physiological mechanisms, frequently including inheritance of the RGR of the faster growing parent and sometimes overdominance for RGR. At other times seed or embryo weight is primarily responsible. However, small seeds frequently exhibit higher RGR than larger seeds, and reciprocal differences in F, seed size, dependent upon maternal parent, complicate the inheritance studies. Additional complicating factors include changes in RGR during germination, and vegetative, reproductive, and final (just prior to maturity or senescence) growth. Resultant variety x time interactions make interpretation difficult as already noted for NAR and other components of growth analysis (Section 11, A). Kheiralla and Whittington (1962) and Peat and Whittington (1965), used covariance on variance regression to minimize these problems. They reported, respectively, on inheritance of RGR in F,, and F, and F, populations from all possible crosses among five tomato varieties. Inheritance of RGR was largely additive with appreciable dominance; i.e., hybrids tended to have RGR intermediate between their parents but closer to that of the parent with highest RGR. Heterosis of RGR was only apparent during initial and final growth, the latter resulted from slower decline of RGR on approaching maturity. These authors concluded that NAR varied too erratically and extensively with time to permit genetic analysis. Besides variety x stage of growth interactions, variation in incident radiation under field conditions was a major factor causing this variation. The destructive sampling required, with adequate guard plants and consequent large space requirement, also make it difficult to study inheritance of NAR and RGR using controlled environments. Allison (1971) grew two pairs of corn inbreds and their Fl’s in pots, out of doors using sand culture. He found both NAR and RGR of the hybrids to be heterotic, exceeding
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values of the parents by about 1 5 % . NAR of hybrids decreased less rapidly after flowering than that of inbreds. D.
LEAF AREA
Leaf area ( L ) , a major physiological component of yield and RGR, is itself a complex character. Its immediate components are leaf number and leaf size. Duarte and Adams (1963) crossed a bean variety having few large leaves with another having many small leaves. The F, had the leaf number of the many leaved parent and leaf size intermediate between parents; i.e., gene action for leaf number was mostly dominant while action for leaf size was mostly additive. Interaction of these components gave overdominance (heterosis) for L ; mean L of the F, was 80% larger than L of the parent with largest L . F2 segregation indicated quantitative inheritance. Fowler and Rasmusson (1969) studied inheritance of L of flag leaves and the two adjacent lower leaves of barley. Mean L of F,’s was usually intermediate to parental means. NSH obtained by regression of F, progeny on F, individual plant means ranged from 18 to 73%. Edwards (1970) made all possible crosses between four lines derived from a population of Loliurn perenne. One was selected for large leaf size, a second for small size, a third for fast rate of leaf appearance, and a fourth for slow rate. Data from F, and F2 generations indicated entirely additive genetic control for rate of leaf appearance, duration of elongation of a single leaf and for time interval between maturation of a leaf and unfolding of the next leaf on the same side of the apex. Nonadditive effects for total leaf area, individual leaf size and its components length and width, and in rate of leaf elongation were associated with tendencies toward heterosis. Rate of total leaf area formation and leaf size exhibited the most heterosis. These characters have the most components, suggesting that heterosis was due to interaction between components of complex characters. Selection for either individual leaf size or rate of appearance of new leaves was accompanied by a negative response in the other. This was shown to occur because connecting the vascular system of the stem to that of an unfolding leaf, which occurs at a specific developmental stage, permits that leaf to begin rapid growth and simultaneously inhibits further growth of lower leaves on the same side of the apex. Edwards and Emara ( 1970) also crossed Loliurn rnuftifioruin selections and obtained BSH and NSH values for length, width, and area of leaves. BSH values were 40-70%, while NSH ranged from 7 to 40%. Lupton et al. (1967) found BSH of L and its components to vary in wheat from nil to 70%. Hsu and Walton (1970) found components of L of wheat to have highly significant additive gene effects and various degrees of dominant effects.
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127
LEAFANGLEAND LIGHTINTERCEPTION
The short stiff upright leaf habit of rice is controlled by an apparent single gene (Aquino and Jennings, 1966). This unique gene has many pleiotropic (simultaneous) effects; the recessive allele gives a reduced number of short internodes, shorter, wider, and more erect leaves, and a larger number of shorter panicles (Morishima et al., 1967). This gene has been responsible for spectacular yield increases (Section 11, B) because of better distribution of light over total leaf canopy surface, better nitrogen response, and higher harvest index (Jennings and Aquino, 1968). Tall and short phenotypes are easily distinguished, but both are altered by modifying genes, so that short plants range from very short to intermediate height. Similar genetically controlled modifications exist for the other characters controlled by the major gene. Morishima et d. (1967) found BSH of 9435% for the genetic variation imposed by the modifying genes. The same habit exists in other rice germ plasm, but is controlled by quantitative genes with marked environmental influence (Aquino and Jennings, 1966). The simultaneous control over several characters suggests that the major gene may actually be a block of genes which behave as a single gene because of tight linkage. Control by a single gene corresponds with high heritability. Barker (1970) found NSH of leaf angle in three barley crosses, in advancing from F, to F, to vary from 20 to 40% when compared on an F, plant basis and from 30 to 60% on an F, family basis.
F. NET CO, EXCHANGE RATE Ojima ,and Kawashima (1970) presented the most complete data showing genetic segregation in crosses between parents with high and low NCE. Differences among soybean parents and derived F, progenies were highly significant at saturating or near saturating light intensities (Fig. 12). F, means ranged from above and below the parents, most being intermediate. This agrees with bean data from crosses of M-62 X RED KIDNEY (Izhar and Wallace, 1967) and M-62 x REDKOTE (Martin, 1970). There was significant variation in mean NCE of F, plants and highly significant variation among F, progeny means. F, means ranged from near the low to near the high parent. Both the soybeans and beans segregated as expected for quantitative inheritance. Both studies used infrared analysis of CO, uptake of single attached leaves and demonstrated more than adequate sensitivity for identifying NCE differences among plants and progenies of segregating populations. McDonald (1971) presented data for gross photosynthesis of rice, but indicates similar results for NCE. F, variation was large and significant
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--
nu
-)cI-
c
N
E 30
0
U
w
** ** **
P//A*
I
20
1
30
-1 40
Light intensity
FIG. 12. Light-photosynthesis curves of Ft lines. *, and 1 % , respectively. (From Ojima and Kawashima, 1970.)
**,
Significant at 5%
at high light intensities and much less with low light. F, rates were heterotic, i.e., higher than the high parent, which contrasts with the intermediate F, means for M-62 x RED KIDNEY, M-62 x REDKOTE, and for crosses between Mitnulus ecotypes (Hiesey et al., 1968) and Phalaris species (McWilliam et al., 1969). Heichel and Musgrave (1969a) and Wu (1971) also reported positive heterosis for F, NCE rates in corn as did Holmgren (1968) for crosses between Solidago ecotypes, but Bjorkman et al. (197 1) found negative heterosis in Atriplex crosses. The F, of A . patula X A . roseu had much lower NCE than either parent. Hiesey et al. (1968) and Bjorkman et af. (1971) present limited data showing F, segregation. Ojima and Kawashima (1970) found correlation of F, progeny means with parental F L plant means to be low, indicating low NSH. Similarly, in advancing from the F, to F, generation, NSH of the M-62 X REDKOTE cross was only 7 % (Martin, 1970). BSH estimates among F, plants and F, progenies were both 6 7 % . McDonald (1971) found BSH of rice crosses to be 60% or higher. Wilson and Cooper (1970b) found BSH of 70-90% among progenies of a 6 X 6 half-diallel cross in ryegrass and NSH ranging from 17% to 71%, with most values being about 60%. The lowest NSH was from variance component analysis and the highest from regression of F, mean on midparent value. Ryegrass is cross-pollinated and parental populations are heterogeneous, while beans are self-pollinated and homogeneous, which imposes differences in methods for obtaining
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NSH. In the opinion of the reviewers, the NSH estimates of 60% and above are higher than would be obtained in advancing from F, to F, or F, to F, generations (see Section 111, J ) . High NSH would also be obtained for the regression of midparent value on F, means of the bean crosses studied, since the F, means were intermediate and close to midparent values; high values would not have been obtained from the rice and corn crosses exhibiting F, heterosis.
G.
STOMATAL NUMBERAND RESISTANCE
Heichel (1972a) found an F, corn hybrid, from a cross between inbreds with low and high stomata number to show dominance for low epidermal cell number per unit of leaf area and partial dominance for low stomatal number. F, and backcross segregation suggested control of both by few rather than many genes. Martin (1970) obtained similar data from the bean cross M-62 X REDKOTE. Because stomate number per unit leaf area varies with leaf expansion, Martin expressed the data as stomatal index (stomatal number/stomatal number plus epidermal cell number). The F, showed dominance and overdominance (heterosis) , respectively, for stomatal indices of upper and lower leaf surfaces. Stomata1 index, as indicated above, is a complex character with components of stomatal and epidermal cell number. The dominance and overdominance for stomatal index actually resulted from interaction between components, from dominance of small number of epidermal cells and from stomatal number intermediate between the parents. BSH for both upper and lower stomatal indices were about 60%, and the respective NSH values for advancing from F, to F, were 25 % and 11% . Statistical analysis indicated highly significant differences in mean stomatal index among F, lines and among plants within some F3 lines. Instances of single gene control over stomate opening and closing were cited in Section 11, C.
H. ENZYME ACTIVITY Studies of Huffaker et al. ( 1970) and Andersen et al. (1970) were cited in Section 11, C, as respective examples of single gene control over quantity of RUDPase synthesized in alfalfa and quality (specific activity per unit of protein) of RUDPase in tomato. Keck et al. (1970) found plastoquinone pool size to be larger in plants heterozygous for a single gene conditioning chlorophyll deficiency with the double recessive being lethal, than in the normal genotype. Turnover time for plastoquinone is considered to be the rate-limiting reaction for electron transport in the light mediated energy transfer processes of photosynthesis. On a chloroplast protein basis, the
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heterozygous genotype had photophosphorylation rates 1.5-2.5 times faster than normal homozygous plants. Hageman et ul. (1967) reported that corn inbreds differed in photophosphorylative activity and F, hybrids had intermediate or lower activities than the parents. I.
DARKAND PHOTORESPIRATION
Martin (1970) presents data on respiration rate for the bean cross M-62 x REDKOTE. He found the F, to be intermediate between parental rates. F, plant means differed at the 0.01 level of significance, as did F, progeny means and plant means within some F, progenies. BSH was 26%, and NSH was only 3%. The results indicate that infrared CO, analysis is sufficiently sensitive to be used in selection, but the low NSH indicates that selection in the F, is almost completely ineffective. That high dark respiration ratc is associated with high RGR and NCE in the parental bean varieties, while low rate was correlated with these same factors in some corn inbreds (Heichel, 1971c), and dark respiration rates of another pair of corn inbreds differing in NCE were equal (Wu, 1971) clearly indicates that this rate is not an overriding factor. It is but one among many factors that influence RGR and NAR and it may or may not be limiting. It also indicates that further information is needed relative to coupling of respiration to phosphorylation, energy transfer, and growth before one can say whether high dark respiration rate is desirable or undesirable and use it as a selection criterion. Segregation for these and related factors could be responsible for the low NSH. Indications of intraspecies variation in photorespiration are very recent. The only study of genetics known to the reviewers is for the interspecific cross A triplex rosea X A . putula (Bjorkman et al., I971 ) . The results are presented in Section V because they contribute toward ultimate understanding of the role of photorespiration. .f.
CONCLUSIONS
RELATIVETO HERITABILITY
Many of the estimates for BSH were close to 60%; a few were near 20% or above 70%. This clearly demonstrates genetic control over the physiological components of yield and opportunity to select for desired levels of expression. The low values indicate difficulty in controlling environmental influence and reducing it so that genetic effects are effectively isolated. Estimates of NSH were also variable. Many were 20% or lower, and only a few as high as 60%. Comparison of cited NSH values is complicated because they were from both self- and cross-pollinated species; some
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were derived from regression of F, mean on midparent value, some from regression of F, progeny on F, plant means, some from regression of F, progeny on F, plant means, etc., some from regression of progeny on progeny means, and all were under different environments. The F, generation is most heterozygous and therefore segregation within progenies is most extensive in F, and decreases on the average, because heterozygosity decreases, with each succeeding generation derived from self-fertilization. Correspondingly, because the F, generation has the most segregation it is most heterogeneous, and progenies derived from self-fertilization of individual plants are, on the average, less and less heterogeneous (more homogeneous) in each successive generation. A consequence of decreasing segregation and heterogeneity with advance from F B to later generations is that BSH is highest in the F, and decreases as heterogeneity decreases. A corresponding consequence is that NSH is lowest in advancing from the F, to F, and F, to F,, because segregation is greatest, and becomes higher with each succeeding generation because segregation decreases and homogeneity increases. When heterogeneity within a progeny has been eliminated, i.e., when the parent of that progeny is homozygous rather than heterozygous for all relevant genes, NSH will approach 100% and BSH will be 0%. Selection will then, in correlation with the high NSH, be highly effective in terms of predicting progeny performances because they will be almost identical with parental performance, but the consequence of selection within a progeny will be nil because all subsequent progeny performances will be identical as a result of homogeneity. NSH is causally correlated negatively with extent of heterozygosity and consequent segregation, while BSH is causally correlated positively with the extent of heterozygosity and consequent heterogeneity. Accuracy of determination of both is much affected by the extent of environmental influence on phenotypic variation. Environmental influence affects accuracy of estimating NSH, but the prime factors causing NSH to be low are the segregation of many genes, and the interactions of the many physiological processes controlled by these genes. Since the genes control specific component processes and do not act directly on yield, or even directly on complex physiological components of yield, NSH is (on the average) negatively correlated with the number of contributing physiological component processes. Biological yield and RGR are extremely complex characters; almost all biochemical and physiological processes occurring in the plant are relevant physiological components, i.e., almost all genes affect growth through controlling rates of photosynthesis or altering photosynthate partitioning. Economic yield may be an even more complex character because more genes that affect partitioning of photosynthate become relevant rather than neutral. NCE
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is less complex, but 15 or more components were identified in Section 11, C , and this listing is far from complete. Segregation of each relevant gene (for simplicity assume that each physiological component is controlled by a single gene) will introduce plus and minus effects on expression of the complex character. Since neither the magnitude, direction (i.e., plus or minus effect on rate of the physiological process), or expected interactions of the many component processes is normally determined, it is obvious that mean performance of plants heterozygous for the many genes controlling a complex character will not predict mean progeny performance. Heterosis for biological and economic yield in some but not all hybrids is the best recognized evidence of this unpredictability. F, means for NCE that are intermediate, positively heterotic, and negatively heterotic, as discussed in Section 111, F, provide additional evidence. Heterosis for leaf area and stomata1 index as discussed, respectively, in Sections 111, D and 111, G, illustrate how the immediate components interact unpredictably, when the genetics and developmental sequences (Adams and Grafius, 1971; Thomas et at., 1971) are not known, to give ultimate expression of a complex character. These unpredictable physiological interactions correspond to the epistatic effects (interactions between different genes) identified by statistical genetics, which Jana (1971) fmds to be more extensive than commonly used methods of analysis have indicated. Ikehashi and Ito ( 1971) discuss heritabilities for selecting parents on the basis of indices obtained as a quotient of two traits (such as harvest index). They report that heritabilities of such indices are predictable and depend upon statistical properties of the components.
IV.
Relative Importance of Physiological Components
Engledow and Wadham (1923) were among the first to advocate studies of the physiological basis of yield. During the intervening 50 years such studies have evolved from considerations of morphological components of yield (for example, number of pods, number of seeds per pod, and average weight per seed), to growth analysis, to growth analysis combined with light interception, to gas exchange measurements of net CO, exchange rates and dark and photorespiration, and most recently to measures of enzyme quantities. The data accumulated, many of them in the last 5 years, suggest that genetic variability probably exists within all species for most if not all of the many different physiological components of yield. Upon learning of a varietal difference in NCE or other physiological character related to yield, almost everyone asks, “Is it highly correlated with yield?” and is disappointed when usually it is not. It is unlikely that
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any factor will be found to have a high correlation with yield if one considers at random a large number of varieties within a species. If high correlation exists between a physiological component and yield, this indicates that all genetic variability affecting yield is centered in that component, which is unlikely in view of information presented in this review. High yield can be achieved through high L, high LAR, high NCE, efficient translocation, and many other contributing factors. Each variety achieves yield via its own combination of physiological pathways. Hageman et al. (1967) indicate that expressed yield results from balance of biochemical and physiological processes. Need for balance implies that elimination of just one component process may fully eliminate expression of yield (or of other complex characters). It also implies that excessive activity of a component may sometimes be both directly wasteful, and indirectly harmful by limiting another component to below optimum activity. For example, excessive partitioning of photosynthate to leaves after flowering may cause mutual shading and simultaneously reduce photosynthate partitioned to the economically important seed. A need for balance also implies that yield potential will be increased by improving the currently limiting physiological process, then the process that next becomes limiting, etc. Having identified genetic variability in many different physiological components, we are just approaching the capability of determining the frequency with which each is limiting yield in various species under various environments. This information is needed for intelligent and efficient application of physiology to breeding for higher yields. The task will not be accomplished easily but some guidelines can be suggested. First, duration of growth should be eliminated as a variable when trying to establish the relative importance of other components. It is common experience that within the range of varieties that mature in the available growing season of a given region, the later the maturity the higher economic yield. The effect of duration of growth can be so large as to obscure the effect of other components on economic yield. Therefore economic yield should be expressed on a unit time as well as unit area basis, e.g., kilograms per hectare per day. This would greatly facilitate comparisons of yields from different regions and studies. For example, economic yields of dry beans in the Philippines are only about two-thirds of those in New York State, but since the respective times required for identical varieties to mature are 60 and 100 days, the yields per hectare per day are nearly the same (Wien, 197 1 ) . Expression of economic yield on a yield per hectare per day basis is comparable to the crop growth rate (CGR),which expresses biological yield.on this basis. Expression on the basis of accumulated incident radiant energy (Wien, 1971 ; see Section 11, B) may be even more precise, but the data are much more difficult to obtain.
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Second, biological yield should be regularly considered along with economic yield and harvest index ( H I = economic yield/biological yield). ahere is in many crops a tendency for modern high yielding varieties to have higher harvest indices (and sometimes less biological yield) than the older varieties thcy replaced, even when there was not conscious selection for high HI.NAR, LAR, LAD, RGR, NCE, and dark and photorespiration, and light interception all act more directly upon biological than economic yield. The relationship of all components to economic yield could become clearer if biological and economic yield were commonly determined simultaneously. Determining biological yields in routine yield trials, or total plant weight at maturity which is a good approximation thereof, and calculating harvest indices would provide much insight as to the physiological basis of yield differences among commonly used and potential varieties. This easily obtained information would greatly assist the plant breeder to select for crosses parents with complementary physiological capabilities. Determining the physiological basis of sink capacity, of which HI is one measure, has been indicated as a frontier of current yield physiology which should be extensivcly investigated. Of all physiological components of yield besides maturity and harvest index, the only one so far demonstrated to have relatively large influence on yield is the short upright leaf habit in rice, which is controlled by a single gene (see Sections 11, B and 111, E). Jennings and Herrera (1968) studied populations from a cross between a dwarf and a tall variety. Homozygous dwarf and tall bulk F, . . . F, populations were each derived from nearly 1 0 0 F, plants and “considered as near-isogenic populations differing only in height alleles and closely linked genes.” It is instructive to note that it was not easy, even with monogenic control, to demonstrate precise and significant effects upon yield (see also Hicks et al., 1969; Sinclair, 1971 ). In Jennings and Herrera’s first experiments, the dwarf bulk had only slight superiority in yield over the tall bulk, even with high nitrogen fertilization. Subsequently, with closer plant spacing and even higher nitrogen levels, 35 randomly selected dwarf lines yielded about 76% more than comparable tall lines. Yield data from many countries and genetic backgrounds have verified both the influence of this gene on yield and the high nitrogen requirement for its expression. The more uniform light distribution over total canopy leaf area permitted by the leaf orientation conditioned by this gene is the major physiological character conferring high yielding capability upon dwarf habit of rice (Section 11, B ) ; it causes high net photosynthesis per unit of leaf area (NAR). More equal distribution of light over total canopy leaf area, which need not always require association with dwarf habit in rice and other grasses, or especially in dicotyledonous species, seems likely to give yield improvement in many crops.
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This is particularly so because of the attendant increased response to nitrogen fertilization. This review has repeatedly (but with exceptions) shown correlation (Sections 11, B and 11, C) between: thickness of leaves and NCE, leaf nitrogen content and NCE, leaf thickness and nitrogen content, number of cells per unit leaf area and NCE; enzyme content and NCE, and between leaf thickness (also enzyme content) and responsiveness of both NAR and NCE to increasing light intensity. These correlations suggest that increased nitrogen, in protein and enzyme form, is essential to expression of any genetic or environmental factor which will increase net photosynthesis and biological yield. This is supported in that most of the plant protein is in the leaves, and about half of it is the photosynthetic enzyme RUDPase. Determination of the contribution of a physiological component to yield requires presence of all genetic, physiological, and environmental factors essential to expression of that physiological capability, as does benefit from incorporation into new varieties. The use of isogenic lines (or isogenic populations as used by Jennings and Herrera, 1968) is one of two techniques available for determining importance of specific physiological components. After crossing parents with contrasting expressions of a component, isogenic lines may be obtained by repeated backcrossing to one parent while selecting for the level of expression of the other or by selecting for heterozygosity of the trait through successive generations of selfing. In either case after 6-8 generations, progenies selected for contrasting phenotypes will have essentially the same background genotype except for genes closely linked with those controlling the component under selection. The difference in yield associated with variation in the yield component is thereby largely freed from being obscured by effects of other components that vary at the same time. Development of isogenic lines is easy for characters controlled by single genes, and by incorporating monogenic characters into several background genotypes, their average contribution to yield can be assessed. For NCE, and other characters controlled by many genes and having low heritability, developing isogenic lines and isogenic populations will not be easy. In this event determination of contribution to yield can be achieved by advancing the progenies of a cross to the F,-F, generation, with possible selection for other characters, so that homozygosity will exist at most loci. This can be followed by obtaining the correlation between levels of expression of the physiological component and yield over a relatively large number of selections. Isogenic lines or populations, and correlation between character expression and yield, are the only means of demonstrating that a physiological component makes an important contribution to yield. An aspect of relative importance of physiological components of yield deserving special mention is subdivision of growth into periods commonly
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referred to as vegetative and reproductive growth (Bunting, 197 1) . Specifically, we are concerned with growth prior and subsequent to development and growth of organs that are of economic interest, whether they are reproductive (seeds or fruits) or vegetative (storage roots or tubers or other organs, or sugar, starch, or other specific chemical compounds). We shall, however, illustrate the problem by contrasting vegetative and reproductive growth. Need for this division is suggested by indications from growth analysis that vegetative and reproductive growth patterns differ (Kheiralla and Whittington, 1962). It is emphasized by findings that most of the photosynthate partitioned to seed of wheat (1970), rice and similar cereals comes from the flag leaf (Lupton, 1969; Evans and Rawson, 1970; Ryle, 1970) and that of corn comes mostly from the ear leaf and leaves above it (Allison and Watson, 1966). Thus, comparison during reproductive growth of relative NCE, translocation rates and other components of growth of different varieties may be most relevant to understanding physiological-genetic bases of varietal variation in economic yield (Bingham, 1969). However, it is during vegetative growth that both the size of photosynthetic organs functional during reproductive growth and potential number of flowers and seeds is determined. Thus, vegetative growth determines both sink capacity (potential number of seeds) of the economically important organs and the photosynthetic capacity for sustaining, during reproductive growth, the growth and development of these economically important organs. V.
Using Genetic Differentiation for Elucidation of Physiological and Biochemical Pathways
Genetic differentiation of microorganisms has been used extensively for determining how genes control biochemical and metabolic pathways. Because of the complex component-process interactions illustrated in this review, genetic differentiation of higher plants has been used much less (Nelson, 1967). Recent studies indicate that genetic differentiation of plants in combination with vaned environments can assist in elucidation of physiological and biochemical pathways. One example of using genetic differentiation of plants that will surely lead to elucidation of physiological and biochemical pathways is the work of Bjorkman ef al. (1971) on photosynthetic characteristics of C, and C , species. The following characteristics have been associated with species that fix CO, via the C , as contrasted with C , pathway: higher concentration of phosphoenolpyruvate carboxylase ( PEPase) , higher light saturation points, faster and more complete translocation from the leaf, presence of large chloroplasts in bundle sheath cells, lack of photorespiration, lower
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CO, compensation points, lack of oxygen inhibition of photosynthesis, efficient use of low CO, concentrations, higher temperature optimum, and high stomata1 but low mesophyll resistance. Bjorkman and co-workers crossed Atriplex rosea, a C4 species, with A . patula, a C , species. F, fertility was poor, but a limited number of F, and F, plants were obtained. Negative F, heterosis for NCE (Section 111, F ) occurred in spite of 14C0, fixation through both C, and C, photosynthetic pathways. Low NCE could not be attributed to low capacity of carboxylation enzymes since activity of RUDPase was as high in the F, as in the C, parent and activity of PEPase was higher than that of the C, parent. The F, was less efficient in use of low CO, concentrations than either parent, indicating low affinity for CO,. But, with high CO, concentrations its NCE approached that of the parents. As temperature was raised above 15OC, NCE of the F, leveled off and then decreased while that of the C , parent increased up to 25OC and that of the C, increased up to 3OOC. Responses to light intensity indicated photochemical steps were not responsible for the differences in NCE. The results suggested that the F, had higher resistance to CO, transport within the leaf, but anatomical comparisons indicated F, internal leaf structure to be intermediate between the parents. NCE rates of 9 F, plants were all low, and their mean did not differ from the negatively heterotic mean of the F,, although they had great variation in growth habit, leaf shape, leaf anatomy, and biochemical characteristics. Oxygen inhibited photosynthesis of the F, and all F, plants as it did that of the C, parents. CO, compensation points of F, and all F, plants were intermediate or like those of the C, parent. Stomata1 resistance of F, and all F, plants was less than that of either parent. While these parental, F, and F, progenies have not yet provided understanding of the C, and C, photosynthetic pathways and relationships to photorespiration and correlated characteristics, this work demonstrates that the “correlated characteristics” are not inherited as a tightly linked syndrome as suggested by data limited to comparisons between c, and c, species. The results reemphasize conclusions of Hageman et al. (1967) that balance and coordination among physiological components are essential to optimum expression of complex characters. Similar results were obtained from attempts to determine if photorespiration is responsible for the higher NCE of M-62 as compared with RED KIDNEY beans. Martin et al. (1972) compared these varieties with respect to CO, compensation points, zero CO, intercepts of CO, response curves, 0, suppressions of NCE, differential lzCO, and 14C0, uptake, and 14C0, efflux into CO, free air. These phenomena are all in the syndrome considered to be correlated with C, and C3 photosynthetic pathways. Differential performance by the two varieties under vaned intensities of
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CO, and 0, indicated that genetic variation for these characteristics exists between the two bean genotypes, i.e., within a C, species. The best illustration of using plant material in elucidating biochemical pathways of photosynthesis is work of Levine (1969) and others with a lower (Chlamydomonas reinhardi) rather than higher plant. Single-gene mutants, controlling a series of biochemical steps, were used to show that the photosynthetic electron transport chain is driven by two separate light reactions. Stepwise deletion of components of biochemical pathways by mutation is a more natural way, with perhaps fewer side effects, of affecting enzyme activity than the use of chemical inhibitors. Both methods give similar conclusions. That similar elucidation of photosynthetic pathways be gene mutations of higher plants is possible is indicated by recent work on regulation of enzyme activity in photosynthetic systems as reviewed by Preiss and Kosuge (1970). As examples, work with a barley mutant has demonstrated that although efficiency is altered, photosynthesis can proceed in the absence of chlorophyll b (Boardman and Thorne, 1968). Treharne and Eagles (1970) found that RUDPase of a Norwegian population of Dactylis glomeruta was most active at low temperatures, while that of a Portuguese strain was more active at high temperatures, providing at least partial explanation of variety x temperature and variety X lightintensity interactions discussed in Section 11, A. Charles-Edwards and Charles-Edwards (1970) showed that NCE of the tropical grass species Cenchrus cifiuris decreased above optimum temperature because of protein denaturation, while the NCE of ryegrass decreased because respiration increased more with temperature than photosynthesis. A light-activating factor which affects RUDPase activity has recently been identified, along with tomato mutants which do not respond to the light stimulation (Andersen et ul., 1970). Chlorophyll-deficient mutant pea (Pisurn sativum) and tobacco (Nicotianu tubacum) plants were found to have CO, uptake rates per unit of chlorophyll as much as two times that of normal plants (Highkin et al., 1969; Schmid, 1967). The wilty mutant tomato described in Section 11, C exhibits normal phenotype after application of abscisic acid (Imber and Tal, 1970). VI.
Summary and Applications in Plant Breeding
In the long run, the most effective approach to breeding for higher yield would seem to be to identify physiological components causing varietal differences in economic yield and acquire understanding of their genetic control. The evidence indicates that genetic variability probably exists for all such components. Broad sense heritabilities indicate that frequently more than half of the variation is genetic in F, populations from crosses
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between parents with high and low expressions of such components, Presumably this proportion can be increased with improved control of environment. F, and F, data provide evidence that instrumentation and assay techniques are sufficiently sensitive to identify genetic segregation, i.e., to identify small but statistically significant differences among individual plant and progeny means. These factors support feasibility of applying physiological-genetic studies to the breeding of higher-yielding varieties. Among factors making application difficult are the large number of physiological components of yield, and their further division into subcomponents, etc. Like yield, subdivisible physiological components are complex characters; they result from interaction of many component processes and environmental influence on the individual processes or the genes controlling them. Complex characters lack genes controlling them per se. Genes control them indirectly by controlling either the component processes or their subcomponents. A consequence of this indirect control, through genetic control over the many components, is that narrow sense heritability (NSH) in segregating generations is low, i.e., mean performance of a selected plant is poorly correlated with its progeny’s mean performance, making selection relatively ineffective. Further, determination of plant and progeny mean performance is usually expensive, requiring sophisticated equipment or considerable expenditure of time, or both. As a result it may not be much easier to select for these physiological components than for yield itself. Experience with bean and soybean progenies segregating for NCE and bean progenies segregating for dark respiration rates illustrate the problems of selection for a complex component. Infrared COz analysis was highly sensitive; it identified significant differences in net CO, exchange (NCE) and dark respiration rates among F, plants and particularly among F, progenies. However, the NSH estimates, 7 and 3%, respectively, for NCE and dark respiration of beans, indicate that only slight progress will be made by selecting in the F, generation. This is comparable to the situation long recognized with respect to selecting for yield in the F,. Similar low heritability and selection progress can be expected for other complex physiological components, especially when the number of components contributing to the character is large. The above considerations suggest that for NCE and dark respiration at least, and probably for other complex characters, selection should not begin until the F,. This is the first generation for which selection can be based on both progeny and individual plant means, which should raise NSH to the highest possible value. If selection for NCE or dark respiration rate has priority over selection for other characters, it should begin in F., rather than F, or later because the F, has a smaller number of progenies
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and individual plants to be evaluated than will any subsequent generation, and it will be possible to assay all or most F, progenies. Some germ plasm will have to be eliminated from evaluation in F, and later generations, because of population size in excess of equipment capability, and testing in F, permits elimination on the basis of performance rather than chance. Even with selection on the basis of both progeny and individual plant means, NSH will be relatively low. In order to emphasize progeny performance and test as large a population as possible, it is probably best to base selection on relatively few readings on many plants from many F, progenies, rather than many readings on few plants and progenies as done in the first-time-ever research of Martin (1970) and Ojima and Kawashima ( 1 970). It is intuitively obvious that high NCE rates can contribute toward high yield. It is also obvious that dark respiration utilizes photosynthate, and at first consideration low rates seem desirable. As discussed in Section 11, D, however, the optimum rate of dark respiration depends upon its coupling to energy transfer. It is imperative that interactions between dark respiration and other physiological components of yield be understood before the rate of dark respiration can be used intelligently in selection. The high yields achieved in rice by incorporating short, erect, thick, dark-green leaves and short-stiff stems clearly demonstrate the merit of including physiological-component traits in plant breeding programs. It seems likely (Section 111, E) that this gene is really a tightly linked block of genes, and that control of so many characteristics which combine to exert their physiological effects upon yield through numerous mechanisms as discussed in Section 11, B, would be by quantitative inheritance. Indeed, control of these same characteristics by many genes, as for a complex character, is common in rice. However, the release of high yielding varieties only 5-6 years after initiation of the breeding program (IRRI, 1967) demonstrates the rapid progress possible when physiological components with significant effects are simply inherited. Further benefit from this gene’s ability to improve distribution of light over total canopy leaf area, to improve effectiveness of nitrogen fertilization, to prevent lodging, and to divert photosynthate to the economically important rice grain (Section 11, B) should be realized when it is combined with other genes conditioning the highest possible KCE, optimum dark respiration and coupling to energy transfer and growth, and other desirable traits. Further, in the long run and depending upon limitations imposed by genetic linkages, maximum expression of all these characteristics may be available only when breeding is by the slower complex-character approach. All these considerations suggest that even in crops for which spectacular progress has already been achieved there is still much potential for increasing yield.
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Extensive use of physiological genetic data as criteria for selection within segregating progenies will be limited by the expensive instrumentation required, the large time expenditure for assaying many plants, the low narrow sense heritability, and difficulty of simultaneously combining all these physiological components with commercially acceptable quality, disease and insect resistance, etc. Many researchers agree that the most effective use of physiological components will be in the selection of parents for crosses. In contrast to segregating progenies, genetic differentiation between relatively homogeneous populations is identifiable with fewer plants, and succeeding generations behave similarly to parent. It can be relatively easily determined that one variety achieves high biological or economic yield because of high leaf area, another because of better distribution of intercepted light over total canopy leaf area, and still another because of high harvest index. Parents for crosses can then be selected on the basis of potential physiological complementation, assuming that genetic recombination of recognized physiological components will, on the average, give more high-yielding progenies than crosses between parents for which nothing is known about these component capabilities. Larger populations of fewer crosses can be grown and selected more extensively. Upon beginning such programs it will be wise to screen a large number of genetic lines and select for parents those with superior expressions of physiological components. If a breeding program is started with germ plasm lacking in genetic diversity, it may be necessary to begin again when lines superior to those currently considered best are ultimately identified. It should be recognized that superior genotypes may ultimately arise from transgressive segregation, sometimes in crosses where both parents had only poor performance. The possibility of transgressive segregation emphasizes the ever present role of chance as associated with genetic segregation and unknown interactions of physiological-component processes. Selecting parents on the basis of complementarity of physiological components simply improves the probability of finding superior segregates. Knowledge of the physiological bases of yield will not substitute for and replace standard plant breeding procedures. Breeding for higher yield will continue to require much time and effort. There is vast genetic variability available to use in this effort, but its effective and efficient exploitation requires understanding of the physiology and associated genetics by which yield is achieved, Rice and wheat provide examples of the merit of considering physiological components since yield improvements in these crops has been based on photoperiod insensitivity and better distribution of light over total leaf area. The harvest index of many crops has been improved over time without attention to physiology, and Ojima and Kawashima ( 1970) have found that superior soybean varieties selected from crosses
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made without knowledge of parental or progeny levels of NCE, usually have the NCE of the higher parent. These results support the view that if physiological genetic data are used in selecting parents, it should be possible to select directly for yield, using standard selection and breeding procedures. Knowledge of the physiological genetics of yield will of course greatly improve the plant breeder’s understanding of desirable plant type and habit, and of appropriate selection procedures and breeding methods. REFERENCES A d a m , M. W.. and Grafius, J. E. 1971. C r o p Sci. 11, 33-35. Allard, R. W. 1960. “Principles of Plant Breeding.” Wiley, New York. Allison, J. C. S. 1971. Ann. A p p l . B i d . 68, 81-92. Allison, J. C. S., and Watson, D. J. 1966. Ann. Bot. ( L o n d o n ) [N.S.] 30, 365-381. Andersen, W. R., Wildner, G. F., and Criddle, R. S. 1970. Arch. Biochem. Biophys. 137, 84-90. Apel, P., and Lehmann, 0. 1969. Pliotosynthetica 3, 255-262. Aquino, R. C., and Jennings, P. R. 1966. C r o p Sci. 6, 551-554. Barker, R. E. 1970. M.S. Thesis. University of Minnesota. Berdahl, 3. D., Rasmusson, D. C., and Moss, D. N. 1972. C r o p . Sci. 12, 177-180. Bingham, J. 1969. A g r . Progr. 44, 3 0 4 2 . Bjorkman, 0. 1968. Physiol. Plant. 21, 84-99. Bjorkman, O., Pearcy, R. W., and Nobs, M. A. 1971. “Annual Report.” Dept. Plant Biol., Carnegie Institution, Stanford, California. Blackman, G. E. 1950. Ann. Bor. (London) [N.S.] 14, 487-520. Boardman, N. K., and Thorne, S. W. 1968. Biochim. Biophys. Acts 153, 448-458. Bunting, A. H. 1971. Ann. A p p l . Biol. 67, 265-285. Carlson, G . E., Pearce, R. B., Lee, D. R.,and Hart, R. H. 1971. C r o p Sci. 11. 35-37. Chang, T.-T., Li, C. C., and Vergara, B. S. 1969. Euphytica 18, 79-91. Charles-Edwards, D. A., and Charles-Edwards, J. 1970. P / a n f a 94, 140-151. Clark, D. G . , Hecht, H., Curtis, 0. F., and Shafer, J. I., Jr. 1941. A m e r . 1. Bot. 28, 537-541. Donald, C. M. 1962. 1. Aust. Z m r . A g r . Sci. 28, 171-178. Dornhoff, G. M., and Shibles, R. M. 1970. C r o p Sci. 10, 42-45. Downes, R. W. 1970. Aust. 1. Biol. Sci. 23, 775-782. Dreger, R. H., Brun, W. A., and Copes, R. L. 1969. C r o p Sci. 9, 429-431. Duarte, R., and Adams, M. W. 1963. C r o p Sci. 3, 185-186. Dubetz, S. 1969. Can. J. Bor. 47, 1640-1641. Dvorak, J., and Natr, L. 1971. Pliotosyntl~etica5, 1-5. Eagles, C. F. 1967. Ann. Bol. (London) [N.S.] 31, 31-39. Eagles, C. F. 1969. Ann. Bot. ( L o n d o n ) [N.S.] 33, 937-946. Eagles, C. F. 1971a. Ann. Bot. ( L o n d o n ) [N.S.] 35, 63-74. Eagles, C. F. 1971b. Ann. Bot. ( L o n d o n ) [N.S.] 35, 75-86. Eagles, C. F., and Treharne, K. J. 1969. Photosynthetica 3, 29-38. Edwards, K. J . R. 1970. Genet. Res. 16, 17-28. Edwards, K. J . R., and Emara, Y. A. 1970. Heredity 25, 179-194.
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Pearce, R. B., Carlson, G. E., Barnes, D. K., Hart, R. H., and Hanson, C. H. 1969. Crop Sci. 9, 423-426. Peat, W. E., and Whittington, W. J. 1965. Ann. Bot. (London) [N.S.] 29, 725-740. Pendleton, J. W., Smith, G. I., Winter, S. R., and Johnston, T. J. 1968. Agron. J . 60, 422-424. Preiss, J., and Kosuge, T. 1970. Annu. Rev. Plant Physiol. 21, 433-465. Radford, P. J. 1967. Crop Sci. 7, 171-175. Reitz, L. P., and Salmon, S. C. 1968. Crop Sci. 8, 686-689. Rhodes, I. 1971. I. Brit. Grassland Soc. 26, 9-15. Rosario, E. L. 1967. M.S. Thesis, College of Agriculture, University of Philippines, Los Banos. Ryle, G. J. A. 1970. Ann. Appl. Biol. 66, 155-167. Sasahara, T. 1971. lap. J. Breed. 21, 61-68. Schmid, G. H. 1967. PIanta 77, 77-94. Sinclair, T. R. 1971. PkD. Thesis, Cornell University, Ithaca, New York. Singh, I. D., and Stoskopf, N. C. 1971. Agron. 1. 63, 224-226. Singh, M., Peters, D. B., and Pendleton, J. W. 1968. Agron. J. 60, 542-545. Slatyer, R. 0. 1970. Planta 93, 175-187. Syme, J. R. 1970. Aust. 1. Exp. Agr. Anim. Husb. 10, 350-353. Takahashi, R., and Yasuda, S. 1971. In “International Barley Genetics Symposium” (R. A. Nilan, editor), p. 388. Washington State Univ. Press, Pullman. Takano, Y., and Tsunoda, S. 1971. Jap. I . Breed. 21, 69-76. Takeda, T., and Fukuyama, M. 1971. Proc. Crop Sci. Soc. Jap. 40, 12-20. Tal, M. 1966. Plant Physiol. 41, 1387-1391. Tanaka, A., Navasero, S. A., Garcia, C. V., Parao, F. T., and Rameriz, E. 1964. Int. Rice Res. Inst., Tech. Bull. 3. Tanaka, A., Kawano, K., and Yamaguchi, J. 1966. Int. Rice Res. Inst., Tech. Bull. 7. Tanaka, T. S., Matushima, S., Kojyo, S., and Nitla, H. 1969. Proc. Crop Sci. SOC.lap. 38, 287-293. Tanner, J. W., Gardener, C. J., Stoskopf, N. C., and Reinberg, E. 1966. Can. I. Plant Sci. 46, 690. Thomas, M. D., and Hill, G. R. 1949. In “Photosynthesis in Plants” (J. Franck and W. E. Loomis, eds.), p. 19. Iowa State Univ. Press, Ames. Thomas, R. L., Grafius, J. E., and Hahn, S. R. 1971. Heredity 26, 423-432. Thorne, G. N., and Blacklock, G. C. 1971. Ann. Appl. Biol. 78, 93-1 11. Thorne, G. N., Welbank, P. J., and Blacklock, G. C. 1969. Ann. Appl. B i d . 63, 241-251. Treharne, K. J., and Cooper, J. P. 1969. J . Exp. Bot. 62. 170-175. Treharne, K. J., and Eagles, C. F. 1970. Photosynthetica 4, 107-1 17. Tsunoda, S. 1959. lap. 1. Breed. 9, 237-244. Van Dobben, W. H. 1962. Neth. J. Agr. Sci. 10. 372-389. Verhagen, A. M. W., Wilson, J. H.. and Britten. E. J. 1963. Ann. Bot. (London) [N.S.] 27, 627-640. von der Pahlen, A., and Goldberg, J. B. 1971. In “International Barley Genetics Symposium” (R. A. Nilan, ed.), p. 434. Washington State Univ. Press, Pullman. Waggoner, P. E., and Simmonds, N. W. 1966. Plant Phvsiol. 41. 1268-1271. Wallace, D. H. 1958. Ph.D. Thesis, Cornell University, Ithaca, New York. Wallace, D. H., and Munger, H. M. 1965. Crop Sci. 5,343-348. Wallace, D. H., and Munger, H. M. 1966. Crop Sci. 6, 503-507.
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Watson, D. J. 1952. Advan. Agron. 4, 101-145. Watson, D. J. 1958. Ann. Bot. ( L o n d o n ) [N.S.] 22, 37-54. Watson, D. J. 1968. A n n . Appl. Biol. 62, 1-9. Watson, D. J., and Witts, K. J. 1959. A n n . Bor. (Loridon) [N.S.] 23,431-439. Welbank, P. J., French, S. A. W., and Witts, K. J. 1966. Ann. Bot. (London) [N.S.] 30, 291-299. Whaley, W. G. 1952. I n “Heterosis” (J. W. Gowen, ed.), p. 98. Iowa State Coll. Press, Arnes. Wien, H. C. 1971. Ph.D. Thesis, Cornell University, Ithaca, New York. Wein, H. C., and Wallace, D. H. 1971. Unpublished data. Williams, W., and Gilbert N. 1969. Heredify 14, 133-149. Wilson, D., and Cooper, J. P. 1969a. Ann. Bof. ( L o n d o n ) [N.S.] 33, 951-965. Wilson, D., and Cooper, J. P. 1969b. New P h y f o l . 68, 627-644. Wilson, D., and Cooper, J. P. 1969c. New Plzyfol. 68, 645-655. Wilson, D., and Cooper, J. P. 1970a. New Pliytol. 69, 233-245. Wilson, D., and Cooper, J. P. 1970b. Heredity 24, 633-649. Wilson, D., Treharne, K. J., Eagles, C. F., and DeJager, J. M. 1969. J . E x p . But. 20, 373-380. Wu, B-F. 1971. Ph.D. Thesis, Cornell University, Ithaca, New York. Zelitch, I., and Day, P. R. 1968. Plant Physiol. 43, 1838-1944.
ZINC IN SOILS AND PLANT NUTRITION
.
W . 1 Lindsay Department of Agronomy. Colorado State University. Fort Collins. Colorado
I. Introduction .................................................... I1. Zinc in Soils .................................................. A . Zinc Content ............................................... B . Distribution of Zinc in the Profile ............................. C. Zinc Minerals in Soils ........................................ D . Adsorption of Zinc in Soils ................................... E. Zinc Interactions with Organic Matter ......................... F . Solubility of Zinc in Soils ..................................... G. Stability of Synthetic Zinc Chelates in Soils . . . . . . . . . . . . . . . . . . . I11. Availability of Zinc to Plants ..................................... A . Patterns of Zinc Deficiency .................................. B . Factors Affecting Zinc Availability ............................ C . Movement of Zinc to Plant Roots ............................ D. Absorption of Zinc by Plants ............................... E . Distribution of Zinc in Plants ................................ F. Function of Zinc in Plants ................................... IV. Diagnosing Zinc Deficiencies . . . . . . . . . . . . . . . . ................. A . Plant Analysis . . . . . ................. B . Soil Tests for Zinc ........................................... V. Correction of Zinc Deficiency ..................................... A. Sprays and Injections ........................................ B. Zinc Fertilizers .............................................. C . Residual Effects of Zinc Fertilizers ............................ VI. Summary and Future Research Needs ............................. References ......................................................
147 148 148 148 148 149 151 153 154 158 158 159 163 164 167 168 169 169 170 173 173 174 179 180 181
I . Introduction
Zinc deficiency in agricultural crops is one of the most common micronutrient deficiencies. Since Thorne (1957) reviewed the subject of zinc deficiency and its control. zinc deficiencies have become even more widespread (Wets. 1966; Mortvedt et al., 1972) . The use of zinc fertilizers to correct zinc problems has widespread interest in the fertilizer industry. Inclusion of zinc in commercial fertilizer recommendations is a common practice throughout the world. Introduction of atomic absorption spectrophotometry as a convenient and accurate 147
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W. L. LINDSAY
method of measuring zinc has stimulated interest and research with this nutrient element, and soil tests to assess available zinc in soils are as commonplace today as major nutrients soil tests were a few decades ago. Several good reviews have been published that summarize much of the general information that is known about zinc in soils and plant nutrition (Thorne, 1957; Seatz and Jurinak, 1957; Stiles, 1961; Hodgson, 1963; Mitchell, 1965; Viets, 1966; Chapman, 1966; Mortvedt et al., 1972). No attempt will be made in this review to repeat much of that published information. Instead, attention will be focused on those areas where the most significant interest and progress appears to have been made. Readers new to the field may wish to refer to the review by Thorne (1957) as a reference to much of the earlier work.
It.
Zinc in Soils
A. ZINC CONTENT The average zinc content of the lithosphere according to Goldschmidt (1954) is approximately 80 ppm of zinc. The zinc content of soils according to rather extensive surveys (Swaine, 1955; Jensen and Lamm, 1959) is generally in the range of 10-300 ppm. Certainly zinc, because of its concentration, can be considered as a trace element in soil. B. DISTRIBUTION OF ZINC
IN THE PROFILE
I n general, the total zinc is quite uniform throughout soil profiles and does not accumulate at the surface to any great extent (Swaine and Mitchell, 1960; Follett and Lindsay, 1970). In contrast, Alston and McConaghy ( 1965 ) reported that EDTA (ethylenediaminetetraacetic acid) -extractable zinc decreased sharply with depth in the profile. This finding agrees with the findings reported by Swaine and Mitchell (1960) using 2.5% acetic acid and 1 N ammonium acetate buffered at p H 7. Follett and Lindsay ( 1970) also reported that DTPA (diethylenetriaminepentaacetic acid) -extractable zinc declines rapidly with depth in 37 profiles of Colorado soils, whereas total zinc content was fairly constant. C. ZINC MINERALS IN SOILS
Generally, zinc does not form independent silicate minerals in igneous rocks, nor does it occur to any great extent in quartz and feldspars. Krauskopf (1972) reviewed the geochemistry of zinc and pointed out that zinc
ZINC IN SOILS AND PLANT NUTRITION
149
occurs most frequently in the lithosphere as the mineral ZnS (sphalerite) . The Zn2+with an ionic radius of 0.83 A substitutes to some extent for Mg2+(0.78 A) and Fez+(0.83 A) (Goldschmidt, 1954). Jenne (1968) proposed that zinc, along with several other heavy metal ions, may be occluded and coprecipitated with hydrous oxides of manganese and iron, and that these oxides form the principal matrix in which the less abundant heavy metals are held. Naturally, more work is needed to examine this hypothesis. The chemistry of zinc is simpler than that of many other heavy metals because it shows only the single valence state of Zn2+in natural environments. Most commonly, Zn2+shows 4-coordination in mineral structures, but in some minerals 6-coordination occurs with oxygen (Krauskopf, 1972). Zinc forms a silicate mineral (sauconite), but the pure mineral is rare. Most of the simple compounds like ZnO (zincite) , ZnCOs (smithsonite) , etc., which form with the common anions in soil are too soluble to persist in soils, as will be demonstrated later. Under reducing conditions where H,S is produced, ZnS (sphalerite) can form, but under normal oxidizing conditions S2- is too low for this mineral to be stable. Zinc appears to be scattered throughout the mineral fraction of soils. It is probably held in crystal lattices by isomorphous substitution and as occluded ions. Since it is a trace element, it is usually surrounded by many other solid phases. Nevertheless, the soil matrix of iron, aluminum, manganese, and other oxides, carbonates, and silicates do impose some control on the solubility relationships of zinc in soils. The relative stabilities of the commonly known zinc compounds in soils are depicted in Fig. 1. The zinc-soil line represented in this diagram is that reported previously (Lindsay and Norvell, 1969a; Norvell and Lindsay, 1969; Lindsay, 1972a). It is apparent that the common zinc minerals represented here are far too soluble to persist in soils. If these minerals are added to soils they can be expected to dissolve to supply available zinc to plants. The minerals, ZnSiO, and Zn2SiOa (Willemite), would lie on this diagram parallel to the zinc-soil line, but uncertainty regarding their solubility preclude includifig them at this time. D. ADSORPTION OF ZINC
IN
SOILS
Not only is zinc present in soils as minerals, but it can also be held by exchange sites and adsorbed to solid surfaces. Separation of zinc reactions into those of precipitation or adsorption is most difficult, and very few studies permit a clear conclusion on this point. One of the major problems in studying adsorption reactions of zinc has been failure to consider
150
W. L. LINDSAY
FIG. I . The solubility of various zinc materials in soils. CO, = 0.0003 atm; P = lo4 M. From Lindsay (1972a). with permission to reproduce this copyrighted material from the Soil Science Society of America.
which of the various hydrolysis and complex species of zinc in solution are adsorbed. Some have conjectured that retention of zinc in excess of the exchange capacity capacity of soils may be due to precipitation of Zn(OH),, but the possibility of its precipitation was not critically examined (DeMumbrum and Jackson, 1956; Bingham et al., 1964). Elgabaly and Jenny ( 1943 ) concluded that some adsorbed zinc becomes nonextractable by entering the octahedral layer of montmorillonite. Later Elgabaly (1950) suggested that Zn" might be fixed in holes normally occupied by A13+in the octohedral layer. Hodgson (1963) and more recently Ellis and Knezek (1972) have reviewed some of the earlier works on zinc adsorption on soils and clay minerals. Nelson and Melsted (1955) studied the reactions of zinc with montrnorillonite and concluded that strongly bound zinc was desorbed according to first-order chemical kinetics. Tiller ( 1967) concluded that the interaction between zinc and silicic acid is probably due to adsorption rather than formation of a separate zinc silicate phase. Udo et al. (1970) found that the adsorption of zinc by calcareous soils could be explained
ZINC IN SOILS AND PLANT NUTRITION
151
by the Langmuir adsorption equation. Further investigations are necessary to examine the adsorption reactions that occur in soils.
E. ZINC
INTERACTIONS WITH
ORGANIC MATTER
High levels of organic matter in the upper horizon of soil are believed to be important in keeping zinc more available in the surface horizon of soils. Numerous studies have demonstrated a high correlation between organic matter and chemically extractable or available zinc (Follett and Lindsay, 1970; Martens ei al., 1966). First of all, zinc is essential to all forms of life-both plants and animals. Naturally all forms of organic matter and bio-residues contain zinc that will be released during decomposition and made available to plants and other organisms in the soil. Zinc deficiencies are frequently found in areas where the surface soil has been removed. Farmers have found that liberal application of manure and other organic wastes are often effective in correcting zinc deficiencies. On the other hand, old corral sites that are high in organic matter often cause zinc deficiency. Why this apparent contradictory behavior? Apparently organic matter can interact with zinc in two important ways. First, soluble zinc can be mineralized and made available to plants. Second, zinc can be bound into organic constituents that are immobile in soils and constitute a fixation mechanism by which zinc is not readily released. DeRemer and Smith (1964) showed that incorporation of sugar beet residues can cause zinc deficiency during the early stages of decomposition. The slow release of available zinc in soils in the cool spring season before the microbiological activity flourishes is also often associated with zinc deficiency. The presence of soluble zincorganic complexes in soils was demonstrated by Hodgson et al. ( 1965, 1966). They concluded that on the average about 60% of the soluble zinc in soils is complexed. The degree of complexing of zinc was correlated with soluble organic matter ( r = 0.88). It should be emphasized that complexing was related to soluble organic matter, not to total organic matter content of the soil. Soluble organic matter had no relationship to total organic matter in the soils studied by Hodgson et al. (1966). Stevenson and Ardakani (1972) reviewed the reactions of organic matter with micronutrients. They concluded that insoluble metal combinations are most likely bound to the humic fraction, particularly humic acids, while soluble metal complexes are mainly associated with individual biochemical molecules, for example, organic acids and amino acids. Metal complexes with fulvic acids also have high water solubilities. Many of the soluble
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W. L. LINDSAY
organic constituents in soils have only transitory existence. The amount present at any moment represents a balance between those synthesized and those destroyed by microorganisms. High concentrations may be found near decomposing plant residues. Root exudates, according to Riviere (1960) and Vancura (1964), contain many of the common organic acids found in plants. These undoubtedly play an important role in the chelation of metal ions in the vicinity of plant roots. Webley and Duff (1965) found rather large quantities of sugar acids such as 2-ketogluconic acid in habitats rich in organic matter where a large number of microorganisms were thriving. Mortensen (1963) made a rather extensivc review of numerous ligands in soils that are capable of complexing zinc and other metal ions. Randhawa and Broadbent (1965a,b) studied the adsorption of zinc by humic acids. The least stable fraction that accounted for most of the zinc was believed to be associated with phenolic -OH and weakly acidic -COOH groups. The more stable fraction of zinc was bound by strongly acidic -COOH groups. In their studies strongly bound zinc represented less than 1% of the total zinc retained, but is considered to be of great significancz because of its preferential adsorption. (0
/7
8
I
% 6 rn
X
0 J
1
2
4
6
8
PH FIG.2 Effect of pH on stabilities of Zn(II)-organic matter complexes. The stability constants shown by the symbols represent results from different workers. The vertical clashed line indicates the range of values reported by Matsuda and Ito. From Stevenson and Ardakani (1979, with permission to reproduce this copyrighted material from the Soil Science Society of America.
ZINC IN SOILS AND PLANT NUTRITION
153
Because of their high water solubility, metal complexes of fulvic acids are of considerable importance in soils. Geering and Hodgson (1969) reported a log K = 4.3 for the formation constant for a nondialyzable zinc complex from displaced soil solution of the A horizon of a Williamson silt loam. Stevenson and Ardakani (1972) summarized some of the reported values for the log K of formation of zinc-organic matter complexes. The results are reproduced in Fig. 2. The pH-dependent stability of the zinc-organic matter complex and the wide divergence in measured stability of zinc complexes is apparent. Although much remains to be learned about the interaction of zinc with organic matter in soils, obviously both soluble and insoluble zinc-organic matter complexes are formed and play an important role in soils. Further work in this area is to be encouraged.
F. SOLUBILITY OF ZINC IN SOILS 1 . Concentration of Zinc in Soil Solution Zinc is only sparingly soluble in soils. Solid phase minerals and adsorption reactions prevent a high concentration of zinc from persisting. Hodgson et al. (1966) measured total soluble zinc in several New York and Colorado soils. They reported an average of about 75 ppb of zinc in several New York soils and < 2 ppb in 20 Colorado soils. They interpreted these results as indicating one reason why zinc deficiencies are more common on alkaline than on acid soils. 2 . Zn2+Activity-pH Relationships Until recently no activity measurements of Zn2+ in soils had been reported. Hodgson et al. (1965) measured the competition between added complexing agents and those naturally present in the soil solution. By partitioning added B5Znbetween aqueous and organic phases, they were able to estimate the degree of complexing of Znz+in soil solution. Similar measurements were extended to include the more alkaline soils of Colorado (Hodgson et al., 1966). Their overall findings showed that the average concentration of free Zn2+in several New York soils was about 20 ppb whereas in the calcareous soils from Colorado free Zn2+was <0.8 ppb. Norvell and Lindsay (1969, 1972) allowed ZnEDTA (zinc ethylenediaminetetraacetate) and ZnDTPA (zinc diethylenetriaminepentaacetate) to react with soils of different pH and were able to calculate the free Zn2+ concentration necessary to achieve the solubility relationship they ob-
154
W.
L.
LINDSAY
served. Their results fit the equilibrium relationships worked out by Lindsay and Norvell ( 1969a) and Lindsay ( 1972a). Zn2+
+ soil
Zii-soil
+ 2FI+
(1)
for which log K = -6.0. The solubility relationship of Zn2+in soils can then be expressed by the important and useful relationship (ZI12+) =
1 0 6 (11+)2
(2)
Initially, Lindsay and Norvell ( 1969a) hypothesized that ZnSiO, and amorphous silica could account for this solubility relationship, but they later showed that the reported value in the literature for the solubility of ZnSiOJ is in error (Norvell and Lindsay, 1970). The exact solid phases responsible for the solubility of Zn" in soils as defined by Eqs. ( 1 ) and ( 2 ) are not known. Nevertheless, these equations provide an extremely important reference to which Zn?+ solubility relationships in soils can be compared. These equations emphasize a significant pH relationship of Zn2+ solubility in soils. More specifically, Eq. (2) shows that Zn2+activity decreases 100-fold for each unit increase in pH.
3. Ionic Species in Soil Solution Soil solution contains numerous ionic and complexed ionic and molecular species including the hydrolysis species of Zn2+ (Lindsay, 1972a). A more detailed diagram showing the relative importance -of the various inorganic zinc species expected in soils is given in Fig. 3. The solubility line for Znya is based on Eq. (2) discussed above. The formation constants used to construct this figure were taken from Sillen and Martell (1964). There has been considerable conjecture about thc importance and significance of zincate ions in soils. It is obvious from Fig. 3 that neither Zn(0H) .3- nor Z n ( 0 H ) ,z- contributes significantly to total soluble zinc in the pH range of soils. For most purposes they can be ignored. The ZnSO,(aq) complex can also affect the solubility of zinc depending upon the level of SO$$-in soils as indicated by the dashed lines in Fig. 3. In addition to the inorganic zinc species in soil solution, it should be pointed out again that zinc-organic complexes and chelates are also present and will raise the total zinc in solution above that shown here. G . STABILITY OF SYNTHETK ZINC CHELATES IN SOILS Synthetic chelating agents can be used to prevent the precipitation of Zn'+ and other metal ions from solution. They are often included in ferti-
I55
ZINC 1N SOlLS AND PLANT NUTRITION
(6
4
5
6
7 PH
8
9
10
FIG.3. Soluble zinc species in soils in equilibrium with soil Zn*+.SO?- = Calculated from Sillen and Martell (1964).
lizers for this purpose. The question naturally arises: Are synthetic chelates stable in soils, or is Zn2+displaced from the metal chelates by other cations in the soil? I. Stewart and Leonard (1956) shook solutions of ZnEDTA with soil and analyzed the supernatant for water-soluble zinc and iron at various intervals. In Lakeland soil of pH 5.3 the exchange was rapid initially but slowed down after 2 days. There was approximately 50% exchange in 13 days. W. B. Anderson (1964) found that about 25% of the zinc added as ZnEDTA or ZnDTPA remained soluble in a calcareous soil after 15 days. Lindsay el al. (1967) made the initial attempts to develop theoretical chelating agents in soils. The predicted stability diagrams for ZnEDTA and ZnDTPA in soils were developed later by Lindsay and Norvell (1 969a) and are given in Fig. 4. At low pH, Fe3+displaces Zn2+from
156
W. L. LINDSAY 1
100 r
"
5
6
7
8
PH
FIG.4. A summary diagram showing the stability of zinc ethylenediaminetetraacetate (EDTA) and zinc diethylenetriarninepentaacetate (DTPA) complexes in soils as affected by pH and CO1. From Lindsay and Norvell (1969a).
these chelates whereas at high pH, Ca'+ displaces it. ZnEDTA has a maximum stability at pH 6.5 where approximately 70% of the EDTA ligand is chelated with Zn2+.ZnDTPA, on the other hand, has a maximum stability at pH 7.3. In the presence of CaC03, increasing CO, increases CO,?-, depresses Ca'+, and allows more Zn'- to be held by these chelating agents. For example, in a calcareous soil of pH 8 increasing CO, from that of the air (0.0003 atm) by 10-fold, increases from 37% to 90% the fraction of ZnDTPA present. Even much larger changes in CO1 are known to occur in soils. The increased stability of ZnDTPA over ZnEDTA below pH 6 is based on the formation of a Zn,L- complex where two Zn2+are chelated by the same DTPA ligand {Li-). The zinc stability diagrams in Fig. 4 were later tested by allowing ZnEDTA and ZnDTPA to react with soils of diffcrent pH for periods up to 30 days (Norvell and Lindsay, 1969, 1972). The results they obtained corresponded very closely to those predicted by the stability diagrams. ZnEDDHA [zinc ethylenediaminedi (o-hydroxyphenylacetate)] is unstable in soils and results in a series of cation displacements before final equilibrium is attained. The results reported by Lindsay et a!. (1967) show that Zn'+ is rapidly displaced from the chelate by Mn'+ and then Mn2+ is later displaced by Fe3+. Since FeEDDHA is stable throughout the pH
ZINC IN SOILS AND PLANT NUTRITION
157
range of soils (Lindsay et al., 1967) other metal ions are unable to displace Fe3+from this chelate. Norvell (1972) in his review of metal chelate equilibria in soils considered additional chelating agents and metal ions that may be present in soils. His calculations for the stability of 11 chelates under conditions expected in soils are redrawn in Fig. 5 showing the ratio of chelated Zn to Zn2+.In the pH range of calcareous soils the effectiveness of various chelating agents to chelate Zn2+is in the order: DTPA > CDTA, HEDTA, EDTA > NTA > EGTA > citrate, EDDHA, oxalate. At low pH, all chelates are less effective to chelate Znz+because of displacement by Fe3+. At higher pH as the activity of Fe3+ is depressed by precipitation of Fe(OH), more Zn2+becomes chelated. Considerable progress has been made in understanding the stability relationships of metal chelating agents in soils. The significance of these h d ings with regard to the movement of zinc in soils and its availability to plants will be discussed in later sections of this paper. 'O
r
1
PH
FIG.5. Effectiveness of various chelating agents for increasing the solubility of zinc in soils. DTPA, diethylenetriaminepentaacetate; EDTA, ethylenediaminetetraacetic acid; NTA, nitrilotriacetic acid; CDTA, cyclohexanediaminetetraacetic acid; EGTA, ethyleneglycol-bis(2-aminoethy1ether)tetraacetic acid; HEDTA, hydroxyethylethylenediaminetriacetic acid; EDDHA, ethylenediamninedi(o-hydroxyphenylacetic acid). From Norvell (1972) with permission to reproduce this copyrighted material from the Soil Science Society of America.
158
W. L. LINDSAY
Ill.
A.
Availability of Zinc to Plants
PATTERNS OF ZINC DEFICIENCY
During the early 1900’s a number of disorders in agricultural crops were reported that can now be documented as zinc deficiencies (Stiles, 1961). The first sign of zinc deficiency is usually an intervenal chlorosis. In crops like corn (Zea mays L.) the intervenal areas broaden into chlorotic bands on either side of the midrib of the leaf. In trees, instead of normal-sized leaves evenly distributed along the shoots, there develop unevenly spread rosettes or clusters of small, stiff leaves. Thus, zinc deficiency is often referred to as rosette, little leaf, mottle leaf, or yellows. Chapman (1960, 1966) has summarized much of the detailed information available on the diagnosis and control of zinc deficiency. He has summarized the visual zinc deficiency symptoms and levels of zinc associated with healthy and deficient plants for various crops. Crops differ in their sensitivity to zinc deficiency. Fruit trees, particularly citrus, peaches (Prunus persica), and apples (Malus spp) , are good indicator crops. Pecan (Carya illinoensis) and pineapple (Ananas comosus) also show the deficiency. In the annual crops, corn, beans (Phaseolus vulgaris), onions (Allium), and potatoes (Solanum tuberosum) are sensitive to zinc deficiency (Viets et al., 1954b). Generally, grasses, legumes, and small grain crops are rather insensitive to zinc deficiency. Beeson { 1957) indicated areas in the United States where zinc deficiencies have been reported for various crops. Later Berger (1962) showed that at least 30 states in the United States have reported zinc deficiencies. If accurate maps were drawn today, they would most likely show zinc deficiencies in nearly all states. Considerable interest has been given to zinc problems in several parts of this country (Viets, 1951; Viets et al., 1953; Baskett and Scott, 1949; Judy et al., 1964, 1965; Barnette and Warner, 1935; Barnette et al., 1936; Pumphrey and Koehler, 1959; Reuss and Lindsay, 1963). One of the most spectacular trace element problems is that of the 90-mile trace element desert in Southern Australia where over 300 million acres of land are affected. Zinc along with copper and molybdenum were the trace elements that were most often found deficient (Anderson and Underwood, 1959). In an F A 0 report, Ryan et al. (1967) concluded that low zinc levels occur in 10 of 15 European countries and in Israel. As the search continues, the areas of zinc deficiency throughout the world are expected to expand.
ZINC IN SOILS AND PLANT NUTRITION
159
B. FACTORS AFFECTING ZINC AVAILABILITY Lucas and Knezek (1972) recently reviewed the climatic and soil factors that seem to promote zinc deficiency in crops. Trace element problems in 15 European countries and Europe were also summarized in a report of The Food and Agriculture Organization (Ryan et al., 1967). Factors associated or contributing to zinc deficiency are numerous. Some of these factors are discussed below.
I . Soils of Low Zinc Content Very often, sandy soils are deficient in available zinc. Since quartz is generally low in zinc content, such soils are inherently low in total zinc content, often 10-30 ppm. Although water-soluble zinc is often as high in sandy soils as in finer textured soils, the labile zinc content is generally much lower so that more extensive depletion zones of zinc occur in the immediate vicinity of growing roots. Sometimes peats and mucks (histosols) are also deficient in zinc (Lucas and Knezek, 1972). Here, again, total zinc content is often the contributing factor because the surface layer where plant roots feed becomes isolated from the mineral forms of zinc in the soil below. In high rainfall areas where acidic conditions prevail, weathered minerals release zinc that is soon removed by leaching. Zinc deficiencies on acid soils are generally associated with low soil zinc content (Schroo, 1959; Jackson et al., 1967; Lucas and Davis, 1961). Sometimes, however, acid soils may show rather high total zinc and low available zinc because that which is released by weathering is rapidly lost from the profile.
2. Soils with Restricted Root Zones Zinc deficiencies are frequently found on soils with restricted root zones. These may be caused by hardpans, by high water tables, or by other factors. Also, plants grown in containers, such as in the greenhouse, may often show zinc deficiencies. Compacted areas caused by tractor wheels may also be the cause of zinc deficiency in certain soils (Lucas and Knezek, 1972). 3. Calcareous Soils Calcareous soils generally fall in the pH range of 7.4 or higher. As shown in Fig. 1, the solubility of Zn2+in soils decreases with increase in pH. Hence a greater incidence of zinc deficiencies would be expected in calcareous soils and, indeed, such has been reported (Thorne, 1957; Navrot and Ravikovitch, 1969; Vets, 1966). The zinc content of calcareous soils is often no less than that of noncalcareous soils. In fact, it may even be higher (Thorne et al., 1942). Adsorption of zinc by carbonates
160
W. L. LINDSAY
(Leeper, 1952; Jurinak and Bauer, 1956) has also been given as a reason why zinc availability may be low on calcareous soils. Udo et al. (1970) showed no significant correlation between zinc content and calcium carbonate ( r = 0.428, not significant), but the adsorption maximum calculated from the Langmuir adsorption equation was correlated zinc carbonate content ( r = 0.755). Grunes el al. (1961) reported severe zinc deficiencies on corn and potatoes growing on exposed subsoils. 4 . Soils Low in Organic Matter
One of the most frequent places where zinc deficiencies are found is on leveled sites where the surface soil has been removed (Viets, 1951; Grunes et al., 1961). There are probably at least two major reasons for this: the exposed subsoil is generally much lower in organic matter and is frequently higher in pH and carbonate. Several workers have shown a positive correlation between chemically extractable zinc and organic matter content. For example, Follett and Lindsay (1970) showed a high correlation ( r = 0.76) between organic matter content and DTPA-extractable zinc. Both DTPA extractable zinc and organic matter decreased rapidly with depth in the profile while the correlation of the two parameters remained fairly constant ( r = 0.71 for all horizons). Zinc deficiencies are often found on old tile drains where the surface soil has been buried and the subsoil exposed.
5 . Microbial Inactivated Zinc A frequent observation is that zinc deficiency in a crop like corn is much more severe following a crop of sugar beets (Beta vulgaris) than following corn or many other crops. DeRemer and Smith (1964) showed that incorporation of sugar beet tops in soil reduced available zinc to the succeeding bean crop. Further studies by Boawn (1965) failed to show that sugar beet residues had much affect on zinc deficiency in the succeeding crop. Factors such as high rates of phosphorus fertilizer generally applied to beets may be the major contributing factor causing greater zinc deficiency after sugar beets. Zinc deficiency is often quite pronounced on old corral sites and barnyards. It is believed that rapid growth of microorganisms may at least temporarily tie up available zinc. 6. Cool Soil Temperatures
Zinc deficiencies are often encountered on field crops during the early growing season, and then disappear by midseason (Ferres, 1949; Millikan, 1953; Pumphrey and Koehler, 1959). In Colorado, zinc deficiencies are often severe during cool, wet spring seasons and disappear by mid-July.
ZINC IN SOILS AND PLANT NUTRITION
161
Several explanations might be offered to account for these observed zinc deficiency patterns. One is that the root system of the plants is not well established in cool soils so that their feeding zone is restricted. A second explanation is that available zinc may come from organic matter, and in cool soils the reduced microbiological activity is such that insufficient available zinc is released. When the soil warms up, sufficient zinc is often released. Bauer and Lindsay (1965) using a short-term uptake technique showed that soils incubated at 43OC for 1-3 weeks released available zinc to corn plants. However, they were unable to detect the temperature-released zinc by chemical soil test extractants. This is not surprising when it is recognized that 10-40 times more zinc was extracted by the chemical methods than was extracted by the plants. Martin et al. (1965) showed that high phosphorus induced zinc deficiency in tomatoes at low temperatures but not at high temperatures. Often in Colorado corn appears to be normal until the weather suddenly becomes wet and cool. The new growth during the cool, wet spells is often chlorotic and severely yellow, sometimes with an abrupt contrast between yellow and green on the leaf.
7 . Plant Species and Varieties Plants differ widely in their ability to obtain zinc from soils. Not only do these differences occur among species, but also among varieties. In Michigan it has been clearly shown that the Saginaw variety of pea bean does very well on zinc-deficient soils whereas the Sanilac variety does very poorly and easily becomes zinc deficient (Lucas and Knezek, 1972). A detailed review of the differential responses of plant genotypes to micronutrients including zinc has recently been published (Brown et al., 1972).
8. High Levels of Phosphorus High levels of available soil phosphorus have been implicated as contributing to zinc deficiency (Barnette et al., 1936; West, 1938; Boawn et al., 1954, 1957; Stuckenholtz et al., 1966; R. Ellis et al., 1964; Navrot et al., 1967; Ganiron et al., 1969; Rudgers et al., 1970; Boawn and Brown, 1968). Barnette et al. (1936) found that application of superphosphate with zinc fertilizer reduced the effectiveness of the zinc. In Utah, Thorne and Wann (1950) found that high levels of C0,-soluble phosphorus were often associated with zinc deficiency on fruit trees. In contrast, Viets et al. (1953) found no depressing effect of added phosphorus on zinc corn in central Washington.
162
W. L. LINDSAY
Olsen (1 972) reviewed the problem of zinc-phosphorus interactions in soils and plants in considerable detail. For this reason, a detailed review will not be repeated here. The solubility diagram in Fig. 1 demonstrates that Zn3(P0,)2.4H,0 is much more soluble than soil zinc. This means that Zn,(PO,),-4H,O would dissolve in soils and release zinc to plants, not make it less available. Addition of phosphorus to soils does not generally decrease the extractable zinc as measured by water (Bingham and Garber, 1960), by 0.1 N HC1 (Stuckenholtz et al., 1966), or by dithizone (Brown et al., 1970). Thus, it would appear that precipitation of an insoluble zinc phosphate in soils is not the cause of a phosphorus-associated zinc deficiency. Applied phosphorus has also been implicated as interfering with the uptake, translocation, or utilization of zinc (Thorne, 1957; Boawn and Brown, 1968; Adriano et al., 1971 ) . In other cases, phosphorus reduced zinc content in the tops, but total uptake was not reduced (Millikan, 1963; Boawn and Leggett, 1964; Watanabe et al., 1965; Jackson et al., 1967). Additions of phosphorus are known to depress the uptake of zinc (Boawn and Leggett, 1964; Brown et al., 1970). Somehow an excessive concentration of phosphorus interferes with the metabolic function of zinc at certain cites within plant cells; nevertheless, the zinc concentration per se is not the direct cause of the disorder. The actual cause of phosphorus-induced zinc deficiency is still unknown. Fortunately, the disorder can be alleviated by modest applications of zinc in the order of 5 to 10 kg/ha of zinc. Further investigation into the cause and effect relationship might logically be directed at the biochemical level within plant cells. 9. Eflect of Nitrogen
Nitrogen has been reported as affecting zinc availability, but its effect has been much less dramatic than that of phosphorus. Ozanne (1955) observed an increase in zinc deficiency in subterranean clover as the nitrogen supply increased and noted that the effect was not due to increased growth. He suggested that increased nitrogen resulted in greater protein nitrogen, which retained more zinc in the roots as a zinc-protein complex. Viets et al. (1957) studied the effect of various nitrogen sources on zinc availability. They found that pH changes accompanying the use of acidifying nitrogen fertilizers had the greatest effect on zinc uptake and growth. Boawn et al. (1960) compared (NH,)?SO,, NH,N03, and Ca(NO?), as nitrogen carriers and found that pH changes exerted the greatest effect-that is, nitrogen sources that reduced soil pH increased zinc availability. Nitrogen fertilizers sometimes increase the uptake of zinc quite effec-
ZINC IN SOILS AND PLANT NUTRITION
163
tively. Drake ( 1965) has suggested that nitrogen increase could exchange capacity of roots which in turn increases the absorption of zinc from luxury concentrations in soil.
c.
MOVEMENT OF ZINC
TO
PLANT ROOTS
1. Convection and Diflusion
Generally, two processes are involved in the movement of nutrients to plant roots-convection or the movement of nutrient with the soil solution and diffusion or the movement of nutrients through the soil solution. Olsen and Kemper (1968) made a detailed review of this subject, and more recently Wilkinson (1972) reviewed the principles of diffusion with regard to the micronutrients. The amount of nutrient that moves to plant roots by convection can be estimated by multiplying the concentration of that nutrient in the soil solution by the amount of water transpired by a plant (Barber, 1962). Using this method, Elgawhary et al. (1970a) estimated that less than 5% of the zinc required by a plant such as corn would normally be transported by convection, leaving diffusion to account for the remainder. Obviously, diffusion of zinc is an important factor that must be considered in accounting for the movement of zinc to plants. Using autoradiographs, Barber et al. (1963) demonstrated that zinc is depleted in the immediate vicinity of plant roots, demonstrating that diffusion gradients are established. Plants differ in absorbing nutrients from dilute solutions (Carroll and Loneragan, 1968, 1969), so they can be expected to establish different diffusion gradients. This may be one factor involved in explaining why some plants thrive and others become deficient in identical environments.
2. Intensity-Capacity Factors Not only is zinc in the soil solution diffusible to plant roots but also the labile solid phase zinc that is easily solubilized must be considered as potentially diffusible. Elgawhary et al. (1970a) used 65Znto measure the labile zinc in a Platner loam. They obtained a value of 571, indicating that for each unit of soluble zinc there were 571 units of labile zinc ready to replenish that removed from solution. Thus, labile zinc makes a very significant contribution to available zinc. If it were not for this labile zinc that buffers the soil solution zinc as zinc is removed by roots, soils would not constitute a fertile medium for plant growth. The movement of zinc to olant roots is denendent. therefore. both on
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W. L. LINDSAY
intensity factors (concentration) and on capacity factors (ability to replenish). Increasing the pH decreases the solubility of zinc in soils (Lindsay and Norvell, 1969a), and thereby reduces the concentration, the concentration gradient and, hence, the uptake and availability of zinc to plants.
3. Role of Chelates in Zinc Movement Chelating agents increase the concentration of metals soil solution. The extent to which they combine with zinc determines the solubility of zinc. Elgawhary et al. ( 1970a) showed a direct relationship between the amount of chelated zinc in soil solution and the apparent diffusion coefficient of zinc in soil. Chelates can increase the movement of zinc both through convection and through diffusion. The increased concentration means that more of the chelated metal is transported with the transpiration stream and also that greater diffusion gradients are established between the bulk solution in the soil and the level of nutrient at the absorbing root. Hodgson et al. (1967) demonstrated that citrate at 2 X M increased the transport of zinc across an agar block 100 times that of a water control. Elgawhary et al. (1970b) showed that chelating agents acting as root exudates in a simulated root greatly increased the rate of diffusion of 65Zn from soil into the simulated root. Similar processes are expected to increase the availability of metal ions in soils (Lindsay 1972b).
D. ABSORPTION OF ZINC
BY P L A N T S
1 . Rates of Zinc Uptake
Carroll and Loneragan (1969) measured uptake rates of zinc as low as 2 ng atoms per gram fresh root per day. They used continuous-flow nutrient solution techniques to ensure that zinc was not depleted during uptake. Schmid et al. (1965) reported uptake rates as high as 4000 ng atoms per gram of fresh root per day with excised barley (Hordeurn) roots. These rates, if continuous, would lead to deficiencies on the one hand and toxicities on the other. Hewitt (1966) reported optimum levels of zinc for plants grown in nutrient solution to be from 0.3 to 3.0 @I. Carroll and Loneragan (1968) grew several species over a range of zinc concentrations. All plants grew well at 0.01 pM zinc and gave maximum growth at 0.25 pM zinc or less. Uptake of zinc was linear with concentration to about 10 p M zinc in the nutrient solution. Toxic effects were encountered in the 1-6 p M level, which is considerably below toxic levels previously reported. Apparently the higher toxic levels previously reported were initial concentrations that were not replenished during uptake.
ZINC IN SOILS AND PLANT NUTRITION
165
2. Active vs. Passive Uptake Moore (1972) reviewed the broad aspects of micronutrient uptake by plants. Considerable controversy exists as to whether zinc uptake by roots is active or passive. Much of this controversy arises because early workers did not differentiate between passive exchange adsorption and active accumulation by cells (Epstein and Leggett, 1954). Passive absorption arises from electrostatic adsorption on cell walls and other surfaces within the apparent free spaces of roots. This binding is nonselective and nonmetabolically activated. In contrast, active accumulation is highly selective and metabolically activated (Schmid et al., 1965). In short-term uptake experiments, exchangeable zinc may constitute 90% or more of the total zinc held by the roots. Since exchangeable zinc reaches saturation readily, it constitutes a smaller and smaller percentage of the total zinc uptake as absorption continues. As a result, many of the short-term uptake experiments in which exchangeable zinc was not removed must be discounted as proof that zinc uptake is passive (Broda et al., 1964; Rathore et al., 1970). Other studies in which sufficient calcium was not used in the medium to maintain the cell membrane integrity must also be discounted. Tiffin (1967) and Ambler et al. (1970) reported zinc concentrations in xylem exudates from decapitated tomatoes (Lycoperiscon) and soybeans (Glycine soju) that were considerably higher than the nutrient solution. This would suggest active absorption only if the internal Zn is not chelated or present in forms different than that in the external solution. 3. Eflect of Chelating Agents
Chelating agents increase the solubilization of zinc from soils and facilitates its more rapid transport to plant roots as pointed out in Section 111, C, 3. In nutrient solutions chelating agents often have a detrimental effect on zinc uptake. DeKock and Mitchell (1957) reported that zinc uptake by mustard (Brassku) plants was decreased by the addition of DTPA to nutrient solutions. J. C. Brown (1961) and J. C. Brown et al. (1960) reported that soybeans became chlorotic with shortened internodes. They attributed the symptoms to iron deficiency, but the symptoms are even more suggestive of zinc deficiency. Guinn and Joham (1962) showed that EDTA and HEEDTA (hydroxyethylethylenediaminetriacetic acid) substantially decreased the zinc content of cotton (Gossypium) plants. Halvorson (1971) grew corn plants in nutrient solutions supplied with zinc at 1.54 p M and either EDTA or DTPA as an iron source. When the nutrient solutions were maintained at pH 5, growth was normal, but at pH 7.5 the DTPA treatment caused severe zinc deficiency. This deficiency was confirmed by response of plants to ZnSO,. To seek an explana-
166
W. L. LINDSAY
tion for these observations, Halvorson calculated the free Zn2+concentration as a function of pH for the nutrient solution. The results are plotted in Fig. 6. Both chelates reduced the concentration of Zn2+similarly below pH 7, but above this pH DTPA continued to depress Zn2+,rendering it unavailable to the plants. At pH 7.5 DTPA had depressed Zn*+to about lo-”.’ M or 25-fold below that of EDTA. The EDTA treatments produced normal plants, but the zinc content was considerably below that of plants grown at pH 5 . Chelate equilibrium calculations help in understanding the competition between chelating agents and plants for the uptake of zinc (Lindsay and Norvell, 1969a; Halvorson, 1971; Halvorson and Lindsay, 1972).
6
7
e
+
?
C
N
u I7
_t 0
I
IC
ii
I2
I:
FIG.6. The concentration of unchelated Znz+ in a nutrient solution with either ethylenediaminetetraacetate (EDTA) or diethylenetriaminepentaacetate (DTPA) at 100 pM and a total zinc concentration of 1.54 From Halvorson (1971).
a.
ZINC IN SOILS AND PLANT NUTRITION
167
4 . Eflect of Other Zons Chaudhry and Loneragan (1972) working with wheat (Triticum) showed that alkaline earth cations strongly depress zinc uptake over a wide range of concentrations. The order of effectiveness was Mg2+> Ca2+= Ba2+= Sr2+. Bowen (1969) and Schmid et al. (1965) showed that Cu2+depresses the uptake of Znz+ and appears to be absorbed at the same uptake cite. MnZ+was less competitive with Zn2+. Khadr and Wallace (1964) indicated that addition of phosphorus depressed the uptake of zinc by citrus seedlings. However, large additions of K+ (10,000 /.M) included with the phosphorus may have contributed to the depression. When cation concentrations were kept low and constant (Pearson, 1951), phosphorus affected zinc uptake similarly to C1-, NO,-, or SO,2-.
E. DISTRIBUTION OF ZINC
IN PLANTS
1 . Content The usual levels of zinc in plants range from 10 to 100 ppm for most crops and pasture plants (Chapman, 1966; Gladstones and Loneragan, 1967). Zinc content is generally highest in very young seedling and decreases with age (Riceman and Jones, 1958; Gorsline et al., 1965; Carroll and Loneragan, 1968). Reported decreases in zinc content with age often reflects depletion of available zinc in the soil or nutrient solution. Plant species and varieties differ widely in their ability to accumulate zinc even from the same nutrient solution or soil (Carroll and Loneragan, 1968; Gladstones and Loneragan, 1967). These differences are undoubtedly important in determining the susceptibleness of various crops to zinc deficiencies. 2 . Distribution within Plants Zinc is intermediate in its mobility within plants compared to that of other nutrients. When 65Znis introduced into the rooting media of plants, translocation of the radioactive tracer to other parts of the plant usually occurs within a few hours. Thorne (1957) reviewed some of the earlier work on the effects of phosphorus, iron, nitrogen, and light on the translocation of zinc in plants. Roots often show much higher zinc contents than tops, particularly if the plants are grown in media of high available zinc. Riceman and Jones
168
W. L. LINDSAY
(1956) showed that reducing the zinc supply to plants such as subterranean clover (Trifolium subterraneum) causes zinc to move from the roots to the tops until the zinc level in the roots approaches that of the tops. Roots usually accumulate luxury levels of zinc if the supply is adequate (Carroll and Loneragan, 1968). If the plants grow into zinc deficiency, much of the zinc is redistributed and utilized by the top. Bukovac and Riga (1962) followed the distribution of zinc and other nutrients from the cotyledon of bean. On day zero 94% of the zinc was in the cotyledons and 6% in the other tissue. On day 6, 43% of the zinc was in the cotyledons and 57% in other tissue. Since some plants shed their cotyledons at an early stage, rapid export is important. In the case of zinc, half the zinc was exported in 6 days. Massey and Loeffel (1967) studied the distribution of zinc in corn. One strain showed an uptake of 3 mg zinc per plant between tasseling and maturity. The accumulation of zinc in the ear was greater than 3 mg, indicating transfer of zinc from other parts. The stalk contributed most, with portions above and below the ear each losing about half their zinc. Many biennial and perennial plants conserve nutrients by translocating them from senescing leaves and other vegetative tissues.
3. Transport Mechanisms for Zinc Most nutrients absorbed by roots are transported to other parts of the plant through the xylem while leaf metabolites and some mineral elements in the leaves are exported from leaves through the phloem. Most evidence indicates that the load of zinc ascending the xylem exceeds that translocated out of the leaves in the phloem (Tiffin, 1972). Electrophoretic evidence indicates that zinc is not bound to highly stable ligands in the xylem fluid as are other metal cations such as Ni2+,Cu'+, and Fe3+.Tiffin (1967) found that zinc in xylem exudates of tomatoes to be slightly cathodic. The possibility of a zincxitrate complex was dispelled on the basis that synthetically prepared zinc-citrate complexes were anodic. F. FUNCTION OF
ZINC IN
PLANTS
The relationship of zinc in soils and plant nutrition will never be fully complete until we know the exact role that zinc plays in the metabolism of plants. Just a few years ago carbonic anhydrase was the only authenticated zinc metalloenzyme in living organisms (Price et al., 1972). Thorne (1957) reviewed some of the research that showed zinc plays an important role in auxin formation and in other enzyme systems. Presently zinc is recognized as an essential component in several dehydrogenases, proteinases, and peptidases (Vallee and Wacker, 1970). For plants
ZINC IN SOILS AND PLANT NUTRITION
169
these include: ( 1) carbonic anhydrase, (2) alcohol dehydrogenase, ( 3 ) glutamic dehydrogenase, (4) L-lactic dehydrogenase, ( 5 ) D-glyceraldehyde-3-phosphate dehydrogenase, (6) malic dehydrogenase, (7) D-lactic dehydrogenase, ( 8 ) D-lactic cytochrome c reductase, and (9) aldolase. Price et al. (1972) cited several investigators to show that one of the earliest events in the onset of zinc deficiency is a sharp decrease in the levels of RNA and the ribosome contents of cells. Prask and Plocke (1971) found that ribosomes of Euglena gracilis become extremely unstable with zinc deficiency. As pointed out by Price et al. (1972), if it is confirmed that zinc is a component essential to the stability of cytoplasmic ribosomes, this specific function of zinc for normal plant growth will be received with considerable interest. IV.
Diagnosing Zinc Deficiencies
The first methods used to detect zinc deficiencies were visual observations of deficiency symptoms response to foliar application of Zn. Plant analyses and soil tests for zinc are now becoming more widely used. Atomic absorption spectrophotometry (Isaac and Kerber, 1972) and direct reading emission spectroscopy (Jones, 1972; Brech, 1968) have greatly simplified analytical procedure for zinc and have largely replaced other methods. Patterns of zinc deficiency symptoms were considered briefly in Section 111, A. A.
PLANTANALYSIS
According to Jones (1972) over 400,000 plant samples are analyzed annually in the United States as a service to farmers and growers. Plant tissue analysis should reflect the ability of the soil to supply a nutrient element under the conditions of plant growth. In this respect, soil tests may not accurately reflect this soil-plant interaction, which may even change during the course of the growing season. Plant analysis must be accompanied by proper collection and handling of plant samples. Several comprehensive reviews are available on the sampling procedures useful for plant analysis (Chapman, 1964; Kenworthy, 1964). Floyd and Ragland ( 1966) showed that dust contamination affects the analysis of zinc in plant tissues less than it affects elements, like iron and manganese, that are more concentrated in dust. Labanaukas (1968) washed citrus leaves in detergent and rinsed in 3% HC1 to reduce foliage spray contamination of zinc and other heavy metals. Various plants and parts of plants differ considerably in the levels of zinc that accompany zinc deEciencies. Neubert et al. (1969) have pub-
170
W. L. LINDSAY
lished interpretation data for diagnosing nutrient deficiencies in 25 crops. The zinc content of sugar beet leaves was found to be about 3-fold that in the petioles (Jones, 1964). Jones and Mederski (1964) found zinc in soybean leaves to increased slightly with age, but decreased with age in the stem. Viets et al. (1954a) proposed using the upper mature leaves of bean plants to diagnose zinc deficiency. They later classified 26 crop plants as to their responsiveness to zinc fertilizer based upon tissue analysis and yield response (Viets et al., 1954b). Apparently alfalfa (Medicago sativa) requires about one-third to one-half the amount of zinc required by many crops (Lo and Reisenauer, 1968). Goodall and Gregory (1947) reviewed much of the early work on plant analyses as a means of diagnosing zinc deficiency in plants. The most significant advances in this field have been made for tree fruit crops (Kenworthy, 196 1;Chapman, 1961 ). There is no general agreement as to a critical level of zinc in plants below which zinc deficiencies occur and above which they do not. In fact, there is ample evidence to show that plants differ widely in the level of zinc required for maximum growth (Chapman, 1960, 1966). Wets et al. (1953) concluded from field experiments with corn that 15 ppm in the mature leaves (sixth leaf from the base or second leaf from the nodes below the upper ear node), selected when the pollen is shedding, was adequate for a crop of at least 100-125 bushels per acre. B.
SOILTESTSFOR ZINC
Farmers may wish to apply fertilizer when no information is available on zinc deficiency symptoms or plant tissue analyses. Reliable zinc soil tests can be extremely useful under these conditions. Recently two detailed reviews on micronutrient soil tests have appeared (Cox and Kamprath, 1972; Viets and Lindsay, 1972). No attempt will be made here to repeat the detailed material in these reviews. Instead, some additional insights and discussion regarding zinc soil tests will be presented. The prime objective of a zinc soil test is to determine whether a given field will show zinc deficiency for certain crops. If it meets this criterion, the test will automatically be a good indicator of residual value as the extractable zinc approaches the critical level once again. Most crops require only small quantities of zinc, usually less than 0.4 kg/ha. The question of how much zinc fertilizer should be used is of secondary importance. Usually a modest application rate of 4-10 kg/ha of zinc is adequate for several years. Considerable discussion has ensued as to the various forms of zinc that are present and available in soil. For example, Viets (1962) considered
ZINC IN SOILS AND PLANT NUTRITION
171
five pools: ( 1) water soluble; (2) exchangeable; ( 3 ) specifically adsorbed, chelated, or complexed; (4) secondary clay minerals or oxides; and (5) primary minerals. However, plants and most of the chemical extractants used to assess available zinc are unable to distinguish among these discrete forms. Diffusion theory is useful in showing that both intensity factors and capacity factors af€ect nutrient availability in soils (Olsen and Kemper, 1968). Elgawhary et al. (1970a) measured a labile zinc value of 571 for a soil with which they worked. This means that 571 units of zinc are ready to replenish each unit of zinc in solution as it is removed during absorption. A good soil test for zinc should reflect both the intensity factor and capacity factor associated with both soluble and readily labile zinc. Chelating agents that react with zinc to form soluble complexes offer unique advantages over many other soil test methods (Trierweiler and Lindsay, 1969; Viets and Lindsay, 1972). First of all, chelating agents combine with free Zn2+according to the reaction Zn2+
+ Ln-
ZnL(n-2)-
where L represents the chelating ligand and n is the negative charge of the free ligand. During the reaction, chelated Zn accumulates in solution as the chelating agent combines with Zn2+,causing more zinc to be released from the labile solid phases. Soluble metal chelates are easily separated from the solid phase matrix of the soil by filtration, and zinc in the filtrate can easily be measured by direct atomization of the filtrate into an atomic absorption spectrophotometer. An advantage of chelate extractants over strong acids is that the pH of the extracting media can be carefully selected and controlled. This prevents gross destruction of acid-soluble soil minerals such as carbonates and oxides. In calcareous soils especially, the destruction of carbonates may release occluded zinc that is normally not accessible for use by plants. Nelson et al. (1959) proposed to include titratable alkalinity as a significant variable to achieve a better separation of calcareous soils into deficient and nondeficient classes. Viets and Boawn (1965) concluded that only two zinc soil test methods had been sufficiently calibrated against plant uptake in either field or greenhouse to be useful at that time. These were the NH,OAc-dithizone and the 0.1 N HCl methods. Stewart ,and Berger (1965) used MgCI, to extract water soluble and exchangeable zinc. Trierweiler and Lindsay (1969) used EDTA buffered at pH 8.6 with 1 M (NH,)zCO, to prevent the destruction of CaCO,. They concluded that the EDTA extraction excelled the 0.1 N HC1 procedure and was much more convenient than the dithizone procedure. (Brown and Krantz, 1960; Brown et al. 1962; Brown et al. 1970; Shaw and Dean, 1952).
172
W. L. LINDSAY
Later Lindsay and Norvell (1969b) reported the use of DTPA (diethylenetriaminepentaacetic acid) as an extractant for the simultaneous determination of zinc, iron, manganese and copper deficiencies. The extractant consists of 0.005 M DTPA, 0.01 M CaCI,, and 0.1 M TEA (triethanolamine) buffered at pH 7.30. In the extraction, 10 g of soil are shaken with 20 ml of extractant for 2 hours and filtered. The concentrations of zinc, iron, manganese, and copper are determined directly on the extracts by atomic absorption spectrophotometry. Using this test, Lindsay and Norvell were able to successfully separate 77 Colorado soils into the proper zinc-deficient and nondeficient categories. The method has the advantage that not only zinc but also iron, manganese, and copper can be determined simultaneously (Follett and Lindsay, 1970, 1971). Brown ct al. (1971) examined 92 fields in California with four different zinc soil tests. They reported predictive values of 83% for DTPA, 79% for dithizone, 73% for 0.1 N HCl, and 72% for EDTA. They indicated an overall preference for the DTPA method with a critical level of 0.5 ppm of zinc, which separated the deficient from the nondeficient soils. Dolar and Keeney (1971 ) reported poorer results with DTPA compared to 0.1 N HCl and EDTA buffered at pH 7.0. It should be pointed out, however, that they buffered their DTPA extracting solution at pH 10.9 rather than 7.3 as proposed by Lindsay and Norvell. Lauer ( 1971 ) recently reported some interesting results with respect to DTPA, 0.1 N HCl, and labile soil zinc. He labeled 30 soils with "Zn and subjected them to alternate wetting and drying cycles for approximately 1 month. He grew corn on the labeled soils and extracted them with various chemical extractants. The measured mean labile zinc values for the soils were 4.6 by the corn plants, 4.3 by DTPA, and 7.7 ppm of zinc, by 0.1 N HCl. The labile zinc values reflected by the corn plants and DTPA were almost identical and highly correlated ( r = 0.98). The larger labile zinc values for 0.1 N HC1 were interpreted to indicate that 0.1 N HCl was extracting zinc that was not available to the plants nor extracted by DTPA. Martens et al. (1966) similarly concluded that much of the zinc extracted by 0.1 N HCl over dithizone is not extractable by plants. Wear and Evans (1968) compared several chemical extractants for zinc. Their best correlation coefficient between extractable zinc and zinc uptake by corn ( r = 0.89) was obtained with 0.05 N HCI 0.0025 N H,SO,. Their study was restricted to acid soils with coarse to medium texture. Massey (1957) found a multiple correlation coefficient of 0.80 when both pH and dithizone-extractable zinc were correlated with zinc uptake. Again, the study was restricted to 34 acid Kentucky soils. In acid soils it would appear that the 0.1 N HCl method and dithizone
+
ZINC IN SOILS AND PLANT NUTRITION
173
are about equally effective in predicting zinc deficiency. When soils containing CaC03 are included, the EDTA, DTPA, and dithizone methods excel the 0.1 N HCl method. The EDTA and DTPA procedures are much more convenient to use than dithizone. The DTPA method has the advantage that zinc, iron, manganese, and copper can be extracted simultaneously. In conclusion, the DTPA micronutrient soil test appears to be one of the more promising general soil tests for zinc. A careful comparison has not been made of the various EDTA extraction procedures and the importance of a buffer at pH 7.0, pH 8.6, or with no buffer at all. Such comparisons would seem worthwhile.
V.
Correction of Zinc Deficiency
The widespread occurrence of zinc deficiency could make it a serious nutritional problem. Fortunately, zinc deficiencies are relatively easy to correct, either with sprays or with soil application of zinc fertilizers. Usually soil applications in the range of 5 kg/ha of zinc will last 3-8 years (Boawn et al., 1960; Brown et al., 1964; Viets, 1961). Thorne (1957) reviewed some of the early work on the correction of zinc deficiency by means of soil treatments, trunk injections, and foliage sprays. These methods include driving zinc-coated nails into trunks of trees, painting pruning wounds with ZnSO,, dipping root transplants into zinc solution, and applying foliar and dormant sprays. Numerous field and greenhouse trials have been conducted to evaluate the most effective methods of correcting zinc deficiencies. Murphy and Walsh (1972) have recently reviewed this subject. Often in a rapidly developing area of science, it is difficult to see the forest because of the trees. This has been the case in the evaluation of micronutrient fertilizers. In this section no attempt will be made to include all the details of the many studies that have been conducted with zinc fertilizers. Instead, attention will be focused upon the basic principles that emerge as a guideline for the use of zinc fertilizers. A.
SPRAYSAND INJECTIONS
Sprays have been most successful for correcting zinc deficiency on trees (Chapman, 1966). Typical recommendations for foliar zinc sprays are 0.9-4.5 kg of ZnSO,.7H,O per 378 liters of water (2-10 lb/lOO gal) applied in the early summer and repeated as necessary. Often hydrated lime is included to neutralize acidity and avoid leaf and fruit damage. Foliar sprays are most commonly used on pineapple (Ananas comosus), citrus,
174
W. L . LINDSAY
deciduous fruit, and nut trees. Dormant sprays, which are applied in late winter or early spring, are also used. Typical sprays range from 4.5 kg to 23 kg of ZnSO,.7H,O per 378 liters of water (10-50 lb/100 gal) (Chapman, 1966). Zinc chelates have also been used as foliar sprays. In field and vegetable crops, foliar zinc sprays are used mostly as emergency treatments after deficiency symptoms have appeared. For corn, a typical treatment consists of 920 liters per hectare (100 gal per acre) of 0.5% ZnSO,.7H,O applied at the 3-leaf and again at the 5-leaf stage of growth (Lingle and Holmberg, 1957). The exact concentration and application rates vary from crop to crop based on experience, Generally, zinc deficiency in field and vegetable crops are corrected with soil-applied zinc rather than with sprays (Viets, 196 1 ) . Placing zinc-coated nails in tree trunks has been used as a means of correcting zinc deficiency in trees (McWhorter, 1945 ) . However, this practice has never been widely used, nor has it been entirely successful.
B. ZINC FERTILIZERS The efiectiveness of various zinc fertilizer sources in supplying zinc to plants can be best explained from the chemical reactions and solubility relationships of these materials in soils. These reactions and the rates at which they occur are highly dependent upon soil pH and how the fertilizers are placed. For this reason, fertilizer sources and placement will be discussed together. 1 . Inorganic Zinc Sources From the zinc solubility diagram in Fig. 1 it is obvious that inorganic zinc compounds such as ZnO, ZnCO,, and Zn, (PO,) 2 . 4H,O are sufficiently soluble to supply available zinc to plants. The Znz+ level maintained by these compounds is approximately 10' times greater than that normally found in soils indicated by the soil-Zn line in Figs. 1 and 2 (see Section 11, C and F). Nevertheless, many fertilizer experiments show that under some conditions these compounds do not always correct zinc deficiencies (Boawn er al., 1957; Brown and Krantz, 1966). The question is asked, why? First, physical accessibility of the fertilizers to plant roots is essential. When zinc compounds of intermediate solubility are granulated or banded, they remain localized so that only a small fraction of the total soil volume is affected. Brown and Krantz (1966) elegantly demonstrated that granulation or localization of inorganic zinc sources generally reduces their availability. On the other hand, mixing these zinc sources with as little as % s of the total soil volume was adequate to correct zinc deficiency. The effect of granulation in restricting zinc movement is most pronounced
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at high pH because most of the common inorganic zinc compounds used as fertilizers decrease in solubility with increase in pH (Fig. 1). Zinc sulfate is perhaps the most commonly used inorganic zinc fertilizer. This material is highly water soluble and dissolves readily in soils. The released zinc can precipitate as the oxides, hydroxides, carbonates, or silicates, or it can be adsorbed onto the soil matrix. Band-applied ZnSO, frequently shows slightly greater availability than similar applications of granular ZnO or ZnCO,, particularly in high pH soils. In acid soils the difference between ZnSOi vs ZnO and ZnCO, is usually very slight because ZnO and ZnCO, are also too soluble to persist. Other inorganic sources of zinc follow similar patterns. Materials such as ZnS and many zinc frits have not been successful as zinc fertilizers because they dissolve so slowly that their reaction zone is restricted to the immediate vicinity of the fertilizer granule. If these materials are finely ground and mixed into the soil, their availability to plants is generally increased. They are also generally more effective in acid than in alkaline soils. Furthermore, rather large applications are necessary to compensate for their slow release. 2 . Zinc Chelates Numerous studies have been made to compare the effectiveness of zinc chelates with inorganic zinc fertilizers (Lingle and Holmberg, 1957; Boawn et al., 1957; Judy et al., 1965; Vinande et al., 1968; Brown and Krantz, 1966; Benson et al., 1957; Stewart and Leonard, 1957; Chesnin, 1963). Generally, zinc chelates are more available than inorganic sources per unit of zinc, but numerous exceptions are frequently encountered that are difficult to explain without some basic understanding of metal chelate equilibria in soils. These equilibrium relationships, as they relate to zinc, were discussed in Section 11, G and will be referred to here to demonstrate their usefulness. Anderson (1964) examined the effectiveness of several zinc chelates for correcting zinc deficiency in corn in a calcareous soil. His results are summarized in Fig. 7. This study was conducted in the greenhouse and all fertilizer materials were applied in a band. The relative effectiveness of the zinc chelates for supplying zinc were in the order ZnDTPA > ZnEDTA > ZnEDDHA > ZnSO, > ZnRayplex (polyflavinoid). The slight superiority of ZnDTPA over ZnEDTA in a calcareous soil near pH 8 is predicted from the chelate stability diagram in Fig. 4. For soils in the pH range of 6 to 7 no differences would be expected between ZnEDTA and ZnDTPA. ZnEDDHA was a poorer source of zinc because it is unstable in soils where Zn2+is displaced by Fe3+ (Lindsay et al., 1967). The low availability of ZnSO, reflects its restricted movement from the band.
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i
00 ~
t
2
3
4
5
Z n Added-ppm
FIG.7. Effectiveness of various zinc chelates as fertilizers for corn when banded in a calcareous soil. ZnDTPA, zinc diethylenetriarninepentaacetate; ZnEDTA, zinc ethylenediaminetetraacetate; ZnEDDHA, zinc ethylenediarninedi(0-hydroxyphenylacetate). From Anderson ( 1964).
ZnRayplex also rapidly dissociates in soils and its zinc is released (Anderson, 1964). The effectiveness of other zinc chelates in increasing zinc solubility in soils can be predicted similarly from Fig. 5 and from Norvell (1972). The decreased effectiveness of most chelating agents for Zn?- at low pH is caused by displacement of Zn?+ from the chelate by Fez+.Equilibrium pH diagrams of zinc chelates in soil provide an extremely useful tool for understanding and predicting the response that may be expected from the use of various chelates in different soils.
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Attempts are often made by well-meaning fertilizer dealers, extension workers, and farmers to arrive at an efficiency factor that will establish a cost basis of comparing chelated and inorganic zinc fertilizers. Such factors are often very misleading because soil pH and methods of placement are so important. If chelated and inorganic zinc sources are compared in band placement, the chelated zinc is favored (Fig. 7 ) . If they are mixed into a sufficient volume of the soil, the differences are greatly reduced because the inorganic sources become more accessible to roots (Brown and Krantz, 1966). If comparisons are made in calcareous soils, the differences are greater than in acid soils. If the chelated sources are used in extremely acid soils, they revert to iron chelates, and often chelated sources are no more effective than inorganic sources. Here, again, differences among chelates must also be considered (Fig. 5 ) . Many natural organic products, often referred to as chelates, readily dissociate in soils and behave essentially like water-soluble inorganic sources. Zinc chelates that are not readily dissociated in soils are most useful where fertilizers are granulated or applied in bands, especially in high pH soils. The greater mobility of the chelated zinc enables it to move into a greater volume of soil when the probability of root-fertilizer contact is increased. If chelated fertilizers are mixed or placed in acid soils, the advantage over inorganic sources is much less.
3. Zinc in Macronutrient Fertilizers It is often desirable to incorporate zinc in macronutrient fertilizers to reduce costs and eliminate separate fertilizer applications. The question naturally arises: Is the efficiency of the zinc affected by such combination? Silverberg et al. (1972) reviewed the technology related to the incorporation of micronutrients in macronutrient fertilizers including solid, fluid, and suspension fertilizers. Lehr (1972) reviewed the subject of chemical interaction between added micronutrients and macronutrient carriers. Such interactions are important because it is the reaction products of the fertilizer that react with the soil into which they are placed. Since these two review papers are rather detailed and up to date, much of the information therein will not be repeated here. Giordano and Mortvedt (1972) reviewed investigations relating to the agronomic effectiveness of zinc in macronutrient fertilizers. Most of these studies have been made by the Tennessee Valley Authority (TVA) and their cooperators during the past several years. A few additional findings not included in their review will be presented here. Zinc fertilizers are frequently combined with various nitrogen carriers. From the work of Boawn et al. (1960), Terman et al. (1966), and Mortvedt and Giordano (1967), it appears that nitrogen carriers such as
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NH,NO, and (NH,),SO, are among the most advantageous combinations. With or without macronutrients, granulation decreases zinc solubility and distribution in soils. Lauer (1969) examined the possibility of forming soluble zinc-NH, complexes in the vicinity of fertilizer granules that would increase the mobility of zinc into the surrounding soil. The results indicate that zinc-NH, complexes are not sufficiently stable to increase the solubility of zinc significantly in the pH range below 7.5. The formation of soluble ZnSO,(aq) increases the soluble zinc to some extent and may be an important factor in accounting for the beneficial effect of SO,?- salts in enhancing the availability of zinc. This point needs further investigation. When zinc fertilizers are combined with phosphate fertilizers, numerous reaction products are formed (Lehr, 1972). Some of these products contain NH,' or K+ when their salts are present in the mixture. Most of the reaction products, including Zn,( PO,) 2 . 4H,O, are sufficiently soluble in soils to furnish available zinc to plants (see Sections 11, C and 111, B, 8 ) . Some of the more insoluble products retard the reactions of zinc with soil
,
0
I
I
I
4
Zn
Added, ppm
FIG.8. Ineffectiveness of liquid polyphosphate for increasing zinc availability to corn. A , zinc ethylenediaminetetraacetate plus monoammonium phosphate (MAP) ; 0, ZnSO, plus MAP; D, ZnSOI plus polyphosphate (25-100%). From Truelson (1967).
ZINC IN SOILS AND PLANT NUTRITION
179
and tend to restrict its movement away from the fertilizer granule or band. This effect is more noticeable in localized placement than mixed placement. Incorporation of zinc in liquid ammoniated polyphosphate fertilizers (APP) has been evaluated as a zinc source (Mortvedt and Giordano, 1967). Often liquid carriers are superior to their granular counterparts because of better distribution in the soil. Again under mixed placement the differences are less pronounced. There was considerable conjecture when liquid 1 1-33-0 ammoniated superphosphoric acid (APP) was first used that zinc-polyphosphate complex was sufficiently stable to increase zinc solubility and availability in soils. Truelson (1967) compared the effectiveness of zinc incorporated with monoammonium phosphate (MAP) vs. polyphosphate (APP) . The results of a greenhouse trial with corn are summarized in Fig. 8. Obviously, that ZnS04 incorporated with polyphosphate had no beneficial effect over its incorporation with MAP. Various combinations of orthophosphate and polyphosphate varying from 0 to 100% polyphosphate were included in the study and gave almost identical zinc uptake as the zinc-MAP combination. ZnEDTA was a much superior zinc source to zinc polyphosphate. Truelson further showed that the zinc-polyphosphate complex is unstable in soils and contributes very little in keeping zinc more soluble. The calculations of Norvell (1972) relative to zinc-P,O, and zinc-P,OIo complexes in soils also suggest that the complexes are sufficientlyunstable to be discounted as beneficial in chelating or complexing zinc in soils. They are beneficial in the liquid fertilizer in permitting easy additions of zinc to liquid phosphorus fertilizers.
c.
RESIDUAL EFFECTS OF
ZINC
FERTILIZERS
Most soil test extractants and plant-uptake trials indicate that zinc fertilizers have a significant residual effect that persists for several years, depending upon the crop, the soil, and the rates at which the fertilizers are applied. Boawn et al. (1960) reported that approximately 35% of an 18 kg/ha application of zinc was still extractable with 0.1 N HC1 after four cropping seasons from the upper 20 cm of a Ritzville fine sandy loam. After 5 years no significant differences were found from plots receiving up to 4.5 kg/ha over plots receiving no zinc fertilizer. Brown et al. (1964) found that 2.5 ppm of zinc as ZnSO, was adequate for 6 or 7 successive crops. When 12.5 ppm of zinc was added, 10 successive crops were grown in the greenhouse on a previously zinc-deficient soil without zinc deficiency recurring. Dithizone-extractable zinc declined gradually with cropping, and when it reached 0.55 ppm, there was a plant response to further addition of zinc. Follett and Lindsay (1971) showed that DTPA-extractable zinc
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on 11 soils in the greenhouse declined rapidly during the first week and gradually thereafter. More recently, Boawn (1971 ) reported similar declines in DTPA-extractable zinc over a 3-year period in field investigations. Present results indicate that zinc fertilizers will show a residual effect for 2-8 or more years depending upon the crop, the soil, and the rate of fertilizer addition. Furthermore, soil test extractants such as 0.1 N HC1, dithizone. and DTPA appear to be successful in monitoring available zinc levels and determining when additional zinc fertilizer is required. VI.
Summary and Future Research Needs
Zinc deficiency in agricultural crops is increasing in many parts of the world. It is one of the most frequently encountered micronutrient deficiencics. Our knowledge of the chemistry of zinc in soils is increasing. Recent measurements of Zn'- concentrations in a limited number of soils permit some broad generalizations so that mineral solubility diagrams, ionic species in solution, and stability-pH diagrams for zinc chelates can be developed. Such equilibrium relationships are extremely valuable in helping to understand the behavior of zinc in soils and fertilizers. Further investigations are necessary to characterize more fully the solid phase matrix of soil responsible for controlling Zn'+ concentration in soils and for testing the initial measurements of Zn'- presented herein. The importance of diffusion and mass flow as transport mechanisms for moving zinc to plant roots has been clearly demonstrated. Chelates play an important role in these transport processes that are most frequently the rate-limiting step in zinc uptake by plants growing in soils. These processes need further critical examination. Perhaps the most fascinating development from the author's point of view is the development of a theoretical basis for understanding the equilibrium relationship of zinc and other metal chelating agents in soils. Such advances have proved to be extremely useful in explaining quantitatively the exchange of metal ions from chelating agents. These relationships provide a sound basis for understanding and interpreting the behavior of chelating agents in soils and their effect on zinc availability to plants. Further investigations are needed to characterize the natural organic complexes of zinc so that they, too, can be handled more than on an empirical basis. The use of zinc fertilizers has expanded greatly during the past twenty years. Hopefully, our increased knowledge of zinc chemistry in soils and fertilizers will enable continued advances in this area without needless repetition of empirical approaches too often used as the research models of the past.
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Labanaukas, C. K. 1968. Calif. Agr. 22. 12-14. Lauer. D. A. 1969. Unpublished M.S. Thesis, Colorado State University, Fort Collins. Lauer. D. A. 1971. Ph.D. Thesis. Colorado State University, Fort Collins. Univ. Microfilms, Ann Arbor, Michigan (Diss. A b s t r . ) . Leeper, G. U’. 1952. Antw. Rex,. P/arif Physio/. 3, 1-16. Lehr, J. R . 1972. I n “Micronutrients in Agriculture” (I. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 459-503. Soil Sci. Soc.Amer., Madison, Wisconsin. Lindsay, W. L. 1972a. In “Micronutrients in Agriculture” ( J . J . Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 41-57. Soil Sci. SOC. Amer., Madison, Wisconsin. Lindsay, W. L. 1972b. I n “The Plant Root and Its Environment” (E. W. Carson, ed. ), Chapter 17. University Press of Virginia, Charlottesville (in press). Lindsay, W. L., and Norvell, W. A. 1969a. Soil Sci. SOC. A m e r . , Proc. 33, 62-68. Lindsay, W. L., and Norvell, W. A. 1969b. Agron. Abstr. p. 84. Lindsay, W. L., Hodgson, J . F., and Norvell, W. A. 1967. Trans. Cornm. I1 and IV, pp. 305-316. Int. SOC.Soil Sci., Aberdeen. Lingle, J . C., and Holmberg, D. M. 1957. Proc. Atner. SOC.Hort. Sci. 70, 308-315. Lo, S. Y . , and Reisenauer, H. M. 1968. Agron. J . 60., 464-466. Lucas, R. E., and Davis, J . F. 1961. Soil Sci. 92, 177-182. Lucas, R. E., and Knezek, B. D. 1972. I n “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 265-288. Soil Sci. SOC.Amer., Madison, Wisconsin. McWhorter, 0. T. 1945. Better Fruit 39(10), 1 1 . Martens, D. C . , Chesters, G., and Peterson, L. A. 1966. Soil Sci. SOC. Amer., Proc. 30, 67-69. Martin, W. E., McLean, J. C., and Quick, J. 1965. Soil Sci. SOC. Amer., Proc. 29, 411-413. Massey, H. F. 1957. Soil Sci. 83, 123-129. Massey, H. F., and Loeffel, F. A. 1967. Agron. 1. 59, 214-2 17. Millikan, C. R. 1953. Dep. Agr., Victorin, Aust., Tech. B u / / . 11. Millikan. C. R. 1963. Artsf. 1. Agr. Res. 14, 18Cb205. Mitchell, R . L. 1965. Itr “Chemistry of the Soil” (F. E. Bear, ed.), 2nd ed., pp. 320-368. Van Nostrand-Reinhold, Princeton, New Jersey. Moore, D. P. 1972. In “Micronutrients in Agriculture” ( J . J . Mortvedt, P. M. Giordano. and W. L. Lindsay, eds.), pp. 171-198. Soil Sci. SOC. Amer., Madison, Wisconsin. Mortensen, J . L. 1963. Soil Sci. SOC.Arner., Proc. 27, 179-186. Mortvedt, J . J., and Giordano, P. M. 1967. J . A g r . Food Cliem. 15, 118-122. Mortvedt, J . J., Giordano, P. M., and Lindsay. W. L., eds. 1972. “Micronutrients in Agriculture.” Soil Sci. SOC.Amer., Madison, Wisconsin. Murphy, L. S., and Walsh, L. M. 1972. 117 “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 347-387. Soil Sci. SOC. Arner., Madison, Wisconsin. Navrot, J., and Ravikovitch, S. 1969. Soil Sci. 108, 30-37. Navrot, J., Jacoby, B., and Ravikovitch, S. 1967. Plnrif Soil 27, 141-147. Nelson, J . L., and Melsted, S. W. 1955. Soil Sci. SOC. Arner., Proc. 19, 439-443. Nelson, J. L., Boawn, L. C., and Viets, F. G., Jr. 1959. Soil Sci. 88, 275-283. Neubert, P., Wrazidlo, W., Vielerneyer, H. P., Hundt, I., Gullmick, F., and Bergrnann, W. 1969. Research Report, Institut f u r Pflanzenernahrung Jena, Berlin.
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Norvell, W. A. 1972. In “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 115-138. Soil Sci. Soc. Amer., Madison, Wisconsin. Norvell, W. A., and Lindsay, W. L. 1969. Soil Sci. SOC.Amer., Proc. 33, 86-91. Norvell, W. A., and Lindsay, W. L. 1970. Soil Sci. SOC.Amer., Proc. 34, 360-361. Norvell, W. A., and Lindsay, W. L. 1972. Soil Sci. SOC.Amer., Proc. (in press). Oliver, S., and Barber, S. A. 1966. Soil Sci. SOC.Amer., Proc. 30, 468-470. Olsen, S. R. 1972. In “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 243-264. Soil Sci. Soc. Amer., Madison, Wisconsin. Olsen, S. R., and Kemper, W. D. 1968. Advnn. Agron. 20, 91-151. Ozanne, P. G. 1955. Aust. 1. Biol. Sci. 8, 47-55. Pearson, G. A. 195 1. Ph.D..Thesis, University of California, Berkeley. Prask, J. A., and Plocke, D. J. 1971. Plant Physiol. 48, 150-155. Price, C. A., Clark, H. E., and Funkhouser, E. A. 1972. fn “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 231-242. Soil Sci. SOC.Amer, Madison, Wisconsin. Pumphrey, F. V., and Koehler, F. E. 1959. Nebr., Agr. Exp. Sta., Circ. 102. Randhawa, N. S., and Broadbent, F. E. 1965a. Soil Sci. 99,295-300. Randhawa, N. S., and Broadbent, F. E. 1965b. Soil Sci. 99, 362-366. Rathore, V. S., Wittwer, S. H., Jyung, W. H., Bajaj, Y. P. S., and Adams, M. W. 1970. Physiol. Plant. 23, 908-919. Reuss, J. O., and Lindsay, W. L. 1963. Colo., Agr. Exp. Coop. Ex!. Ser. Pamphlet 59. Riceman, D. S., and Jones, G. B. 1956. Aust. J . Agr. Res. 7, 495-503. Riceman, D. S., and Jones, G. B. 1958. Aust. J . Agr. Res. 9, 730-744. Riviere, J. 1960. Ann. Agron. 11, 397-440. Rudgers, L. A., Demeterio, J. L., Paulsen, G. M., and Ellis, R., Jr. 1970. Soil Sci. SOC. Amer., Proc. 34, 240-248. Ryan, P., Lee, J., and Peebles, T. F. 1967. “World Soil Resources,” Rep. No. 31. FAO, Rome. Schmid, W. E., Haag, H. P., and Epstein, E. 1965. Physiol. Plant. 18, 860-869. Schroo, H. 1959. Neth. J . Agr. Sci. 7 , 309-316. Seatz, L. F., and Jumnak, J. J. 1957. In “USDA Yearbook of Agriculture” (A. Stefferud, ed.), pp. 115-121. US. Govt. Printing Office, Washington, D.C. Shaw, E., and Dean, L. A. 1952. Soil Sci. 73, 341-347. Sillen, L. G., and Martell, A. E. 1964. Chem. SOC.,Spec. Publ. London 17. Silverberg, J., Young, R. D., and Hoffmeister, G. 1972. In “Micronutrients in Agriculture’’ (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 431-458. Soil Sci. SOC.Amer., Madison, Wisconsin. Stevenson, F. J., and Ardakani, M. S. 1972. In “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 79-114. Soil Sci. SOC.Amer., Madison, Wisconsin. Stewart, I. 1963. Annu. R e v . Plant Physiol. 14, 295-310. Stewart, I., and Leonard, C. D. 1956. Proc. In!. Conf.Peaceful Uses A t . Energy, Is!, 1955 Vol. 12, pp. 159-164. Stewart, I., and Leonard, C. D. 1957. Soil Sci. 84, 87-97. Stewart, J. A., and Berger, K. C. 1965. Soil Sci. 100, 244-250.
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Stiles, W. 1961. “Trace Elements in Plants,” 3rd ed. Cambridge Univ. Press, London and New York. Stuckenholtz. D. D., Olsen, R. J., Gogan, G., and Olson, R. A. 1966. Soil Sci. SOC. Arner., Proc. 30, 759-763. Swaine, D. J. 1955. Commonw. Bur. Soil Sci. ( G t . Brit.), Tech. Commun. No. 48. Swaine, D. J., and Mitchell, R. L. 1960. 1. SoilSci. 11, 347-368. Terman, G. L., Allen, S. E., and Bradford, B. N. 1966. Soil Sci. SOC. Amer., Proc. 30, 119-124. Thorne, D. W. 1957. Advan. Agron. 9, 31-65. Thorne, D. W., and Wann, F. B. 1950. Utah, Agr. Exp. Sta., Bull. 338. Thorne, D. W., Laws, W. D., and Wallace, A. 1942. Soil Sci. 54, 463-468. Tiffin, L. 0. 1967. Plant Physiol. 42, 1427-1432. Tiffin, L. 0. 1972. In “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 199-229. Soil Sci. SOC.Amer., Madison, Wisconsin. Tiller, K. G. 1967. Naiure ( L o n d o n ) 214-852. Trierweiler, J. F., and Lindsay, W. L. 1969. Soil Sci. SOC.Amer., Proc. 33, 49-54. Truelson, D. 1967. Unpublished M.S. Thesis, Colorado State University, Fort Collins. Udo, E. J., Bohn, H. L., and Tucker, T. C. 1970. Soil Sci. SOC. Amer., Proc. 34, 405-407. Vallee, B. L , and Wacker, W. E. C. 1970. In “The Proteins” (H. Neurath, ed.), 2nd ed., Vol. 5. Academic Press, New York. Vancura, V. 1964. Plani Soil 21,23 1-248. Viets, F. G., Jr. 1951. Agron. 1. 43, 150-151. Viets, F. G., Jr. 1961. U S . , D e p . Agr., Leap. 495. Viets, F. G . , Jr. 1962. 1. Agr. Food Chem. 10, 174-178. Viets, F. G., Jr. 1966. In “Zinc Metabolism” (A. Prasad, ed.), pp. 90-128. Thomas, Springfield, Illinois. Viets, F. G., Jr., and Boawn, L. C. 1965. Agronomy 9, 1090-1101. Viets, F. G., Jr., and Lindsay, W. L. 1972. In “Soil Testing and Plant Analysis” (L. M. Walsh and J. D. Beaton, eds.), 2nd ed. Soil Sci. Soc. Amer., Madison. Wisconsin (in press). Viets, F. G., Jr., Boawn, L. C., Crawford, C. L., and Nelson, C. E. 1953. Agron. 1. 45, 559-565. Viets, F. G., Jr., Boawn, L. C., and Crawford, C. L. 1954a. Plant Physiol. 29, 76-79. Viets, F. G . , Jr., Boawn, L. C., and Crawford, C.,L. 1954b. Soil Sci. 78, 305-316. Viets, F. G., Jr., Boawn, L. C., and Crawford, C. L. 1957. Soil Sci. SOC. Amer., Proc. 21, 197-201. Vinande, R., Knezek, B., Davis, J. F., Doll, E., and Melton, J. 1968. Mich., Agr. Exp. Sia., Quart. Bull. 50, 625-636. Watanabe, F. S., Lindsay, W. L., and Olsen, S. R. 1965. Soil Sci. SOC. Amer., Proc. 29, 526-565. Wear, J. I., and Evans, C. E. 1968. Soil Sci. SOC. Amer., Proc. 32, 543-546. Webley, D. M., and Duff, R. B. 1965. Plant Soil 22 307-313. West, E. S., 1938. 1. Counc. Sci. Ind. Res. Aust. 11, 182-184. Wilkinson, H. F. 1972. In “Micronutrients in Agriculture” (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, eds.), pp. 139-169. Soil Sci. Soc. Amer., Madison, Wisconsin.
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Fowden G Maxwell. Johnie N. Jenkins. and William L . Parrott Departments of Entomology ond Agronomy. Mississippi Stote University. and United States Department of Agriculture. Boll Weevil Research laboratory. State College. Mississippi
I. Introduction .................................................. 11. Terminology . . . . . . . . . . . . . . . . . . ............................. 111. Insect Resistance in Selected Field ps .......................... A Leguminosae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Gramineae ................................................ C. Malvaceae .......................... .................. .................. IV Horticulture Crops ...................... A . Rosaceae ................................................. B Cruciferae ...................... ............... C Solanaceae ................................................ D. Convolvulaceae ............................................ E. Compositae ............................................... ............ F . Liliaceae ................................. G Cucurbitaceae . . . . . . . . . . . . . . . . . ......................... H . Leguminosae ............................................. V Forest Trees .................................. ..................... VI. Miscellaneous . . . . . . . . . . . . . . . . . . A Solanaceae ................................ B Chenopodiaceae ........................................... VII . Problems Associated with Breeding for Resistance to .......................... VIII Utilization of Resistant Varieties . . . . IX. Summary . . . . . . . . . . . . . . . .................................. References ............................. ....................
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I . Introduction
The principal objective of this review is to compile the more significant papers in the area of host plant resistance since the review of Painter (1958b) which updated his classical book of 1951. A short review of host plant resistance to selected insect pests was published by Sprague and Dahrns (1972) . Research in the field has expanded tremendously in the last decade and while this review attempts to be comprehensive. it is quickly recognized by the authors that many worthy papers may have been missed or else omitted because of lack of space. However. if this review 187
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can serve in part to bring together pertinent literature for the use of researchers and teachers in this rapidly expanding field, then we feel that a vital void has been partially filled. This review was concluded in November 1971. Approximately 1400 papers were reviewed of which only 555 are cited. This is indicative of the growing importance of this field as one of the primary methods in which the pesticide load in the agro-ecosystem can be greatly reduced. As insect control for the future moves toward the integration of biological, cultural, and chemical suppressant methods into sophisticated pest management systems, host plant resistance to insect pests can and will play a vital role.
11.
Terminology
For a more comprehensive treatment of terminology the readers are referred to Painter (1951, 1958b), Beck (1965) and Beck and Maxwell (1973). Briefly, resistance for the purpose of this review is defined as those heritable characteristics possessed by the plant which influence the ultimate degree of damage done by the insect. From a practical point of view, resistance is the ability of a certain variety to produce a larger yield of good quality than other varieties at the same initial level of infestation and under similar environmental conditions. Resistance therefore is relative and is definable only in terms of other and usually more susceptible varieties. The analysis of why plants are resistant usually indicates that one or more of three basic components or mechanisms are involved. Plants may be noiiprrferrerl for oviposition, shelter or food, primarily because of the lack of or presence of chemical or physical factors. Second, resistant plants may affect the biology of the insect adversely, which is called antibiosis. Third, resistant plants may be tolerant, surviving under levels of infestation that would kill or severely injure susceptible plants. These three components are complex and many times interrelated. They are primarily concerned with effects rather than causes.
Ill.
Insect Resistance in Selected Field Crops
A.
LECUMINOSAE
1 . Alfalfa and Clover
a. Spotted Alfalfa Aphid, TIzerioaphis nzaculata (Buckton). This species was first found on alfalfa in North America in New Mexico in the
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spring of 1954. It now occurs in all the alfalfa-producing areas in the United States (Smith, 1959). It causes losses of millions of dollars annually and is considered to be the most widespread and serious pest of alfalfa. At first it was confused with the yellow clover aphid, Theriouphis trifolii (Monell), but Dickson et al. (1955) indicated that its host range differed from that of the yellow clover aphid. This finding was confirmed and extended by Peters and Painter (1957, 1958) to include a total of 23 species in the genera Medicago, Melilotus, Trifolium, and Trigomella which are favorable for the reproduction and development of this aphid. Some species and varieties of single species exhibited considerable differences in resistance to the aphids. Howe and Smith (1957) reported that the variety LAHONTAN and its parental clones had a high degree of resistance in the seedling stage, showing both antibiosis and tolerance. Howe and Pesho (1960), when evaluating the performance of a large number of varieties in mature field stands, found that LAHONTAN, MOAPA, ZIA, BAM, and SIRSA NO. 9 were resistant. The synthetic variety, ZIA, carrying resistance to the aphid, was released in New Mexico in 1959. The following resistant synthetics were released in California, Arizona, Nevada, and Utah: MOAPA in 1958, SONORA in 1964, CALIVERDE 65 in 1965, and MESA-SIRSA in 1966. WASHOE was released for the Pacific Coast and intermountain states (Hunt et al., 1966). Foliage damage by the aphid was 15-22 times greater on susceptible varieties than on MOAPA (Barnes, 1963a). LAHONTAN, NEMASTAN, and C 104 had the highest rate of survival of 31 varieties and strains tested in the seedling stage after infestation with the aphids. Alfalfas derived from Turkistan appear to have a higher proportion of resistant plants than other varieties with the exception of African (Hackerott et al., 1958). The synthetic variety CODY, selected from the suceptible BUFFALO variety was developed and released by workers in Kansas in 1960 (Harvey et al., 1960). Resistance in alfalfa to the aphid is associated with a physiological process which is sensitive to environmental factors (Graham 1959; Hackerott and Harvey 1959; McMurtry, 1962; Isaak, 1963; Kindler and Staples, 1970). Survival of the spotted alfalfa aphid and damage to leaves on excised trifoliolates of resistant alfalfa were more apparent than on attached plant parts (Thomas et al., 1966; Thomas and Sorensen, 1971). Approximately two years after the first variety resistant to the aphid was released, there were indications that a biotype was developing. Several fields of LAHONTAN showed rather high populations of aphids, suggesting a breakdown of resistance (Stanford and McMurtry, 1959). A biotype developed that was capable of surviving and reproducing on an aphid-resistant variety (Pesho and Lieberman, 1960).
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Nielson et al. ( 1971 ) evaluated 52 experimental lines for resistance to
4 biotypes of spotted alfalfa aphids and found 3 alfalfas highly resistant to all 4 biotypes. Antibiosis, preference, and tolerance alone and in combinations have been found to be responsible for conferring resistance in alfalfa against the spotted alfalfa aphid (McMurtry and Stanford, 1960; Howe et a!., 1963; Kishaba and Manglitz, 1965; Kircher et al., 1970; Sandmeye c’t al., 1971). b. Pea Aphid Acyrthosiphon pisum (Harris). This insect has been estimated to cause about 60 million dollars in losses annually in alfalfa (Carnahan, 1963). Early observations on resistance to the pea aphid have been reported by Painter (1958b). He listed LADAK as a resistant variety although its level of resistance was not considered high. LADAK was first recommended in Kansas in 1937 and represented the only resistant variety until recent years. Ortman and Painter (1960) reported techniques for selection and evaluation of pea aphid-resistant alfalfa plants. A method of testing for resistance in the field included exclusion of prcdators (Smith and Peaden, 1960). Another method used to evaluate resistance was to count parasitized aphids on the upper surfaces of the leaves (Harvey and Hackerott, 1967). Differences in pea aphid populations between resistant and susceptible alfalfa in solid-seeded plots were highly significant when either live or parasitized aphids were counted. Counting parasitized aphids was easier and more accurate. These techniques or modifications have been used since 1966 for development of several pea aphid resistant varieties. WASHOE, resistant both to the spotted alfalfa aphid and pea aphid was released by Peaden et al. (1966) for the irrigated areas of the Pacific Coast and intermountain region. Dawson, developed by Kehr et al. (1968), was released for Nebraska and the North Central States. Another resistant variety, MESILLA, was released by Melton (1968) for the irrigated area of New Mexico. KANZA, carrying resistance to both spotted alfalfa and pea aphid, was released in 1969 by the Kansas Agricultural Experiment Station (Sorenson et al., 1969). The most recent variety, TEAM, also has moderate resistance to the alfalfa weevil (Barnes et al., 1970). This variety represents several years of work by a number of researchers and demonstrates, along with WASHOE and KANZA varieties, the potential success of combining multiple pest resistance in one variety. Studies by Harvey et al. ( 197 1 ) utilizing resistant KANZA and susceptible CODY under a severe infestation of pea aphids showed KANZA forage yields were 167% of CODY, thus indicating that losses can be reduced significantly by resistant varieties. The genes controlling the inheritance of resistance in alfalfa to both the spotted alfalfa aphid and pea aphid are unknown. It is of special signifi-
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cance that breeding for resistance has been accomplished without knowing the genetics of the basic causes of resistance. These successes were possible because selection methods were applied to alfalfa populations containing a low percentage of resistant plants. Antibiosis and preference are the main mechanisms of resistance in alfalfa. Tolerance in some instances is probably involved, but to a much lesser degree. Auclair (1 963) reported that pea aphid resistant varieties of peas were generally deficient in some amino acids and thereby were less nutritious than the susceptible varieties. To date no studies have been able to correlate resistance to the pea aphid or spotted alfalfa aphid with any specific chemical factors in alfalfa. Much work remains to be done in determining the biochemical causes of resistance in alfalfa to these two pests. c. Alfalfa Weevil, Hypera postica (Gyllenhal). This insect was first found in the United States in Utah in 1904 and near Baltimore, Maryland, in 1951 and has since spread throughout most of the eastern portion of the United States. Significant differences in larval damage among broadcast stands of 294 experimental strains and commercial varieties were found by Dogger and Hanson (1963). They attributed these differences to tolerance. Campbell and Dudley (1965) demonstrated that both Medicago sativa var. gaetula Urb. and M . falcata L. contained plants that differed significantly when the weevil was given a choice of plants for oviposition indicating preference as a mechanism of resistance. The alfalfa weevil showed a preference for certain clones for feeding and oviposition (Van den Burgh et al., 1966; Norwood et al., 1967; Busbie et al., 1968). Several techniques have been developed for mass screening of alfalfa for resistance to the alfalfa weevil. Barnes and Radcliffe (1967) devised a leaf disk method of testing for resistance to the adult weevil. A short period later, Barnes et 01. (1969) developed a procedure which involved infesting 8-day-old seedlings with adult weevils. They were able to measure differences in cotyledon damage by the adults. By mass screening alfalfa populations for resistance to larvae, Byrne and Rittersh (1970) made 83 selections from alfalfa fields which had been established for several years and had been subjected to severe weevil infestations for 5 or more years. Of the 83 selections, they found 15 which caused a reduction in larval weight demonstrating antibiosis as the resistance mechanism. The variety TEAM, which shows moderate resistance to the weevil, was released recently (Barnes et al., 1970). d . Sweetclover Weevil, Sitona cylindricollis Fahraeus. This weevil is regarded by most research workers as the limiting factor in the production of sweetclover. Four years of intensified research on the relative resistance of various accessions of the genera Melilotus, Medicago, and Trigonella
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to feeding injury by the sweetclover weevil was reported by Radcliffe and Holdaway ( 1967). Laboratory procedures to evaluate feeding by the sweetclover weevil were developed by Connin et al. (1958). Manglitz and Gorz (1964) reported that under greenhouse conditions the sweetclover weevil fed on 1 8 species of iMelilotus, 12 of Trigonella, and 1 of Medicago, but no feeding was apparent on M . infesta. From 19 species of Melilotus tested for resistance over a 12-year period, Gross and Stevenson (1964) found one species, M . infesta, immune to the weevil during 2 years of testing. Areson et al. ( 1967) developed a bioassay for detecting compounds which either stimulate or deter feeding by the sweetclover weevil. They concluded that the substance responsible for the resistance of M . infesta or the susceptibiiity of M . officinalis was present in the residue of the water-methanol fraction. Later. Akeson et al. (1969) isolated and identified ammonium nitrate as the compound apparently responsible for this resistance. Beland et al. (1970) demonstrated a relationship between nitrate content and age of the plant. As leaf development increased, the nitrate content became progressively higher. e . Miscellaneous Insects. Alfalfa with a high degree of resistance to the meadow spittlebug, Philaneus spiiniarisrs (Linnaeus) , has been devcloped. The resistance mechanism was assumed to include antibiosis, tolerance and nonpreference (Wilson and Davis, 1958). A limited amount of work has been conducted on locating sources of resistane in alfalfa to the alfalfa seed chalcid, Bruchophagus roddi Gussakovskii, the most destructive insect pest of alfalfa seed in the United States. Nielson and Schonhorst ( 1967) evaluated several experimental HAIRY lines and individual plants and concluded that varieties LAHONTAN, PERUVIAN, ZIA, RANGER, and A-224, as well as Medicago tianschanica Vass. var. agropyretorim Vass., a foreign entry, were promising sources of resistance to the seed chalcid. Lehman ( 1967) reported that 4 clones had some tolerance which might be valuable in a ehalcid resistance breeding program. Differences among clones of alfalfa for the period of nymphal development and nymphal feeding preference of the potato leafhopper, Empoasca fabae (Harris) were reported by Newton and Barnes ( 1965). Wressell (1960) also found differences in populations of the potato leafhopper among 9 varieties of alfalfa that were sampled periodically from Jlune through August. From 28 varieties and 4 experimental populations of alfalfa tested in the seedling stage for resistance to the potato leafhopper, Webster et a!. (1968) found that the extremely winterhardy varieties exhibited high seedling survival in contrast to the nonhardy varieties. An evaluation of resistance to the sweetclover aphid, Therioaphis riehrni
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(Boerner), was made by Manglitz and Gorz (1961) among commonly grown sweetclover varieties and the more promising breeding lines. They reported that among most of the materials tested the older plants demonstrated the greatest resistance. After developing a method to rapidly screen Iarge numbers of alfalfa plants for resistance to the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), Lindquist et al. (1967) were able to show differences among varieties. Research by Maxwell and Painter (1959) on the factors which affect the rate of honeydew deposited by the greenbug, Schizaphis graminum (Rondani), and the spotted alfalfa aphid, demonstrated that the rate of honeydew deposition was influenced by the amount of resistance found in the host plant. They suggested the possibility of using the rate of honeydew deposition as (1 ) a measure of the degree of resistance of host plants to aphids, (2) a measure of the rate of ingestion of plant material, and (3) a measure of metabolic activity of the insect. 2. Peanuts The southern corn rootworm, Diabrotica undecimpunctata howardi Barber, is an economic pest on peanuts in certain areas. This insect feeds on the roots and developing pods, causing a reduction in both yield and quality of the nuts. Chalfant and Mitchell (1967) developed a laboratory screening method in which survival of the larvae on the peanut root was used to measure plant resistance. They were able to show highly significant differences among 172 entries tested. Leuck and Harvey (1968) screened 108 lines in greenhouse flats and found that 14 of the entries were avoided by larvae of the lesser cornstalk borer, Elasmopalpus legnosellus (Zeller) , when the larvae were given a choice. From 54 peanut plants representing 3 species, 5 wild plants remained almost free of mites, Tetranychus tumidus Banks, during 5 months of investigations by Leuck and Hammons (1968). The peanut varieties STARR, ARGENTINE, SPANISH CHECK, and NC-2 possessed some nonpreference to leaf feeding by thrips, Franklinelli spp. Damage from foliage feeding by corn earworm, Heliothis zea (Boddie), was significantly greater for two years on Spanish types than for runner and Virginia types (Leuck et al., 1967). Larvae of the fall armyworm, Spodopteru frugiperdu (J. E. Smith), were confined for three generations on foliage of resistant and susceptible cultivars of peanuts. The resistant cultivars caused a reduction in adult emergence and extension of the life cycle (Leuck and Skinner, 1971). Smith and Porter (1971) reevaluated 9 lines of the Virginia type
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peanuts that previously indicated resistance to southern corn rootworm penetration (Smith, 1970). Their results showed high levels of resistance to larval feeding did not exist in the lines. The low level of resistance noted might provide control with reduced rates of insecticide. 3. Soybeans
One of the earliest works with insect resistance in soybeans was conducted by Poos and Smith ( 1931 ). They compared oviposition of female potato leafhoppers, Empoasca fabae (Harris), on red clover and soybean plants having various types of pubescence. More nymphs hatched on glabrous and appressed-hairy pubescent plants than on the rough-hairy pubescent types. Johnson and Hollowell (1935) found that E . fubae heavily infested and seriously injured the glabrous soybean types while the roughhairy varieties were relatively free from leafhoppers and symptoms of leafhopper damage. The differences in degree of E . fubae infestation, stunting of growth, and leaf injury persisted throughout the season, and at the end of the growing season the glabrous plants averaged about 13 inches in height and bore few seed pods while the rough-hairy plants averaged 24 inches in height and bore an abundance of pods. It was concluded that resistance to leafhopper injury was due to the rough-hairy pubescence or to some character controlled by the same hereditary complex as pubescence. Johnson and Holloweli ( 1935) reached simiIar conclusions. Wolfenbarger and Sleesman (1963) found that soybean varieties and plant introductions with normal, semi-appressed, dense, sparse, appressed, or irregular pubescent leaf types did not express the symptoms of “hopperburn” (crinkled leaves with yellow margins), but only normal, dense, or semi-appressed leaf hair types were resistant to leafhopper nymphal infestation. A wide range in soybean resistance to the Japanese beetle, Popillia japonica Newman, was reported by Coon (1946). Twenty-six varieties were subjected to a natural beetle infestation and periodically rated for foliage damage with values from 1 (resistant) to 5 (susceptible). All varieties tested were susceptible to attack and ratings from 2.3 to 5.0 were reported at the height of beetle infestation. The plants that were the least susceptible were those that matured after the peak beetle populations. These late-maturing varieties produced new foliage over the beetle-damaged foliage and consequently produced a higher seed yield than earlier maturing varieties. Mexican bean beetle, Epilachnu varivestis Mulsant, resistance in several plant introduction cultivars was found by Todd ( 1969). Experimental cultivars were planted among rows of susceptible varieties in which a heavy natural infestation of the Mexican bean beetle occurred. Cultivars PI 229358, PI 200489, and PI 171451 exhibited remarkable resistance under
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intense population pressure with the resistance of PI 229358 approaching immunity. The beetles appeared to prefer to starve rather than feed on PI 229358. Similar results with an infestation of Japanese beetle on PI 229358 were reported by Campbell (1969). The amount of damage was estimated to range from 0.7% on PI 229358 to 4.0% on more susceptible cultivars. No attempt was made by either Todd or Campbell to explain the mechanism of resistance expressed by PI 229358. Twenty-one experimental soybean cultivars plus 3 commercial soybean varieties were evaluated by Clark ( 1971 ) for bean leaf beetle, Cerotorna trifurcata (Forster) ; striped blister beetle, Epicauta vittata (Fabricius) ; and Heliothis zea (Boddie) populations. One soybean cultivar, PI 227687, exhibited a high level of resistance to the striped blister beetle and sustained only a very small amount of feeding damage. Cultivars FC 31921, PI 229358, PI 171451, and PI 227687, plus commercial varieties LEE and HARDEE were subjected to a continuous population of H . zea during most of the growing season of 1970. PI 171451 had the fewest eggs deposited on it. PI 227687 had the highest number of eggs deposited on it, but sustained the lowest amount of pod damage, suggesting antibiosis to larvae. Daugherty (1970) indicated that the number of trichomes per unit area of soybean leaf was positively related to H . zea oviposition and damage. Damage to the pods was also related to the denseness of the trichomes. However, use of lyophilized leaf and pod powder of both dense and glabrous soybeans in rearing H . zea larvae suggested that factors other than pubescence contributed to the resistance.
B. GRAMINEAE 1 . Rice Pathak (1964) reviewed the literature dealing with the two most widely distributed species of rice stem borer in Asia, Chilo suppressalis Walker and Tryporyza inotata Walker, and discussed the usefulness of varietal resistance in rice. The results of a field experiment using two of the most resistant varieties and two of the most susceptible varieties indicated that the use of resistant varieties offers a practical way to control the stem borer. Oryza ridleyi L. was more resistant to the rice stem borer, Chilo suppressalis W., than Oryza sativa L. when observations were made on the number of tillers bored using artificial infestations of larvae, and using natural infestations in bird-proof cages (Van and Guan, 1959). The mechanism was not shown, but 0. ridleyi has a much harder stem than 0. sativa.
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The layer of cells from the epidermis to the hollow pith is thicker and has more sclerenchymatous tissue than 0. rativa (Van and Guan, 1959). Infestations of rice stem borer varied significantly and consistently among 33 rice varieties of diversified germ plasm studied by Patanakamjorn and Pathak ( 1967). Varieties were tested under field conditions and exposed to natural infestations. Differences in borer infestation were caused by a combination of factors, and no single factor was responsible for plant resistance. In general, the rice varieties less susceptible to rice borer infestation were those with hairy upper lamina, tight leaf-sheath wrapping, small stem with a ridged surface and thicker hypodermal laycrs. An attempt to determine varietal differences for rice stcm borer attack by rearing larvae on newly germinated seedlings has been made (Tamura and Suzuki, 1963). Body weight of pupae on seedlings of late-heading varieties was heavier than that of pupae on seedlings of early-heading varieties. Body weight of pupae on heavy-ear types was greater than that of pupae on multiear type varieties. This procedure may be useful in mass screening of varieties. Resistance of silicated rice plants to the rice stem borer, C . suppressalis, has been determined by obscrving the condition of mandibles and tracing feeding residue and excrement. A slag fertilizer which accelerates the silication of rice was a preventivc to the rice stem borer; the green rice leafhopper, Nephotettix bipirtlctatii~cititiceps Uhler; and the rice stem maggot, Clilorop\ oryzae Matsumura; but it increased the injury of the rice-plant skipper, Purnara girttutu Brewer et Grey (Sasamoto, 1957). The mandibles of stem borer larvae which fed on the silicated rice plant were diminished remarkably: thus it seems that the silicated rice stem becomes too hard for the larvae to eat. In addition, hungry larvae have shown a preference for normal and nitrogen manured stems over silicated stems. The same preference response of the larvae was observed with water extracts of normal and nitrogen-rich manured stems compared to silicated ones. Preference depends not only on the physical properties of the food, but also on the chemical properties (Sasamoto, 1958). A lower infestation of rice stem borer 5 km from a higher infestation area was shown to be related to the available silicon in the soil. The high infestation area had less available silicon in the soil and less silicon in the plants. Infestations were markedly reduced by adding silicon to the low silicon soil, but no change was made in infestations by adding silicon to high silicon soil (Nakano et al., 1961 ). In a choice situation rice stem borer larvae prefer high nitrogen to low nitrogen plants, and this preference is also exhibited between alcohol extracts of the two kinds of plants. Nitrogen fertilizer apparently affects not only nutritional aspects, but also the synthesis of attractive substances, both olfactory and gustatory for the larvae (Sasamoto, 1960).
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A hypothesis has been presented that the polymerized silicic acids in rice fill up apertures of cellulose micelle constituting cell walls and make up a silicocellulose membrane. This membrane is supposed to be mainly responsible for protecting the plant from some diseases and insects (Yoshida et al., 1959). Not only are there differences in silicon in rice plants due to its availability in the soil, there are inherent differences in the ability of varieties to accumulate silicon (Djamin and Pathak, 1967). Plant silica content and susceptibility to rice stem borer were negatively correlated. The rice plant contains factors that inhibit the growth and development of rice stem borer larvae even though the plant is the principal host of the insect. Two substances, benzoic and salicylic acids, have been identified which inhibit larval growth when either is added to the larval food medium at a concentration of 1.4% of the dry weight. However, this level is much greater than that found in the rice plants. Other more potent factors were observed, but not identified (Ishii et al., 1962). Resistance to the stem borer, Tryporyza incertalas Walk., is heritable (Sampath et al., 1970). Crosses of high-yielding susceptible varieties like TAICHUNG 1 and IR 8 with resistant TKM 6 have produced some high-yielding resistant plants. TKM 6 is resistant to the nematode, Meloidogyne graminicola GOLDEN and BIRCHFIELD, as well as stem borer. Resistance against namatodes and stem borers may be due to similar unidentified biochemical mechanisms. Israel et al. (1965) found a wide range of incidence of stem borer at heading stage in J.B.S. varieties. A positive correlation was shown between the breadth of leaf and stem borer incidence at vegetative and heading stages in different Dalva varieties. In studying resistance to the rice stem maggot, Yushima and Tomisawa (1957) determined that the value of resistance is best indicated by measuring larval mortality in seedlings in nursery beds rather than the percentage of injured ears. In studies by Iwata (1960) the second brood of larvae developed slower on plants beyond the 10-leaf stage than they did on younger plants. Reddy (1966) listed several varieties that suffer less damage from gallfly, even in years of severe infestation in the Andhra Pradish state of India. Resistance in rice to the planthopper Sogatodes oryzicala Muir was expressed throughout the growing season (Jennings and Pineda, 1970a). A survey of 524 varieties showed that about 20% were highly resistant or resistant. Resistant varieties received little or no damage whereas susceptibles ones were killed. All resistant varieties were Indicas from southeast Asia. The resistance appeared highly heritable and was easily recombined with agronomic traits but was not associated with pubescence. Resistance reduced the number of eggs deposited and number of nymphs hatched,
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prolonged the nymphal period, and reduced the adult longevity of this planthopper f Jennings and Pineda, 1970b). Resistance of the MUDGO variety to brown planthoppers, Nilaparvata Zugans Stal, was reported as outstanding by Pathak et al. (1969). The resistance was transferred to more desirable plant types. Resistance resulted from a strong nonpreference for feeding (Sogawa and Pathak, 1970). Insects caged on MUDGO had high mortality, slow growth rate, small body size, and low fecundity. Newly emerged female planthoppers caged on MUDGO had underdeveloped ovaries that contained few mature eggs. MUDGO plants either lacked feeding stimuli or possessed one or more taste repellents for the planthoppers. Lower asparagine content in MUDGO was suggested as a factor responsible for its resistance to the planthopper. Athwal et al. (1971 ) studied the genetics of resistance of several rice cultivars to the brown planthopper and to the green leafhopper, Nephotettix impicriceps (Ishihara), in the greenhousc. The resistance of MUDGO, MANAVARI co 22, and DALWA SANNAM MTU 15 to the brown planthopper was controlled by singlc dominant genes that appeared to be allelomorphic, however, resistance in KARSAMBA RED A S D ~was due to a single recessive gene that was either allelic or closely linked to the locus that conditions resistance in the other three cultivars. Resistance to the green leafhopper in CultiVarS PANKHARI 203, KARSAMBA RED ASD7 and I R ~Was Controlled by single dominant genes that were nonallelic. The planthopper resistance by MUDGO and the leafhopper resistance of PANKHARI 203 were independently inherited, as was the resistance of KARSAMBA RED ASD7 to the two insects. Eight varieties tested for rice water weevil, Lissorhoptrus oryzophilus Kuschel, resistance by Bowling (1963a) did not vary in susceptibility as measured by larval counts using natural infestations. Larval populations showed an increase with each increase in rates of nitrogen from 0 to 120 Ib per acre. The number of rice grains with gaping or broken palea and lemma correlated significantly with number of emerging Angoumois grain moths, Sitotroga cerealella Oliver, in laboratory cages (Cohen and Russell, 1970). Significantly fewer moths and larvae were found in varieties having less “unsound” seeds. There was a trend for higher oviposition and adult emergence of the rice weevil, Sitophilus oryzae L., and the maize weevil, Sitophilus zeamais M., as the proportion of grains with gaping husks increased (Russell, 1968). 2. Wheat a. Hessian Fly, Mayetiola destructor (Say). This pest occurs in most parts of the holoarctic region where winter wheat is grown and in many
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localities where spring wheat is grown. Painter ( 195 1) described the biology and detailed the history and early citations of resistance to this pest, including the development of the resistant varieties POSO 42 and BIG CLUB 43 in California and PAWNEE in Kansas and Nebraska. PONCA, a wheat resistant to Hessian fly in eastern Kansas, was released in 1951 (Painter, 1958b, 1968), and was followed by the development of the resistant varieties, DUAL, BENHUR, and others by workers in Indiana (Caldwell et al., 1966a,b). Since 1956 several varieties of wheat resistant to the Hessian fly were developed and released for use by the states of Wisconsin, Kentucky, Indiana, Arkansas, Nebraska, Georgia, Oklahoma, Missouri, and Illinois. It is currently estimated that over 10 million acres are planted with 23 different Hessian fly resistant varieties in 34 states. Luginbill (1969) reported that the value of increased yield resulting from resistant varieties in the United States was about 238 million dollars. It has been estimated that losses by Hessian fly in wheat have been reduced to less than 1 % because of resistant varieties (Agric. Res., Vol. 1916, 1970). The genetics of Hessian fly resistance have been analyzed by Cartwright and Wiebe (1936), Painter et al. (1940), Caldwell et al. (1946), Suneson and Noble (1950), Shands and Cartwright (1953), and Allen et al. (1959). These workers described 6 genes responsible for resistance to Hessian fly in wheat. Several additional factors have not yet been identified. The primary mechanisms of resistance controlled by these genes are antibiosis and tolerance. The chemical basis of resistance remains largely unanswered. Refai et al. (1955) in studying possible biochemical causes of resistance in wheat to Hessian fly found no differences in protein, ash, cellulose, silica, and other trace mineral contents between resistant and susceptible varieties. Hemicellulose content and degree of resistance was correlated, and larvae were demonstrated to secrete hemicellulose in vitro as well as a material which inactivated or inhibited wheat plant phosphorylase. Miller et al. (1958, 1960) and Laming (1966) found that silica content was related to resistance in some wheat varieties, but they found no relation between plant pigments and resistance. These studies constitute essentially all the biochemical work on the basic causes of resistance in wheat to the Hessian fly. The selection of Hessian fly races has been described by Gallun et al. ( 1961). Genetics of these populations and frequency of occurrence have been reported (Gallun and Hatchett, 1968, 1969; Hatchett and Gallun, 1968, 1970). Gallun and Reitz (1971) have summarized the effect of wheat cultivars resistant to races of Hessian fly. Seven races of Hessian fly have been isolated in the United States. The Great Plains race is most prevalent west of central Kansas. Races A, B, C , D, and E are found in
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the eastern soft wheat region. Race F is a laboratory race developed at Purdue University. Races were made evident by large-scale planting of varieties with limited resistance factors. These instances of infestation of Hessian flies on wheat having genes for resistance are strong evidence that new races are developing in localized areas where wheats having similar genes for resistance have been grown for several years. b. Cereal Leaf Beetle, Oitlerna melanopa (L.). This newly introduced pest to the United States has created great economic losses in wheat, oats, and barley in the central United States. Resistance has been investigated by screening the world cereaf collection (Everson et al., 1966). Based on larval feeding damage, 323 Triticum lines of 14,444 total lines were found to have none or a trace of feeding and were considered resistant. The primary center of resistant germ plasm was found to be a large continuous area of Asia. Asia Minor, and Eurasia. Numerous resistant lines in the Iberian Peninsula and Eastern Africa would indicate other possible gene pools for resistance. Gallun er al. (1966) found that wheats were less preferred than oats and barleys for oviposition and had less damage from adult and larval feeding. Wheats with highly pubescent leaves were largely avoided for oviposition. A 14-chromosome wheat with highly pubescent leaves had high resistance to the beetle. Various techniques have been devised to evaluate resistance to the cereal leaf beetle (Schillinger, 1966, 1969). Schillinger measured larval weight gains and survival to screen for resistant wheat. Later he reported the use of larval tests to measure antibiosis and tolerance, adult preference for oviposition and feeding and a no-choice ovipositing test to evaluate antibiosis. Schillinger and Gallun (1968) reported in detail the behavior of cereal leaf beetle on pubescent wheat. Gravid females were reluctant to lay eggs on densely haired leaves, and the number of eggs laid was reduced by greater density of pubescence. Eggs laid or placed on leaves with dense pubescence were susceptible to desiccation and less than 10% hatched. Only 20% of first-instar larvae survived 3 days, and these had reduced weights compared to those reared on less pubescent wheat. A genetic analysis of the pubescent character was made on wheats. The character was quantitatively inherited, and a high correlation was found between larval weights and pubescence density (Ringlund and Everson, 1968). The cereal leaf beetle is not polyphagous within the family Gramineae according to Wilson and Shade (1966). Several plants, notably species of Setaria, Echinocloa, and Arundinaria, exhibited antibiosis. Generally the small grains were superior food plants. Some grasses were favorable, but larval development was always less than on small grains.
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c. Greenbug, Toxoptera graminum (Rond.) . A seedling screening method was used by Daniels and Porter (1958) to screen winter wheat hybrids for greenbug resistance. The damage index rating system suggested that resistance was controlled by a single recessive gene. A collection of 111 wheats that showed resistance in Oklahoma were retested for resistance at Denton, Texas by Chada et al. (1961), and 11 strains were selected as the most resistant. Wood and Curtis (1967) exposed pure line selections of a cross between greenbug-resistant Dickinson selection 28A and susceptible PONCA wheat to field-induced populations of the greenbug for four years. The resistant selections consistently outyielded susceptible PONCA wheat near Stillwater, Oklahoma. The genetics of greenbug resistance was investigated by Curtis et al. (1960). They found that resistance of Dickinson selection 28A and CI90.58 was conditioned by a single recessive gene. Similar conclusions were reached by Porter and Daniels (1963) with Dickinson selection 28A. Abdel-Malek et al. (1966) determined that greenbug resistance and Hessian fly resistance could be naturally combined into the same synthetic variety. Singh and Wood (1963) found that a greenhouse strain of greenbug responded differently from a field-collected strain. Fecundity and survival of the field strain of greenbug on resistant and susceptible wheat was not affected at lower temperatures, but was greatly retarded at higher temperatures. This was not evident with the greenhouse strain. Furher studies confirmed the existence of a distinct biotype (Wood, Jr., 1961a). Ortman and Painter (1960) found that high infestations of greenbug caused a maximum of 55% reduction of what root systems. The results indicated that root systems and aboveground plant parts were approximately equally damaged. Techniques for testing resistant small grains in the insectary have been described (Chada, 1959; Wood, Jr., 1961a). Essentially, varieties were grown in flats and subjected to uniform infestation, and after 10-14 days the degree of damage was noted and compared with resistant checks. Ten to 20 plants were used to evaluate each variety. At present all greenbugs are either biotype b or c and all varieties are now susceptible. d . Wheat Stem Sawfly, Cephus cinctus Norton. L. E. Wallace and McNeal (1966) summarized a 66-year work program with sawfly in both the United States and Canada. Three species are involved in North America: Wheat stem sawfly, Cephus cinctus Norton, European wheat stem sawfly, Cephus pygmaeus (L.); and the blackgrain stem sawfly, Cephus tubidus (F.). Their summary of resistant varieties is applicable here: “Resistant varieties of wheat offer the best means of controlling sawflies. The
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solid stems of these varieties appear to inhibit the development of the larvae. The solid stem character is often associated with lower yields, and for this reason resistant varieties are not grown universally in the sawfly areas. Resistant varieties grown throughout the sawfly areas would greatly decrease the populations. If new varieties with better yields were available, growers would be more easily induced to use this means of controlling the sawfly. Resistant varieties presently in use are the spring wheats Rescue, Chinook, Cypress and Sawtana and the winter wheat Rego.” Roberts (1960) indicated the three primary types of resistance to sawfly were: ( a ) resistance to egg laying, (b) resistance to development of eggs and establishment of first-instar larvae, and (c) resistance to development of the older larvae. Field data showed that resistance of wheat to attack depended on the stage of development of the plants at the time of oviposition. Wheat plants were usually most heavily infested for a part or all of the period from 1 week before shot blade to 1 week after flowering stage. Resistant strains lost their resistance to development of the eggs and firstinstar larvae some time between flag leaf and flowering. The mortality of the older larvae increased toward maturity. Holmes and Peterson (1960) found that resistance to oviposition was not a factor in the sawfly resistance of any of the resistant varieties tested (mostly solid stem varieties). They determined that the differences in rates of development of varieties accounted for the differences in infestation as indicated by the relative times, amounts, and locations of oviposition by the wheat stem sawfly. Light intensity was found to greatly influence the resistance expression in RESCUE (Roberts and Tyrell, 1961) . High light intensities were required for full resistance expression, and the required intensities could be produced by supplemental lights of 4000 ft-c in greenhouse studies. Six other strains of wheat reacted in a like manner. Because reduced light intensity reduced stem solidness, the loss of resistance of solid stemmed hosts has been attributed to cloudy weather during the early part of the summer (Holmes et al., 1960). Holmes and Peterson (1961) showed that resistance to hatching of the egg of the sawfly depended on stem solidness of its host. Stem solidness was required for resistance to the larvae (Holmes and Peterson, 1962). However, as with resistance to the egg, the effect of the pith on larval mortality varied between internodes of the same stems. The mortality of eggs and first-instar larvae in the pith varied independently, and it was concluded that these responses were caused by different factors in the plant. The evidence indicated that the role of the pith in death of eggs and early instar larvae was either through desiccation or action as a mechanical barrier to passage or both.
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Inheritance of sawfly reaction and stem solidness in wheats has been investigated (McNeal et al., 1959, 1966; McNeal, 1961; McKenzie, 1965; McNeal and Wallace, 1967). Solid sternness is influenced strongly by environment and may be under different sources of genetic control. Lawson and McDonald ( 1964) have produced lines of the solid-stemmed variety S-615 in which whole pairs of chromosomes have been replaced by corresponding pairs from the hollow-stemmed variety APEX. These lines have been useful in cytogenetic studies of the inheritance of resistance. 3 . Oats
The inheritance of resistance to the greenbug, Toxoptera graminurn Rond., in crosses of oats, Avena sativa L. and Avena byzantina C. Kock, was examined by Gardenshire (1 964) , who hypothesized that the inheritance of greenbug resistance in the oat variety RUSSIAN 77 is conditioned by a single gene. Resistance to the frit fly, Oscinella frit L., has been demonstrated in various varieties. The greatest resistance has been found in varieties developed by growers where frit fly attack is serious (Bingham and Lupton, 1958; Penegrin and Catling, 1967). However, it was generally concluded that resistance was due to escape by virtue of different rates of development and nonpreference expression by the adult for egg deposition. Cultivars with red tiller bases and hirsute shoot bases are less susceptible, but infestations would be much greater if varieties with these characteristics were grown as pure crops (Penegrin and Catling, 1967). The variety SUMMER, an early-flowering variety, escaped infestation when planted early but lost this advantage when planted late when damage was measured as fritted stems (Bingham and Lupton, 1958). However, VON LACHON’S YELLOW possessed a heritable resistance to the panicle attack. None of the varieties were considered to possess resistance to an extent to which it could be utilized in a breeding program. Hydrellia griseola Fall is a leafminer of grasses which attacks oats. Stavrakis (1965) found that resistance in oats was biochemical in nature and resulted in smaller larvae and pupae. Early evaluation of resistance in oats to the cereal leaf beetle, Oulema melanopus (L.) began in the field (Gallun et al., 1966). They evaluated 238 varieties, none of which were considered to be resistant. Laboratory methods were later developed (Schillinger, 1966, 1969; Schillinger and Gallun, 1968) which worked equally well with wheat and barley. Schillinger (1 969) developed three tests which effectively measured antibiosis, adult preference, and tolerance. Larval growth and oviposition under standardized laboratory conditions were used to screen varieties.
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4. Barley
The genetics of resistance to Hessian fly, Mayetiola destructor (Say), was studied in barley by Olembo et al. (1966). Three known resistant varieties were crossed with genetic translocation stocks. A common dominant factor (symbol proposed H f H j ) which governed resistance at cool greenhouse temperatures was found in DELTA, NILE, and ABUSIR. At higher greenhouse temperatures, two complementary dominant genes were necessary to explain resistance in DELTA. The second factor pair which was ineffective alone was dcsignated H j - H f , . The aphid, Rhopalosiphurn padi L., is a vector of barley yellow dwarf virus which has promptcd the search for resistance to R . padi in barley. Hsu and Robinson (1962) found that 49 of 136 barley varieties tested in the greenhouse showed some resistance (antibiosis or tolerance) to the aphid. In a later study they found that 78 of 338 barley varieties tested showed some resistance to the aphid (Hsu and Robinson, 1962). When these varieties were tested in field tests, about half were resistant. Of 14 varieties which demonstrated resistance in field tests in 1961 all demonstrated resistance again in 1962. Robinson (1965) in later studies found that resistant varieties from the two previous tests did not demonstrate rcsistance (antibiosis) in a plant growth room. Factors found to influence significantly the reproduction of aphids were temperature, photoperiod, nutrition of plants, and period of year. Dishner and Everly (1961 ) subjected 1 3 varieties of barley to corn leaf aphid, Rhopalosiphum maidiy (Fitch), in the greenhouse by placing one mature aphid on each plant. The most resistant were UTAH S E L CI 10,000, KEARNEY CI 7580, HOODED 16 SEL 3323, and DICKTOO CI 5529. Hormchong and Wood (1963) cvaluatcd a number of barley varieties and clones using the method of Wood (1961a). Among the most resistant varieties was a cross of KEARNEY and ROGERS, They felt that the resistance was not due to antibiosis although the study of Dishner and EverIy (1961) was based on progeny survival which would reflect antibiosis. The gene responsible for greenbug resistance in barley is apparently different from the gene that imparts resistance to the corn leaf aphid. OMUGI, which is highly resistant to the greenbug, is very susceptible to the corn leaf aphid. A world collection of barley comprising 301 1 varieties was screened for corn leaf aphid resistance during 1963-1967 by Murty et al. (1968). The varieties were sown in field plots and damage scored according to the method of Gardenshire and Chada ( 196 1 ) . Resistant material was then intersown in blocks with susceptible varieties in the field to ensure presence of different biotypes of aphids. None of the entries were completely im-
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mune. The resistant varieties were mostly from the Mediterranean areas, such as BALKAN and KRETA, and from Taiwan. Later studies by Trehan et al. (1970) during 1967-1969 evaluated 1300 exotic and indigenuous varieties of 6-row, 2-r0w, and huskless barley. Early maturing varieties escaped infestation. The Mediterranean area again had several resistant varieties along with two from Japan. Highly resistant were OMUGI SHIN (Japan) and GUIDED REAL (Spain), Pathak and Painter (1958a) found that four biotypes of aphids differ in their damage to barley. On SPARTIN barley all four biotypes had almost the same fecundity, but they showed significant differences in damage to the plant. Also biotypes KS-1 and KS-4 weighed less than half that of KS-3. Barley and oats are the cereals most often attacked by the cereal leaf beetle, Oulema melanopus (L.) (Balachowsky, 1963). Studies of resistance in small grains to the beetle were conducted in Michigan during 1963 (Gallun et al., 1966). Field plantings of 172 barley varieties were examined for damage resulting from adult and larval feeding, and the eggs and larvae were counted. Results indicated that some of the varieties were highly resistant. A diallel cross was used by Hahn (1968) to study the genetic basis for resistance in 8 varieties. The resistance to cereal leaf beetle in barley appeared to be recessive. The mechanism was attributed to both nonpreference for the barley plant by feeding larvae and differential egg laying by adults. Expression of resistance in the plant seemed to be greatly influenced by the physiological stage of development and type of vegetative growth, as well as by environmental variations. Tetranychus sinhai Baker was observed as a new pest of wheat, rye, and barley (Wallace and Sinha, 1961). Sinha and Wallace (1963) observed 169 barley varieties from over the world for their reaction to T . sinhai infestation in the field under drought conditions. Fifteen were found to be resistant, 47 moderately resistant, 88 moderately susceptible, and 15 susceptible. In general, the barley varieties grown in arid regions of the world appeared to be more resistant to T . sinhui infestations. The frit fly, Oscinella frit L., is a pest of some importance in the USSR. Emmerikh (1963) evaluated several varieties for resistance to the frit fly, and several showed striking resistance. The greenbug, Schisuphis graminum (Rondani) is also a pest of barley. The varieties ERA, WILL, and KERR have been released as resistant varieties.
5 . Rye A synthetic rye variety, INSARE F.A.., has been developed for resistance to the greenbug Schisuphis graminum (Rondani) , in Argentina (Arridge,
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1954). Arridge and Re (1963) showed that greenbug resistance is inherited as a single dominant gene in rye crosses. A greenhouse technique was utilized by Livers and Harvey (1969) to evaluate 20 varieties of rye for greenbug ( B biotype) resistance. A range of seedling survival followed infestation. The variety CARIBOU was found to contain resistance. Recurrent selection within the variety CARIBOU increased its resistance from 39 to 96%. Harvey and Hackerott (1969) reported INSARE F.A. resistant to biotypes B and C. The CARIBOU selection was susceptible to the C biotype. This difference between the varieties may be the result of different alleles at a single chromosome locus or to 2 distinct genes at separate loci.
6 . Grasses a. Grasshoppers. Food plant preferences of grasshoppers have been reported extensively by numerous authors. Barnes (l963b) reported the feeding rate and frequency of feeding by the differential grasshopper, Melanoplus differentialis (Thomas), on 17 different range and forage plants. Increased density of a food plant in cages increased the amount of feeding on that plant in relation to other plants present. Survival to the adult stage was best on a mixed diet. Hewitt (1968, 1969) in a similar study with Melanoplus sanguinipes (F.) on 13 species of grass and 4 species of legumes found that preferences and antibiosis were demonstrated for grasses. Six species of grasses were not preferred. Grasshoppers actually lost weight on ALTA and GREEN STIPA. A positive correlation between weight change and rating in the preference test was found for 19 grasses. These results agreed with Pfadt (1949), who found that native grasses did not provide favorable diets for the migrating grasshopper Melanoplus mexicanus (Sauss) . Apparently these and some other grasshoppers would not be an economic problem if crops were found that were as resistant as the native grasses. Pickford ( 1963) found that the clear-winged grasshopper, Camnula pellucida (Scudder) , reared on wheat survived better, were larger, and laid 20 times more eggs than those reared on native grasses. A native sod mixture comprising Stipa comata, Boutelona gracilis, Agropyron smithii, and Carex eleocharis was consistently unfavorable during all stages of grasshopper growth and development. The feeding habits of the differential grasshopper are greatly influenced by its own behavior toward light and temperature in addition to the physical features and orientation of the food plants themselves (Kaufman, 1968). Consequently the plants actually used as food in nature may be very different from those the insect prefers. Hansen and Ueckert (1970) found that the food habits of 6 species of grasshoppers were influenced
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by the sex of the insect. Males ate fewer species of food plants than females of the same species, and the diets of males and females of the same species were occasionally less similar than were the overall diets of different species on the same date. Microscopic examination of crop contents was used to determine the diet dry-weight composition and food preferences of adults of the orthopteran adults of Aeropedellus clavatus (Thomas), Xanthippus corallipes Haldeman, and Cirotettix rabula Rehm and Hebard (Ueckert, 1968). The insects fed on as many as 31 species of plants, but percentages of the diet varied with the species. The first 2 species were principally grass and sedge feeders while the last fed principally on forbs of which 69% by weight was Astragalus sp. A seasonal change in the diet and food preferences occurred during the adult instar. b. Grass Grubs, Costelgtra zealandica (White). Adults of grass grubs move to new areas during the flight season. Radcliffe and Payne (1969) examined preference of adults by introducing them into an enclosure containing plots of common pasture grasses, clovers, weeds, radish barley, and bare ground. The number of beetles on the foliage and the number of eggs were counted. More beetles were consistently observed on foliage of radish, Holcus lanatus L., Lolium perenne L., and the smallest number were observed on Paspalum dilatatum Poir. and on bare ground. There was no significant difference in egg count between treatments. c. Fall Armyworm, Spodoptera frugiperda (.0. I.Smith). Leuck et al. (1968a) investigated 1436 pearl millet inbreds for resistance to first instar larvae of the fall armyworm. Approximately 4 % were rated resistant, 28% intermediate, and 68 % susceptible. Some nonpreferred lines were suggested for use in pearl millet hybridization studies. Antibiosis was demonstrated in Tifton No. 153. Plant analysis for the chemicals 6-methoxybenzoxazolinone and benzoxazolinone (resistant factors to European corn borer) content demonstrated these were not related to resistance in the inbreds tested. Leuck et al. (1968b) also evaluated resistance in 441 clones of Bermudagrass, Cynodon dactylon, to the fall armyworm. Among the clones, 11 lines were rated resistant to larval attack. Analysis of plant extracts for 6-methoxybenzoxazolinone and benzoxazolinone content showed these compounds were not indicators of fall armyworm resistance in Bermudagrass. d . Rhodesgrass Scale, Antonina graminis (Maskell). The cosmopolitan Rhodesgrass scale infests large numbers of important forage grasses of the world. Chada and Wood (1960) list 69 hosts; Schuster (1967) lists 28 in the United States; Brimblecrombe (1966), 14 in Australia; Guaglinni (1963), 22 in Venezuela; Williams and Schuster (1970), 92 in Brazil. Schuster (1967) evaluated 64 of the common range grasses of the Rio
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Grande Plain for susceptibility to scale damage. Yields of 38 species were found to be significantly reduced. Grasses were grouped into three classes : (1) grasses with reduced yields; (2) grasses infested but not affected; and ( 3 ) resistant grasses. Scale numbers were not indicative of scale damage. Thin-stemmed grasses such as Bermuda and filly panicum were severely damaged, while grasses with stouter stems such as Rhodesgrass, Chloris gayana knuth, buffel sandbur, Cenchrus citiare (L.) Link., and southwestern bristlegrass, Setaria scheelei (Steud.) Hituhc., although infested with 3 or 4 times as many scale, were less severely affected. Most species in class 2 were lightly infested. Nine species were immune. A breeding program to develop a Rhodesgrass variety resistant to Rhodesgrass scale was begun in Texas in 1952 (Schuster and Dean, 1972). Surviving plants in naturally infested pastures was the source of resistant plants which were tested for general combining ability as openpollinated progeny (Schuster and Dean, 1972). The tolerant variety BELL, was released from this program (Anonymous, 1966).
7. Corn a. European Corn Borer, Ostrinia nubilalis (Hubner). Painter ( 1951) covers rather extensively the early studies on this insect in reference to biology and host plant resistance studies. Brindley and Dicke (1963) published a comprehensive review including all aspects of research on the European corn borer since its introduction into the United States in 1917, including host plant resistance work. These two publications are excellent for a comprehensive look at the literature prior to 1962. The early work on resistance made use of natural infestations. This technique was not completely satisfactory, but some resistant lines were identified even though antibiosis was highly confounded with preference and nonpreference for oviposition. Techniques for rearing the insect in large numbers enabled the development of artificial infestation techniques utilizing egg masses (Guthrie et al., 1965). This technique enabled researchers to find resistance to the first generation in varietal sources. Some degrees of success transferring resistance to standard lines has been achieved by utilizing backcrossing procedures. Recurrent selection, involving the S , testing procedure, was used with 5 synthetic populations by Penny et al. (1 967). Two cycles of selection were sufficient to shift the frequencies of resistant genes to a high level in all varieties. Three cycles produced essentially borer-resistant varieties. The influence of high nitrogen fertilization on survival of first-generation European corn borer has been studied by Scott et al. ( 1965). The addition of nitrogen fertilizer increased borer survival. Also, the percentage of yield loss was highest at the highest plant population level. Cannon and Ortega
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(1966) in a study involving both nitrogen and phosphorous found that survival of first generation larvae on a susceptible hybrid WF9XM14 was 10-fold greater at 220 ppm than at 10 ppm nitrogen. Survival on a resistant hybrid Oh43 x Oh51A was low and was not affected by the amount of supplied nitrogen. Few larvae survived on plants of either hybrid that received 2.5 ppm or less P; survival at 10 ppm was triple that at 2.5 ppm, but did not improve at concentrations of 20-80 ppm. Resistant corn borer plants treated with nutritional substances from an artificial diet increased larval survival (Scott and Guthrie, 1966). It was concluded that ascorbic acid plus one or more of the other diet ingredients was responsible. In another experiment Scott et al. (1966a) treated susceptible plants to the borer with juice and ether extract from a resistant inbred C131A. In both cases the extract reduced survival to approximately half that found on plants treated with distilled water. The addition of juice or extract from susceptible plants to resistant plants had no effect on larval survival. They concluded that a part of the resistance in C131A was caused by a feeding deterrent or toxic substance. Considerable research has been conducted to determine the biochemical basis of resistance to the first-generation borer. Beck (1965) covers in some detail the research conducted at Wisconsin which led to the discovery of resistant factors A, B, and C. Resistant factor A was identified as 6-MBOA (6-methoxybenzoxazolinone). Klun (1965) found a strong correlation between the amount of 6-MBOA produced by 11 maize inbreds at the whorl stage of development and the field rating of resistance of the inbreds to the first-brood European corn borer. Highly resistant inbreds yielded 10 times more 6-MBOA than highly susceptible inbreds. He also suggested that a precursor of 6-MBOA may be more biologically active than 6-MBOA. Klun et al. (1967) in a follow-up study evaluated 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-0ne (DIMBOA) which Virtanen (1961 ) and Wahlroos and Virtanen (1959) reported as the precursor of 6-MBOA. When DIMOBA was incorporated in the diet, it inhibited larval development and caused 25% mortality. It was concluded that DIMBOA is a chemical factor in the resistance of corn to first-brood European corn borer. The adequacy of DIMBOA in accounting for differences in levels of resistance was studied in 11 inbreds and their diallel combinations by Klun et al. ( 1970). Leaf-feeding ratings were obtained under artificial infestation and whorl-leaf samples analyzed for DIMBOA. The correlation between DIMBOA and resistance to leaf feeding was -0.89 at the inbred level and -0.74 at the hybrid level. The variance for general combining ability accounted for 84% of the variation in resistance ratings and 91% of the variation in concentration in DIMBOA.
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The genetics of first-brood corn borer resistance has been confusing. Some studies have indicated a small number of genes conditioning resistance (Penny and Dicke, 1957; Scott and Dicke, 1965; Scott et al., 1964, 1966b). Other studies indicate that adequate levels of resistance tend to be qualitative in inheritance. Scott et al. (1964) found that in a generation mean analysis involving a highly resistant and a highly susceptible inbred parent, additive genetic variance was of major importance. Certain epistatic effects were also important, suggesting that order of pairing of resistant and susceptible lines in double-cross combinations might be of importance. This possibility was explored by Scott and Guthrie (1967) by using 3 permutations of 12 double-cross hybrids. When the hybrid involved 3 or more resistant parents, the hybrids had a high level of resistance. Some differences were observed among hybrid combinations involving two resistant and two susceptible inbred parents. The observed differences were small but suggested epistatic effects. Concentrated efforts to find resistance to second-brood borers have just recently been initiated. Pesho et al. (1965) identified several inbred lines possessing an acceptable level of resistance. Scott et al. (1967) reported a split-plot study contrasting chemicals and resistance as control measures for second-brood borers. The resistant x resistant crosses exhibited a 4 % yield reduction while the yield of the susceptible X susceptible cross was reduced by 12% . The chemical basis of second-brood borer resistance in corn still remains unknown. Because the second brood differs from the first brood in feeding habits, DIMBOA has been found unrelated to second brood resistance. The genetics of second-brood resistance has not been determined at the present time. b. Corrt Eanvorin, Heliothis zea Boddie. Resistance to the corn earworm has been demonstrated repeatedly in corn. Most of the previous studies deal with the effect of husk extension and husk tightness. Douglas and Eckhardt (1957) summarized 10 years of research and reported on a number of dent corn inbreds and hybrids found resistant to the corn earworm in the South. Painter (1951, 1958b) pointed out some contradictions on how much these physical factors contributed to overall resistance. Jugenheimer ( 1958) reported correlations among 28 characters of 145 inbred lines of maize grown in the United States Corn Belt, but he did not record husk characteristics, nor did he obtain any significant correlation between damage and the chemical evaluations of these lines. Recent reports by Luckman et aZ. (1964), Josephson et al. (1966), Cameron and Anderson (1966), Snyder (1967), Starks and McMillian (1967), Widstrom (1967), Widstrom and Davis (1967), and Widstrom et af. (1970b) confirm that silk balling and long, tight husks beyond the
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tip of the cob are effective barriers to the earworm larvae. The presence of larval feeding stimulants in the corn plant and the ability of larvae to utilize corn tissues as diets in relation to host resistance has been reported by McMillian et al. (1967), Starks and McMillian (1967), Starks et al. (1967), and Widstrom et al. (1970a). Other factors implicated as having an influence on the degree of earworm damage have been soil fertility (Klostermeyer, 1950; Gausman and Wene, 1954); nitrogen balance in the corn plant (Douglas and Eckhardt, 1953); lethal silk factor (Walter, 1957; Wann and Hills, 1966); feeding inhibitors, feeding deterrents, and growth inhibitors (Knapp et al., 1967) ; and low nutritional value of diet (Bennett et al., 1967). Schuster and Wolfenbarger (1966) recorded differences in damage to 48 sweetcorn varieties in the Lower Rio Grande Valley of Texas. Three lines, 471-U6, 81-1, and SWEETEX sustained less damage. They did not speculate on the mechanism of resistance. Hamilton (1969) found that an experimental hybrid possessing a moderate level of resistance from a UBL breeding program yielded 93% worm-free ears compared to 64% worm-free ears from a standard commercial hybrid with the same insecticide treatment. Chambliss and Wann (1971) reported an antibiotic type of resistance in inbred corn lines, 471-U6, 81-1, and M11 913, which rated moderately resistant. Resistant inbreds significantly increased larval mortality over susceptible lines. Resistant lines also retarded larval growth, decreased depth to which larvae penetrated the ear, and delayed pupation of the insect. Some exploratory work has been done on the chemical basis of resistance in corn to earworm (Knapp, 1966; Knapp et al., 1966). These studies involved total and reducing sugars, total nitrogen, and amino acids present in resistant and susceptible dent corn. Results were inconclusive, although it was found that susceptible lines had more sugars and were lower in protein. Inheritance studies concerning resistance in corn to the corn earworm have been inconclusive, probably because of differences in parental material and techniques utilized (Walter, 1962; Widstrom and Hamm, 1969; Dicke and Jenkins, 1945; Widstrom and Davis, 1967; Widstrom et al., 1970b). c. Corn Rootworm Complex (Diabrotica s p p . ) . Three species of rootworms are of major significance on corn, the western corn rootworm, Diabrotica virgifera LeConte, the northern, D. longicornis (Say), and the southern, D . undecirnpunctata howardi Barber. The western and northern species are serious pests in the corn belt region of the United States. The host range of the western corn rootworm has been studied by Branson and Ortman (1967a, 1970) and Branson (1971 ). They determined
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that the insect can develop satisfactorily on at least 21 other grass plants, including winter wheat, Triticum aestivum L., and green and yellow foxtail grass, S. lutescens (Wiegel), barley, H . vulgare L., and intermediate wheat grass Agropyron intermedium (Host). Tripsacutn dactylochen (L.) was found to contain a high degree of resistance due to antibiosis and nonpreference. Corn was the most preferred and most suitable host for the western species. Rranson and Ortman (1967b, 1971 ) have also studied host range of the northern corn rootworm on grasses and determined at least 14 grass species other than corn that serve as hosts. Hagan and Anderson (1967) reported a close correlation between the amount of leaf pubescence and amount of leaf injury by adults of the western corn rootworm. They concluded that the pubescence may act as a barrier to the feeding of adult western corn rootworm. Early reports (Bigger et al., 1941; Painter, 1951) indicate no correlation between root injury and leaf damage by Diabrotica spp. Sifuentes and Painter (1963) reported that various inbred corn lines showed a variance in resistance to leaf feeding by the western corn rootworm. They concluded that the inheritance was monogcnic. Derr et al. (1964) demonstrated the presence of a feeding stimulant in various parts of corn plants to both the western and northern species. Eiben and Peters ( 1966) believe that of various evaluation criteria used to measure resistance to rootworms, pounds of pull to extract the stalk from the ground gave the best indication of rootworm related response and that dry weight of roots gave the best correlation with root characteristics. Utilizing these two criteria, they found three lines (B64, HD2271, and B57) which showed tolerance under different levels of infestation at two locations. Ortman et al. (1968) also reported on a vertical-pull technique for evaluating tolerance of corn root systems to rootworms. Fitzgerald and Ortman (1965) field tested over 150 corn-belt inbreds. On a rating scale of 1 (resistant)-5 (susceptible) over a two-year period, the following inbreds were rated 2.0 or better: N38A, HD2187, SD10, C.I.38B, B55, Oh05, A251, M022, H51, M012, A297, and B57. Data from single crosses involving some of these resistant inbreds suggested that the most resistant inbreds were usually involved in the most resistant single crosses. The component of resistance most responsible is tolerance-in this case the ability to regenerate destroyed roots. Antibiosis has not been observed to play a significant role in the performance of any line under rootworm evaluation in field plots. Shank et al. (1965) indicated that the inbred line SDlO was rated resistant over a two-year study in South Dakota and had promise for use in hybrids for rootworm-infested areas. Fitzgerald et al. (1968) evaluated responses of 6 commercial varieties
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of corn to mechanical (simulated rootworm) damage of the roots. Approximately 0, 25, 50, and 75% of the roots were removed by cutting, and highly significant differences in yield and lodging resulted from the treatments. Walter (1965) in Indiana observed that resistance to the northern corn rootworm in sweet corn differed greatly among closely related lines and was sometimes transmitted to the hybrids of a particular inbred. Root examination gave a better indication of resistance than percentage of leaning stalks. Inbred 291 showed a higher degree of resistance which was attributed primarily to tolerance and other unknown factors. The roots of sorghum were found to be toxic to the western corn rootworm in studies by Branson et al. (1969). The toxicity was attributed to hydrocyanic acid which is released during feeding by the action of p-glucosidase on endogenous cyanogenetic glucosides, such as dhurrin. Branson and Ortman (1969) studied feeding behavior of larvae of western corn rootworm utilizing normal larvae and larvae maxillectomized with laser radiation. They found that receptors were present on the palpi capable of detecting feeding stimulants (sugars) and a deterrent material in oats. Ortman and Gerloff ( 197 1 ) have reviewed the entire rootworm program and dwell on major problems in measuring resistance to these pests. d . Corn Leaf Aphid, Rhopalosiphum maidis (Fitch). This aphid is at times a serious pest of barley, corn, and sorghum. Howitt and Painter (1956) tested many of the sorghums available in the United States for their reactions to corn lead aphids and reported Sudan type sorghums to be consistently more resistant. A single plant of PIPER SUDAN 428-1 was found to be highly resistant to the general populations of aphids. Kansas developed a corn variety, KS 1859, which is resistant to the corn leaf aphid. It was first distributed to farmers in 1950 (Painter, 1958b) and is still grown on limited acreages. Biotypes in insects are becoming more evident and an increasing number of biological races are being reported. KS-1 and KS-2 biotypes of corn leaf aphid were first reported by Cartier and Painter (1956). Two additional biotypes, KS-3 and KS-4, were subsequently reported by Pathak and Painter ( 1958a,b, 1959) .I The distinguishing features and significance of the 4 biotypes and their differential amounts of material taken up in resistant and susceptible sorghums are discussed in detail by Painter and Pathak ( 1960). Pathak and Painter (1959) studied the geographic distribution of the 4 biotypes in Kansas and found that the races were well distributed throughout the state. Different biotypes were recognized from the same field and, in a few cases, even from the same colony. Maxwell and Painter (1962) showed that the different biotypes differ in their ability to influence
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the plant hormone levels in their host. This would suggest different feeding sites as well as.different rates of ingestion. The 4 biotypes reacted differently at different temperaturcs, being more alike at 70°F than at 60 or 80°F. KS-4 survived longer at 60°F followed by KS-1 (Singh and Painter, 1965b). The 4 biotypes varied significantly in reproductive rate, body weight and length of life, to low and high levels of nitrogen, phosphorus, and potassium. A highly significant correlation between the carotene content of mature harvested grain and the aphid infestation on the growing corn plant was found by Coon et al. (1948). The correlation between the carotene content of mature grain and the growing plant was not determined, but it was concluded that physiochemical properties of the plant may play an important role in thc aphid resistance response. Seed low in carotene generally come from crosses having fewer aphids. e. Southwestern Corn Borer, Diatrea grandiosella Dyar. Resistance to this insect has been reported from certain synthetic corn varieties in Arkansas (York and Whitcomb, 1963, 1966). Resistance in the synthetic(s) was closely associated with the Lancaster source of resistance to the European corn borer. A synthetic variety had 48% borer-free plants at harvest, compared with 14% for TEXAS 30 which also lodged 3 times as much as the resistant variety. The variety ARK. SWCB Syn. was released as a germ plasm reservoir for stalk invasion resistance. Josephson et al. (1968) evaluated a large number of hybrids, inbreds, and plant introductions for resistance to the southwestern corn borer. The results were variable over a number of years, probably due to the reliance on natural infestation. None of the inbreds or plant introductions appeared to offer a source of resistance. The degree of resistance was apparently not increased by a mass selection procedure with the synthetic ARK SWCB Syn. f. Rice Weevil, Sitophilus oryzae ( L . ) . This insect is an important factor in corn production in the southern United States. Various factors influence rice weevil infestations. Floyd et al. (1958) found a positive correlation for weevil infestation in the field with bird and corn earworm damage. When the husk was removed from resistant varieties they were more susceptible. Singh and McCain ( 1963) demonstrated that kernel characteristics had a bearing on resistance. They found a highly significant positive correlation between hardness and sugar content of the kernels and field infestation, number of offspring and weevil weights. A significant positive relationship existed between starch and field infestation, number of offspring, and weight of weevils after 90 days. Neither fat nor protein content seemed to be related to these characters. Thus hardness of the kernel and sugar content were important factors in weevil resistance after the
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husks were removed. McCain et al. (1964) reported a technique for measuring preference of rice weevil for corn in the laboratory and reported that some hybrids showed nonpreference for attack. The impact of the resistance factors on natural rice weevil populations in South Carolina have been illustrated by Kirk and Manwiller (1964). In the mid- to late-l940’s, overall infestations of the weevil were approximately 65% of the ears, with from 20% to 30% kernel damage. By the 1960’s less than 20% of the ears were infested, with under 5% kernel damage at harvest. In areas where only recommended resistant varieties were grown, infestations were below 5 % , with less than 1 % kernel damage. f. Fall Armyworm, Spodoptera frugiperda ( J . E . Smith). Feeding and injury to corn is through leaf or ear damage in which the young larvae skeletonize the leaves and older larvae strip the leaves, leaving only midribs and the stalk. Ear damage is characterized by shank damage, resulting in dropped ears. Fall armyworm feeding in Guatemala consisted of gouged out areas of stalks and ear shanks as well as feeding damage to leaves and ears (Painter, 1955). He reported differences in damage among lines. The fall armyworm has been studied less from an insect-plant relationship than most other insects that attack corn. Northern inbreds in general are much more subject to fall armyworm damage on the husk, ear, and in the shank than lines with southern corn as parentage (Dicke, 1955). In the director’s report of the Mexican Agricultural program (Anonymous, 1959), it is reported that Guerrero 169 and 115, Cuba 30, and Yucatan 15 were injured less by fall armyworm than other lines tested. Antigua 2D, 8D and Zapalote Chico lines were reported resistant to the fall armyworm in Mexico in 1964 (Anonymous, 1965). Horovitz (1960) failed to find resistance to the fall armyworm. In the United States as early as 1936, differences in injury or infestation by the fall armyworm were shown among strains of corn (Ditman and Cory, 1936). Brett and Bastida (1963) stated that resistance of sweet corn varieties was due largely to tolerance and that fall armyworm larvae preferred succulent plant tissue in good physical condition. In a field study in which 81 Latin-American lines of corn were evaluated on the basis of larval feeding in the area where the leaf-sheath joins the node, Cuba Honduras 465 and Eto Amarillo were the least damaged lines (Wiseman et al., 1967b). In an extensive study of preference of third and fourth instar fall armyworm for extracts of lyophilized plant material from different crops, sorghum ranked first over corn and the two were preferred over cotton, tomato, tobacco, or chinaberry (McMillian and Starks, 1967). Corn was preferred over Tripsacum dactyloides (L.) by first instar larvae of fall armyworm. Preference was measured by degree of damage to
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seedlings after 5 days. Antigua 2D-160-87 was the most resistant of some 1120 lines tested (Wiseman et al., 1966, 1967b). g . Leafhoppers-Dalbulus maidis (DeLong and Woolcott) and GraminelZa nigrifrons (Forbes). These insects have usually been of noneconomic significance on corn except as vectors for corn viruses. Hybrids susceptible to corn stunt virus were not preferred by leafhoppers over hybrids exhibiting resistance when given a free choice. Apparently incidence of virus was not related to resistance or susceptibility of host to the vector leafhoppers (Collins and Pitre, 1969a,b).
8. Sorghum a. Sorghum-Shoot-Fly, Antherigona varia var. soccata. Five sorghum lines selected from Indian germ plasm were evaluated for resistance to this insect. Nonpreference for oviposition, avoiding larval penetration through the leaf sheaths, and resistance of tillers formed after the destruction of the growing apex were found as resistance mechanisms in three lines. The resistant varieties were characterized by ( 1 ) distinct lignification and thickness of the walls of cells enclosing the vascular bundle sheaths within the central whorl of young leaves, and ( 2 ) a greater density of silica bodies in the abaxial epidermis at the base of the first 3 leaf sheaths (Blum, 1967a, 1968). Ten forage sorghum hybrids (sorghum x sorgo) expressed tolerance to the sorghum-shoot-fly when compared to a hybrid grain sorghum (RS-160). Both types were equally infested as seedlings and no differences in tillering capacity were found, but the rate of uninfested tillers was markedly higher in the forage hybrids as compared with the grain hybrid. Consequently, the reduction in stover production was smaller in forage hybrids than in the grain hybrid (Blum, 1967b). b. Greenbug, Schizaphis graminum Rondani. Seedlings of Sudangrass, Sorghum Sudanese (Piper) Stopf., with resistance to greenbug (B-biotype, originally collected on wheat) were tested for resistance to a greenbug ( C biotype, originally collected on sorghum) that extensively damages sorghums in Kansas during 1968. In greenhouse tests Piper Sudangrass was resistant to the B, but susceptible to the C biotype (Harvey and Hackerott, 1969). General susceptibility to greenbug appeared in a wide array of sorghum germ plasm. Tolerance to the C biotype of the greenbug was discovered in Sorghum virgatum (Hach) Stopf. (Hackerott et al., 1969). Resistance appeared to be conferred by dominant genes at more than one locus. Eight of 1761 varieties and hybrids among different species of sorghum were found to have a high degree of resistance to the A, B, and C biotypes of greenbug by Wood (1971). Resistance was attributed to tolerance,
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nonpreference, and antibiosis. Cate and Bottrell (1971) found that 8 hybrids in the field supported different numbers of greenbugs, and these differences appeared to be influenced by the hybrid. Significant variation was found between 23 sorghum varieties for number of corn leaf aphids, Rhopalosiphum maidis Fitch, which developed on the plants after artificial infestation (Ford and Everly, 1960) (see also the Section 111, B, 7, on corn). c. Sorghum Stem Borer (Chilo zonellus Swin). Natural and hand infestation of egg masses of sorghum stem borer were used to evaluate 30 sorghum varieties in the field. Twelve resistant varieties had reduced numbers of borers. There was also variation between 10 varieties for development and survival of freshly hatched larvae reared on split stems in the laboratory. The results indicate the possibility of accumulation of antibiotic factors in leaves and stems of different varieties (Teotia and Sharma, 1967). S. R. Singh (1965) reported that 29 varieties from the world germ plasm have shown less than 10% injury by the stem borer. Kumar and Bhatnagar ( 1962) evaluated 1140 varieties under natural infestation in the field for stem borer resistance and found a broad range of response. Dwarf and early varieties with thin stem, few, narrow and short leaves, short and thin earheads, less weight and threshing percentage were comparatively more resistant than those varieties with the opposite characteristics. Types with white exposed seeds, spreading earheads, and juicy stem were found to be highly resistant. However, Swarup and Chaugale (1962) in an evaluation of 70 varieties found that those varieties which are late and high in sugar content were less damaged by the stem borer than the early varieties and those having low sugar content. They found that stem borer attack was not related to HCN content. d. European Corn Borer, Ostrinia nubilalis, Hbn. Artificial infestation of egg masses of European corn borer followed by splitting the stem from the top seedhead to the top node and counting the cavities in the peduncle area was an expedient method for determining the degree of infestation in sorghum. The basic kafir and feterita varieties of sorghums exhibited low to moderately low levels of infestation in all tests. The basic milo varieties generally were among the more heavily infested entries in all tests. Hybrids derived from kafir and milo varieties generally exhibited a level of infestation intermediate to that of the parent varieties (Dicke et al., 1963). Reductions in grain yield by artificially infesting with European corn borer eggs were measured in 4 grain sorghum strains with the 2 strains which were previously indicated to be resistant, having 3.1 and 4.6% reduction in yield compared to uninfested checks. The 2 strains whicli had been previously classed as moderate and highly susceptible suffered 10.3 and 14.1 96 reductions in yield, respectively (Atkins et al., 1963).
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e. Miscellaneous Insects. In a field study using natural infestation the grain sorghum variety Apache was more susceptible to sugarcane borer, Diatraea saccharalis (Fabricius ) than 10 other varieties during the production of the first of 2 crops. In the regrowth crop KING’S DIAMOND was the least susceptible and there was more variation in response between the varieties (Teetes and Randolph, 1971). McMillian and Starks (1967) infested 30 sorghum lines in greenhouse flats with larvae of fall armyworm, Spodoptera frugiperda, and found significant differences in leaf feeding and number of larvae recovered per plant. Open heads of sorghum had fewer bollworm larvae, Heliothis zea Boddie, than closed heads, probably as a consequence of larvae being more vulnerable to natural enemies (Doggett, 1964). In studies with the lesser rice weevil, Sitophilus oryzae L., resistance was due to relative grain hardness. The harder the grain the fewer were the eggs deposited. An exception was the waxy type sorghum, TEXIOCA 54, which, though hard-grained, was very attractive for oviposition. The high tannin content of the variety SAGRAIN may have been a further deterrent to oviposition. Also the adult life span of the lesser rice weevil was reduced with increasing hardness of the grain (Russell, 1962, 1966). The harder grained varieties were also shown to reduce the number of emerging first generation rice weevils, SitophiIus zeamais (Russell and Rink, 1965). A positive association was found between a low rate of loss from rice weevil, Calandra oryzae, damage and a thick corneous outer endosperm shell in the grain. Using this criterion of selection, crosses between the variety B.C. 27 from the Belgian Congo and the Tanganyika variety WIRU (with corneous endosperm) produced desirable new strains of sorghum with weevil-resistant grains (Doggett, 1957, 1958). The number of adults emerging from larvae of the red flour beetle Triboliutn castaneum (Herbst), grown in the flour of 62 sorghum varieties showed a broad range of response. Apparently sorghum varieties possess some chemical factors in varying degrees which are responsible for the differential behavior toward the larval development (Dang and Pant, 1966). Absorption of silica in the chinch bug, Blissus leucopterus (Say), resistant variety, ATLAS, Sorghum subglabrascens, was more rapid than in the susceptible variety, DWARF YELLOW MILO (Lanning and Linko, 1961). 9. Sugarcane Sugarcane Borer, Diatraen saccharalis F. A. broad range of infestation of the sugarcane borer was observed on alternate hosts and sugarcane, Saccharurn offieinarum L., in Puerto Rico by Quintana and Walker ( 1970). They measured percent larval infestation following gravid female
RESISTANCE OF PLANTS TO INSECTS
219
releases in field cages. Mathes and Charpentier (1963) described the procedure they have used in Louisiana for screening and developing resistant varieties to the sugarcane borer. They have found four main types of resistance in sugarcane to the sugarcane borer: ( 1) Unattractiveness of varieties to moths for egg deposition. Generally varieties with narrow leaves are least attractive. (2) Conditions unfavorable for young borers to become established in the plant. Some varieties have leaf sheaths that remain intact thereby holding water and drowning many young borers. Also, some varieties shed lower leaves, therefore doing away with shelter for young borers. ( 3 ) Inhibition of borer development in the plant. This is usually caused by nutritional and physical qualities of the tissues. For example, high fiber canes are usually less suited to borer development than low fiber canes. (4) Tolerance-these canes yield well in spite of a high infestation. For example, some varieties produce suckers when injured by the borer. Chang and Shih (1959) found that varieties resistant to the top borer, Scripophaga nivella F., generally have thicker layers of lower and upper epidermis and more dense parenchymatous cells of the leaf midrib than susceptible varieties. Aganval and Kandaswamy (1959) found that damage due to the grasshopper, Gastrimarcus marmoratus Thu., was more severe in sugarcane varieties with light green foliage in field plots in India. Further experiments indicated that the grasshoppers probably were attracted by the light green color. Eriophyid mite was found to show a differential effect toward various sugarcane varieties in India (Agarwal, 1965a). Aganval (1959, 1965b) has reviewed the response of many sugarcane varieties to insect pests in India along with characteristics of the sugarcanes which possibly influence the response. Among the insects are the top borer, Scripophaga nivella F. ; early shoot borer, Chilotraea infuscatellus Snell; stalk borer, Chilotraea auricilia Dgn.; Dehra Dun borer, Bissetia steniellus Hmpsn. ; stem borer, Chilo tumidiscostalis Hmpsn.; internode borer, Proceras indicus K.; root borer, Emmalocera depressella Swinh.; Pyrilla, Pyrilla perpusilla Walk.; mites, Paratetranychus indicus Hist. and Schizotetranychus andropogonii Hist.; lygaeid bug, Macropes exacavatus, Distt. ; sugarcane scale, Targionia glomerata Green; and white fly, Aleurolobus barodensis Mask and neomaskellia bergii Sign.
C. MALVACEAE Cotton
The primary insects attacking cotton are the boll weevil, Anthonomus. graridis Boheman; the bollworm complex, Heliothis zea (Boddie) and
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F. G . MAXWELL, J . N. JENKINS, AND W . L. PARROTT
Heliothis virescens (Fabncius) ; the pink bollworm, Pectinophora gossypiella (Sanders) ; the plant bugs, Lygus lineolaris (Palisot de Beauvois) and L . hesperus (Knight) ; jassids, Empoasca spp.; thrips, Frankliniella spp.; aphids, Aphis gussypii (Glover) ; and the twospotted spider mite, Tetranychus urticae (Koch). The primary insects in the rain-grown cotton belt of the United States are the boll weevil and the bollworm complex. The pink bollworm and plant bugs, Lygus, are the primary insects of the irrigated southwest and western United States. a. Boll Weevil. Cotton, Gossypium spp., is the only cultivated host of importance of the boll weevil. Its host range of wild and cultivated plants is restricted to the family Malvaceae. The list of malvaceous plants that are considered host plants of the boll weevil, originally considered monophagous on cotton, has grown considerably. A complete list with references is given by Stoner ( 1968). A number of compounds have been found in cotton which stimulate biological activity in the boll weevil. A water-soluble arrestant and feeding stimulant was the first material (Keller et al., 1962). This material was present in all aboveground portions of the cotton plant, and a concentration gradient was shown in various plant parts (Maxwell et al., 1963a). A technique to bioassay the material was also developed by these authors. Jenkins et af. (1963) reported that the arrestant and feeding stimulant was not present in bean, okra, or cucumber seedlings, but was present in cotton seedlings. The material varied among cotton species with the two Asiatic species, G . arboreum and G. herbaceum containing less than G . hirsutum. These Asiatic species are also less preferred in the field by boll weevil. A material attractive to boll weevils in the laboratory was extracted from the ice from a lyophilizer used to freeze dry cotton buds. The attractive material had a terpine-like odor. Later, the attractant was also extracted from the air surrounding growing cotton seedlings and fruiting plants (Keller et al., 1963, 1965). A repellent material was found in the volatile substances of cotton. The repellent was present in the extract but could be separated from the attractant (Maxwell et a!., 1963b). The presence of a material repellent to the boll weevil in the perianth of okra and in althea was suggested by Everett ( 1964). A biologically active feeding deterrent was found in the water extract of the calyx of althea, Hibiscus syriacus, by Maxwell et al. (1965). A good review of the chemistry of the compounds is given by Hedin et al. ( 1972). A water-soluble component of cotton anthers was found to stimulate oviposition (Everett, 1964). Buford et al. (1967, 1968) reported a differential rate of oviposition on various cotton lines. They reported a factor
RESISTANCE OF PLANTS TO INSECTS
22 1
present in some cottons which suppressed boll weevil oviposition 3 0 4 0 % . Oviposition was suppressed the most in a G. barbadense cotton, SEA ISLAND SEABERRY. They developed a bioassay technique to screen cotton lines for rate of suppression of boll weevil oviposition and found that the factor in SEA ISLAND SEABERRY was under genetic control. Coakley et al. (1969) found that a material present in the water homogenate of second- and third-instar boll weevil larvae caused square abscission. A technique was developed by Jenkins et al. (1964a,b) to measure boll weevil antibiosis in cotton lines independent of square size. The technique was based on lyophilized cotton squares and the insects were reared from eggs implanted in the diet. Davich et al. (1965) developed a technique for implanting boll weevil eggs in cotton squares for use in testing systemic insecticides and host plant resistance studies against the larvae. The effect of numerous plant compounds on boll weevil growth has been investigated. Ascorbic acid was found to be necessary, but in minute amounts (Hudspeth et al., 1969). In an investigation of the effects of tannin, rutin, quercetin, and gossypol on boll weevil development, only gossypol had a major effect, and then only in amounts greater than 3.5% of the diet (Maxwell et al., 1967). In a study conducted to investigate whether resistance could be chemically induced in the cotton plant, Matteson et al. (1963; Matteson and Taft, 1963), found that 0-isopropoxyphenyl methylcarbamate, O-methoxyphenyl methylcarbamate, M-isopropylphenyl methylcarbamate, and 0-ethoxyphenyl methylcarbamate exhibited definite systemically induced repellency in seedling cotton plants and in plants in the 4-leaf stage in the laboratory. They screened plant extracts representing 117 families and 358 species and 400 fermentation filtrates. Many research workers have screened a large number of cotton lines for resistance, with the results shown in Table I. Stephens (1957) suggested that a study of response of the boll weevil to the morphological mutants in cotton should elucidate some of the close interrelationships between this insect and the plant and provide leads as to where to look for resistance. He and his co-workers conducted an intensive study into this aspect with the mutants H,,H,, gl,, R, (Stephens, 1959; Stephens and Lee, 1961; Wannamaker 1957; Wessling 1958a,b). They found that each of these mutants did affect the sensory perception or behavior of the boll weevil. The insect shows a nonpreference for (discriminated against) hairy, glandless leaf, and red plants. Hunter et al. (1965) conducted an intensive screening program with 336 diverse cotton strains during 1957-1961. They expanded to include selected progenies from these after the first year. They reported finding
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F . G . MAXWELL, J. N. JENKINS, AND W. L . PARROTT
TABLE I Sources and Types of Resistance to the Cotton Boll Weevil
Type oE resistance
Cattori line
Gossypiurn arboreurn Antibiosis Antibiosis G . daridsonii Antibiosis G . ihurberi Antihiosis G. ihurberi Antibiosis Red plant, clean seed Experiniental n u n ~ l ~ e r e dAntihiosis strains .4ntihiosis 0. barbadensr Tolerance Rapid fruiting cottons Red plants Hairy plants Frego hract Hopi Russian 5-4 CB2.545 5 Frego lines 4 Red lines
Several S.I.lines rZsiatic spp. Frego bract Frego hract Frego bract and red Presence of glands Presencc of hairs Red hairy
(H?)
MG-9 (HI and 11,) Pilose ( I l l and If,)
111 H a Ri
?Yhere resistance was espressed
Referrnre
Field Field Field Laboratory Field Field
Bailey et al. (1967a,b) Bailey et al. (1967a,b) Bailey et al. (1967a,b) Jenkins el a2. (1964a,b) Bailey et al. (1967a,h) Douglas (1966)
Field Field
Black and Leigh (1963) Hunter et a / . (1965); Hunter and Waddle, 1958 Hunter et al. (1965) Hunter rt al. (1965) Hunter et ab. (1965)
Preference Preferenre Pro ha big preference .intibiosis Antibiosis Antibiosis Preference
Field Field Field
Preference Preference Preference t'nclassified Preference Preference Stimulates oviposit ion Preference Preference, antibiosis Preference Preference Preference
Field Field Field Field Field Field Lahoratory
Hunter et al. (1965) Hunter et (11. (1965) IIunter et al. (1965) Jenkins et al. (1969); Jenkins and Parrott (197la,h) Jenkins et al. (1969) Jenkins et al. (1969) Merkl and Meyer (1963) Lincoln and Waddle (1966) .Jenkins and Parrott (1971a,h) Jones et al. 11964) Stephens (1'1.59)
Laboratory Field
Stephens (1959) Stephens and Lee (1961)
Ficld Field Field
\Vannamakcr (1957) Wannaniaker (1957) Wessling (I958a,b)
Field Field Field Field
tolerance, preference, and antibiosis forms of resistance but none significant enough for commercial production of cotton in the absence of other forms of insect control. They were the first to find that frego bract cotton was less preferred by the boll weevil. Jenkins et al. (1969) developed field plot techniques which allow the simultaneous evaluation of cotton lines which have inherently different
RESISTANCE OF PLANTS TO INSECTS
223
rates of squaring. They used covariance to adjust the squaring rate. It was shown through the use of 12 pairs of glanded and glandless cotton lines in field plots that the glandless character should not cause any increase in susceptibility to the boll weevil (Jenkins et al., 1967). Utilizing these same glanded-glandless lines, laboratory studies on feeding, oviposition, and development also suggested that glandless should not increase susceptibility to the boll weevil (Maxwell et ul., 1966). The current approach to boll weevil control is to use an integration of several biological and chemical methods. A pilot boll weevil eradication experiment is currently underway in Mississippi. Jenkins and Parrott (1971a,b) have explored the use of resistant cotton strains in this eradication program. They showed that frego bract will reduce boll weevil populations 66-94% below that on non-frego bract cottons when both types are used following a reproduction-diapause control program involving chemical insecticides to reduce the numbers of weevils entering hibernation (diapause) in the fall. Studies involving Humpea spp., the dioecious wild host of the boll weevil, have shown that the male trees are susceptible whereas the female trees are nearly immune (Lukefahr and Maxwell, 1969). The mechanisms involved in resistance are preference and antibiosis. In comparison with buds from male trees, the female buds contain less attractant and feeding stimulant plus a repellent (Maxwell et al., 1969). Both male and female buds contain some factor responsible for antibiosis; however, the antibiosis is much more pronounced in the female buds (Parrott et al., 1969). This mechanism of resistance involves quantitative differences in some of the biologically active compounds (attractant, repellent, feeding stimulant, deterrent, and oviposition suppression factors) which have been shown to be present in cotton. b. Heliothis spp. H . zea (Boddie), the cotton bollworm, and H . virescens (F.), the tobacco budworm, are serious pests of cotton. They are generally referred to as the bollworm complex. Both these species feed on a wide range of host plants (Snow and Brazzel, 1965, 1966). A free exchange of hosts occurs between cotton and other hostplants as reported in a study in Mississippi (Snow and Brazzel, 1965). In field and cage studies the plant morphological characters, smooth leaf and stem, and nectariless, both reduce egg laying by moths of the bollworm complex (Lukefahr and Rhyne, 1960; Lukefahr et al., 1965, 1971). The reduced oviposition was usually accompanied by a reduction in damage. The absence of nectaries (nectariless) should reduce the food for the moths, and both H . zea and H . virescens oviposited fewer eggs when no food was available in tests by Lukefahr and Martin (1964). Davis (1969) has developed an experimental strain of ACALA 1517
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F . G . MAXWELL, J . N. JENKINS, AND W. L. PARROTT
which is glabrous and nectariless. It seems to be essentially equal to ACALA 1517 in disease resistance, agronomic, and fiber properties. Lee ( 1971) has written an excellent report on the various smooth leaf genes and their allelic relationships in the various breeding lines now in use by the plant breeders. The Smt81 gene is present in COKER 413. It is allelic but not the same as MEYERS D, smooth. Sm, from a wild dooryard cotton is also similar in phenotype. Sm,", D, Smooth allelomorph of Srn,", and Sm, should each be sufficient for resistance. The sm3 allele, present in DELTAPINE SMOOTH LEAF, is not sufficiently glabrous to confer resistance. Data by Davis (1969) and Jones ef al. (1971) show that the D, Smooth allelomorph of the SrnlP1 reduces lint percentage by 1 or 2 % . This seems to be its major detrimental effect. Probably this and other slightly deleterious effects due to SrnISzcould be ameliorated through breeding (Lee, 1971). This would probably also be true with Sm,. The investigation of antibiosis as a resistance mechanism was aided considerably by the development of a lyophilized square powder testing diet (Oliver et at., 1967). This was a modification of the boll weevil lyophilized square powder diet of Jenkins et al. (1964a,b). The cotton pigments, gossypol, quercetin, and rutin, were incorporated into standard bollworm diet and their detrimental effects on growth were observed by Lukefahr and Martin (1966). They suggested the increase of natural cotton plant pigments as mechanisms of resistance. By the use of the lyophilized square powder bioassay, cotton lines with antibiosis have been found. High gossypol cotton lines (gossypol content 1.7% in X-G-15) have been found in the dooryard cottons (Lukefahr and Houghtaling, 1969). Lukefahr et al. ( 1969) reviewed the smoothleaf, nectariless, and high gossypol work. A high correlation between antibiosis on lyophilized square powder diets and fresh squares was found. Researchers have begun a program of work to locate new sources of resistance in the wild hirsutum races (Lukefahr et al., 1969). Shaver and Lukefahr (1969) incorporated cotton pigments in diets under controlled conditions and confirmed the suggestion of Lukefahr and Martin (1966) that the naturally occurring flavonoid pigments have potential as sources of resistance to Heliothis spp. They also have potential as a source of resistance to the pink bollworm (Shaver and Lukei fahr, 1969). Shaver and Parrott (1970) showed that age had an effect on the tolerance of H . zea, H . virescens, and P . gossypielliu to gossypol. The early instars were the most susceptible. Oliver et al. (1970) studied food utilization and consumption by H . Zea. All ages of larvae tested had a smaller weight gain on the diet from glanded cotton due to a 21-31% reduction in feeding and due to less efficiency of food conversion compared with larvae fed on diet from glandless cotton. Small larvae were less efficient than large larvae. In a similar study,
RESISTANCE OF PLANTS TO INSECTS
225
Shaver et al. (1971) found that food utilization was reduced in H . zea, but not in H . virescens, on diet from the high gossypol cotton. Larval weights were reduced for both species on the high gossypol diet. There are two recessive genes, glp and g13, which produce cotton plants devoid of gossypol glands in the plant and seed. A series of 10-14 pairs of glanded and glandless lines have been studied by numerous investigators for their effects on Heliothis larval weights. The larvae are larger on glandless cotton than on the glanded member of each pair (Lukefahr et al., 1966; Oliver et al., 1971). In contrast, field studies have shown larger larvae, but in general the damage has not been significantly greater on glandless lines (Oliver et al., 1970; Jenkins et al., 1966). However, the glandless lines in some variety backgrounds tended to receive more damage than the glanded counterpart in both these studies. Thus, these authors suggest a careful consideration of the variety-glandless interaction. Wilson (1971a,b) found that H . virescens larvae at 6 days of age could discriminate between the 9 genotypes of the two glandless genes. Their preference was inversely related to gossypol content of the 9 genotypes. He also suggests that it might be possible to select seedlings by counting pigment glands in the petiole and cotyledon without resorting to the bioassay. In a test combining the resistance due to glabrous and nectariless traits with the disease caused by nuclear polyhedral virus, the results compared favorably with those obtained with methyl parathion (Fernandez et al., 1969). Wene and Sheets (1966a) compared two long staple and four short staple varieties of cotton with respect to H . zea infestation in Arizona. Less damage was done on the long staple ( G . barbadense) cottons, apparently because of a reduced larval survival. Other workers have shown a higher gossypol content in the G. barbadense species, and thus it may be the factor involved in antibiosis in these results. c. The Pink Bollworm (Pectinophora gossypiella (Saunders)) . The pink bollworm was described in 1843 from specimens collected in India in 1842 and is probably indigenous to that country (Noble, 1969). In 1904 it was reported as a serious pest of cotton in German East Africa. It has since become an injurious pest of cotton in many cotton-growing regions of the world. The pink bollworm feeds on a wide range of hosts. The preferred host is the genus Gossypium. Schiller et al. (1962) lists the host plants. Noble (1969) has written an excellent review of 50 years of research on the pink bollworm in the United States. An early report of cottons resistant to pink bollworm is that of Woolcot in 1927. He reported that the native and cultivated cottons of Haiti were resistant, whereas the introduced, cultivated cottons were susceptible.
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F. G . MAXWELL, J. N. JENKINS, AND W. L. PARROTT
Researchers at Texas A & M University have screened a large number of cotton lines for resistance (Brazzel and Martin, 1956, 1959; Reed and Adkisson, 1961; Adkisson er al., 1962). Bolls of a G. hirsuturn X G . tomentosum cross possess a high degree of antibiosis to developing larvae. G. thurberi and a G . hirsutum X G . arboreurn cross (called hexaploid 2-64) possessed a lower degree of resistance (Brazzel and Martin, 1956, 1959). Several dooryard race stocks of G . hirsuturn have been reported to contain resistance: Texas 389 race marie galante (Brazzel and Martin, 1959), Texas 275 race palmerii, and Texas 373 race marie galante (Reed and Adkisson, 1961) . The morphology of the cotton plant is important in resistance to pink bollworm. A tight smooth calyx, spikelike flared bracts, and glabrous vegetative parts are important in the resistance of G . rhurberi. These characteristics modify the egg-laying habits of the moths. Lukefahr and Rhyne (1960) found that the presence or absence of nectaries on the plant did not affect pink bollworm populations. Proliferation of cells resulting in crushing or drowning the young larvae was the mechanism of resistance in Texas 373 Marie Galente and the G. hirsutum x G. tomentosum cross. In crosses with Upland cotton the resistance in Texas 373 seemed to be inherited as a dominant character; however the resistant plants always seemed to resemble the wild parent, and thus it would be difficult to use the resistance (Adkisson et al., 1962). The thickness of the boll rind was found to be negatively correlated with incidence of pink bollworm (Singh er al., 1965). In a study conducted by Khalifa (1967) in the Sudan, the first-instar larvae were killed when they fed on boll gland contents. A number of larvae died before chewing through the boll wall. Shaver and Parrott (1970) found that gossypol in laboratory diets of pink bollworms was more toxic to firstinstar larvae than to older larvae. Squire (1939) found that when young larvae feed on young okra pods they drown in the mucilage which the okra secretes. d . Jassids. An excellent, detailed discussion of the host plant resistance work with jassids up through 1951 is given by Painter (1951) in his textbook. He presents a table listing the species, localities, and economic status of the various species of jassids. Three species have caused the most trouble on cotton; Enzpoasca fasciulis (Jac.) in Africa; E. devastans Dist. on the subcontinent of India, and E . terra-reginae Paoli in Australia. Resistant strains of cotton are available against all three species. A high density of leaf hairs seems to be the most generally utilized type of resistance. Most of the host plant resistance work with jassids was completed before 1951 and allowed successful production of cotton in areas infested with jassids.
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227
In the Sake1 variety ( G . barbadense), the hairy lines developed more resistance to E . lybica (Deberg.) than the glabrous lines, but the resistance did not develop until some weeks after breeding of the insect started. The hairy lines were more susceptible to Bemisia tabaci (Gennadius) (Evans, 1965). Muttuthamby et al. (1969) reported that the H , gene induced hair of sufficient length and density to confer resistance. The HlP gene also is sufficient and is present in their local varieties PAK 51, L-11, and AC-134. Sikka et al. (1966) found that length of hair was of prime importance in resistance, followed by density on the lamina; whereas, hair on the midrib was not important in imparting resistance. These conclusions were based on an examination of 106 strains and genetic stocks. In a study with E. devustans, Tidke and Sane (1962) found a high correlation of resistance with thickness of leaf lamina, angle of insertion of hairs, length of hairs, number of hairs per unit of leaf vein, and hairs on the lamina. Jahontov (1955) found a high correlation between osmotic pressure of plant sap and resistance to jassids. Plants with osmotic pressures of 2.64-2.98 were susceptible whereas plants with pressures of 12.37-1 8.71 were almost immune. Fertility of jassids reared on highly resistant plants was greatly reduced. Annappan et al. (1965) reported that what they call the Cambodian cottons (G. hirsutum) are susceptible, whereas the Karunganni cottons (G. arboreurn) are resistant. In 1960 and 1961 a mild incidence of jassids was noted on the G. arboreum cottons. During the next two years this developed into a widespread occurrence. Annappan and co-workers then studied 107 varieties of G . arboreum cotton for resistance. They found that 98% tolerance to jassids in varieties with narrow leaf lobes and 78% in the varieties with wide lobes. They stated that leaf shape and succulence play a role in resistance to jassids. e. Lygus. Only a limited amount of host plant resistance work has been done in cotton with the plant bugs Lygus hesperus and L. lineolaris, the tarnished plant bug. Attempts to develop artificial diets for Lygus have met with only partial success (Auclair and Raulston, 1966; Landes and Strong, 1965; Raulston and Auclair, 1968; Strong and Kruitwagen, 1969; Vanderzant, 1967). Beard and Leigh (1960) reported successful rearing of L. hesperus in the laboratory using fresh green beans. We have successfully reared L. lineolaris on fresh green beans for two years in our laboratory. It was suggested by Butler (1968) that in nature L. hesperus supplements its plant feeding with honeydew from other insects present in alfalfa in Arizona. After flowering, the Lygus obtain sugar by feeding on the secretions of the nectaries. The biology of L. lineoluris on cotton has been described by Bariola (1969). Bech (1967) described the feeding damage on a histological level.
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F. G . MAXWELL, J. N. JENKINS, AND W. L. PARROTT
Lygus feeding affects plant height (increase) and growth pattern. Pricking the terminal of the stem apex with a glass needle and/or feeding by Lygus vosselari Popp caused an increase in cell division (Dale and Coaker, 1958). Young turgid meristematic tissue is preferred for feeding. A material is present in salivary juices which causes blackening of the tissue around the wound (Dale and Coaker, 1958). Polygalactourinase was found in the posterior lobe of the principal salivary gland of L . hesperus (Strong and Kruitwagen, 1968). Strong (1970) reports that injury caused by L . hesperus is due principatly to enzymatic digestion of plant tissues by polygalactourinase excreted during feeding. If feeding results in destruction of specific plant hormone producing sites the resulting damage is in accord with the known physiological responses to hormone manipulation in plants. Heavy levels of L . lineoluris on caged cotton can result in abnormal plants, but not plants typical of the abnormality referred to as crazy cotton (Scales and Furr, 1968). Seedling cotton can be killed by L. hesperus and blank squares can result from early feeding (Wene and Sheets, 1966b). The nature of the damage of L. hesperus to presquaring cotton has been described by Wene and Sheets (1966b). A cooperative program on host plant resistance has begun between the Mississippi Agricultural and Forestry Experiment Station and the Agricultural Research Service of the USDA. We have developed mass rearing techniques using fresh green beans and established procedures for screening for resistance in the seedling stage. A few preliminary plant selections have been made. We have partially completed a study on the behavior of L. lineoluris in the field on cotton and several wild hosts. f. Spider Mite, Ffeahoppers, and Aphids. In the United States the twospotted spider mite, Tetranychus urticae (Koch. ) is cosmopolitan. The strawberry mite, T . turkestani Ugarov and Nikolski, occurs in California and across the northern cotton belt. The tumid spider mite, T . tumidus Banks is found on the Gulf Coast and the Atlantic seaboard. The carmine spider mite, T . cinnabarimus (Boisduval), and the desert spider mite, T . desertorum Banks, occur in the central cotton belt along with the twospotted spider mite (Leigh and Hyer, 1963; Schuster, 1971). Eotetranychus smithi Prichard and Banker was reported on cotton in western Tennessee for the first time in 1967 (Caldwell, 1967). The damage caused by mites feeding on leaves is described by Lieserling ( 1960). Cells collapse, water balance of leaves is disturbed, transpiration is accelerated, and the leaves finally dry out. Respiration increases, assimilation decreases, and photosynthesis is inhibited. Leigh and Hyer (1963) developed a rating scale for field infestations of spider mites ( T . urticae) and found resistance in G . thurberi, G .
RESISTANCE OF PLANTS TO INSECTS
race
229
richmondii, TANGUIS ( G . barbadense) and a 4-42 cross. ACALA 4-42 was intermediate in resistance and ACALA glanded and glandless breeding lines were scored equal to each other. Later Leigh et al. (1968) ranked PIMA S-2 as resistant, ACALA 4-42 and ACALA SJ-1 as intermediate, and AUBURN 56 as susceptible. The hairy cotton BAHTIM 101 was reported to be highly resistant to the spider mite, jassid, and cotton aphid in Egypt (Kame1 and Elkassaby, 1965). Schuster et al. ( 1972a,b,c) have developed mass rearing techniques for T . urticae, as well as screening techniques utilizing cotton seedlings, and have selected some resistant plants. Both tolerance and antibiosis were reported as mechanisms of resistance. There was no major effect of gossypol in a study with a number of glandless and glanded lines. In a study of nutrition, Rodriguez and Hampton (1966) determined the essential amino acids for T . urticae Not much host plant resistance work has been done with the cotton aphid, Aphis gossypii Glover. Auclair (1967) has reared the aphid for two generations on liquid diet. Sucrose content and pH were two important factors in getting the aphid to successfully feed through a feeding membrane. A limited amount of host plant resistance work has been conducted with the cotton fleahopper. Pseudatomoscelis seriatus (Reuter) . Schuster et al. (1969) have described the host plants of the fleahopper in the Rio Grande Valley of Texas. Glabrous cottons were shown to develop smaller populations of fleahoppers than hairy cottons (Lukefahr et al., 1968, 1970). The presence or absence of nectaries or gossypol glands did not affect populations (Lukefahr et al., 1968). It is now suspected that the glabrous cottons are more severely damaged than the hairy ones even though the fleahopper population is less (Lukefahr, 1971). In a study of injury by thrips (Thrips and Franklinella spp. Thysanoptera) to Upland cotton, the varieties with EMPIRE in their background were the most resistant. Varieties were ranked from most resistant to susceptible aS fOllOWS: EMPIRE, DEKALB 108, DIXIE KING, REX, AUBURN 56, PLAINS, STONEVILLE 7, COKER 100A, and DELTAPINE 15 (Hawkins et al., 1966). g . Other Cotton Insects. In Southern Chad, Brader (1966, 1967) found that Podagrica dilecta (Dalm.) and P . uniformis (Jac.) prefer glandless cotton over glanded; however, P . pallida (Jac.) was more abundant on plants with glands. Brader noticed that some varieties of glandless were less damaged than others. The banded wing whitefly, Trialeurodes abutilonea (Roldeman), in Arizona was more prevalent on hairy segregates in a DWARF A (G. barbadense) x LANKART ( G . hirsutum) cross. However, an attempt to rehirsutum TANGUIS
X
ACALA
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F. G. MAXWELL, J . N. JENKINS, AND W. L. PARROTT
late susceptibility to hairiness on 26 species of cotton was not successful (Butler and Muramoto, 1967). However, in the Sudan Gezira the hairy varieties of cotton which are resistant to E. lybica have large populations of sweet potato whitefly, Bemisiu tabaci (Gennodius) Mound, 1965). The cotton leafworm, Prodeniu litura (Auct.), feeds on a large number of plants in Egypt (Moussa et al., 1960). The new hairy variety in Egypt, BAHTIM 101, is more resistant to the cotton leafworm than other commercial lines or G. anomalum. Fewer eggs are laid, less damage results from artificial infestation of eggs, larval mortality is higher, and percentage of pupation is lower on BAHTIM 101 when compared with MENOUFI, a glabrous commercial variety (Kame1 and Elkassaby, 1965 ) . Considerable work has been done in cotton relative to host plant resistance. However, with the exception of resistance to jassids we do not have any resistant varieties which are relied on for control. Cotton is a highly bred and specialized crop. Adaptation includes the usual agronomic factors as well as fiber quality factors. Most of the sources of resistance to major insects have been found in wild cottons. These are difficult to utilize in a practical breeding program. Also, we do not have adequate techniques with many of the insects to reliably select resistance plants in segregating populations. In many cases mass rearing and release of the insects are required to utilize the techniques which have been developed. In the [Jnited States, except in the far West, nearly all the cotton is planted to varieties developed by commercial companies. Researchers have found resistance sources, but have not shown the commercial breeders any practical way to use them at the present time. A notable exception to the above is the use of morphological characters that confer resistance. The two major insects in the rain-grown cotton belt in the United States are the boll weevil and the bollworm. Smooth leaf cottons show great promise for resistance to bollworms. Frego bract shows great promise for resistance to boll weevils. The commercial breeders can handle these traits since both are easily worked with. We should know in a few years if the companies are able to utilize these two resistance mechanisms in commercial varieties and still maintain acceptable agronomic and fiber properties. Their effects on other insects will also be determined during the variety development period. Present indications are that both smooth leaf and f-rego bract can be bred into new varieties presently being developed, but each may be more susceptible to one or more other insects than present varieties. Earliness is a valuable escape mechanism for boll weevil resistance. Walker and Niles (1971) have shown the potential value using early strains and modified cultural practices.
23 1
RESISTANCE OF PLANTS TO INSECTS
IV.
Horticulture Crops
A.
ROSACEAE
Most of the work in host plant resistance in the Rubis spp. raspberry, has been with the rubis aphid, Amophorophora rubi. This aphid is the vector of several viruses which the winged forms spread from location to location and which the apterae spread within a location (Briggs, 1965a,b). Hill (1956) was one of the first to recognize that strains existed in this aphid. Four strains (1, 2, 3, and 4) are presently recognized (Briggs, 1959, 1965a,b; Knight et al., 1960; Keep and Knight, 1967). Winter (1929) was the first to report resistance in raspberries in the cultivator HERBERT. Huber and Schwartz (1938) reported resistance in INDIAN SUMMER, LLOYD GEORGE, PYNE IMPERIAL, and PYNE ROYAL. Table 11 shows the relationships between the genes for resistance and the strains of aphid (Briggs, 1959, 1965a,b; Knight et al., 1960; Keep and Knight, 1967). Aphid strain 4 has been difficult to maintain and thus its virulence on the various resistance genes is unknown. A source of resistance to strain 4 has been found in a seedling of Rubis idaeus var. strigosum (L518) (Keep and Knight, 1967). Genetic control in the aphid involves one dominant and one recessive gene. The dominant gene gives strain 2, the recessive gene strain 3. In
TABLE I1 Relationship between the Various Resistance Genes in Rubis spp. and the four Strains of the Rubis Aphid Reaction to aphid straina Gene in plant
a
3
4
R
S
R
-
S S
R R
R R R R R
S S S
S S S S S
1
R
R
R S
R R
R R
R, resistant; S, susceptible.
R R R R S
-
S
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F. G . MAXWELL, J . N . JENKINS, AND W. L. PARROTT
the absencc of both genes aphids are strain 1, and the presence of both genes gives strain 4, Strain 2 and 4 are rare and occur only on the obsolete variety, MALLING, LANDMARK, and provisions for resistance to strains 1 and 3 are of major importance in Britain (Briggs, 1965a,b). The sources of the 10 genes for resistance are tabulated (Knight et al., 1960; Keep and Knight, 1967) : Resistance gene
Source Raumfortti cultivar Chief cultirar Chief cultivar Chief cultirar Chief cultivar Chief cultirar Chief cultirar R. idaeits r a r . strigositm R. idaeus var. strigosum R. occidentalis 1,.
~
~
_
_
_
____
In Britain strains 1, 2, and 3 of the rubis aphid can be controlled on raspberries by using either a combination of resistance genes A , and A , or A , and A , (Knight et al., 1960). Resistance to strain 4 will require the use of A ; or A , (Briggs, 1965a). The gene A , , , can be used for resistance to strains 1, 2, and 3 (Keep and Knight, 1967). The resistance work in strawberries, Fragaria ananassa Duchense, has been with Tetranychus urticae Koch and T . turkestani Ugarov and Nikolski; most of the work has been published since 1968. Biologically active compounds have been reported in water extracts and essential oil extracts of strawberries by Rodriguez ef al. (1971 ) and by Dabrowski and Rodriguez ( 1971 ). Water-extractable compounds form a part of the biochemical mechanism responsible for resistance of certain strawberry clones to T . urticae and T . turkestani. Attracting, repelling, and gustating responses were different for KY 22-61-9 and Ky 17-16-15 and the susceptible clone CITATION. Some of the essential oils of CITATION have far-reaching effects on the behavior of the two species of mites. Both resistance index and the biochemistry of the strawberry plant change during the season, and Dabrowski and Rodriguez ( 1971) feel that this change in resistance has a biochemical basis. They have found no evidence of antibiosis or morphological characters associated with resistance; however, they do point out that T . urticae is a polyphagous, phytophagous acarine, and it would appear difficult to find a specific biochemical entity that would elicit an attractive type response to this mite.
RESISTANCE OF PLANTS TO INSECTS
233
Chaplin et al. (1968, 1970) discussed the breeding behavior of the resistant clones of strawberries. They found that resistance was partially dominant and controlled by multiple genes. Breeding for mite resistance in strawberries is thus feasible.
B. CRUCIFERAE The cabbage maggot, Hylemya brassicae (Bouche), has received considerable attention from host plant resistance workers. In a study of oviposition preference of cabbage aphid for various plants in the family Cruciferae, significantly more eggs were oviposited during the season on rutabago, Brassica napus var. napobrassica (L.) Reichle, and turnip, B . rapa L., than on radish, Raphanus sativns L., and mustard B. nigra (L.) Koch, and the least were oviposited on cauliflower, B. oleracea var. botrytis. Radish appeared to be particularly susceptible to oviposition for a very short period (Doane and Chapman, 1962). In a study by Mukerjimk (1969) in Ontario, Canada, the ovipositional preference of the cabbage maggot was studied on the early cruciferous crops, brussel sprouts, B. oleracea var. gemmifera, broccoli, B. oleracea var. botrytis, cabbage, B . oleracea var. capitata L., cauliflower, B. oleracea var. botrytis, Chinese cabbage, B . oleracea var. capitata, and kohlrabi, B. caulorapa. Significantly more eggs were oviposited on Chinese cabbage than any other host. In the first generation he found that the percentage mortality for each of the life history stages differed significantly on the various crucifers, but the total generation mortality did not. Pond et al. (1962) studied the relationship between two species of cabbage maggot, H . brassicae and H . cilcrura Rondani, and turnip lines resistant to H . brassicae. Laurentian is a variety susceptible to H . brussicae. This species laid the most eggs around Laurentian, whereas H . cilcrura laid the fewest eggs on this variety. On the other hand, lines selected for a high degree of resistance to H . brassicae seemed particularly attractive to H. cilcrura. Swailes (1959, 1960) found that two factors were involved in resistance to cabbage maggot in rutabagas: ( 1) difficulty in feeding by larvae and ( 2 ) resistance to larval growth. In the variety Wilhelmsburger, the resistance was principally a characteristic of cortex tissue involving resistance to the establishment (initial feeding) of the larvae. Canadian Gem had poor larval establishment and high larval mortality of second and third instar larvae. Varis (1958) in work in Finland reported several varieties of turnip more resistant to cabbage maggot and H . floralis (Fallen), the turnip maggot,
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F . G. MAXWELL, J . N . JENKINS, AND W. L. PARROTT
than the native Big Leaf Turnip. Shape of the turnip was not important to the insects; however, the round and long varieties which generally have strong roots endured damage better and recovered more quickly (tolerance) than the flat and flat-round varieties with thin roots. In a study of the biochemical components of cauliflower and resistance to the cabbage looper, Trichoplusia ni (Hubner) , and the imported cabbage worm, Pieris rapae (L.), the amino acids pipecolic acid (proline) and tyrosine were present in the resistant varieties SAVOY CHIEFTAIN and PI 254000, respectively, but were not detected in the susceptible variety GOLDEN ACRE. Total free amino acids and total soluble nitrogen were higher in susceptible varieties than in resistant varieties (Benepal and Hall, 1967). Ten varieties of cabbage were studied by Matthewman and Lyall (1966) for resistance to cabbage maggot. VIKING GOLDEN ACRE was the most susceptible. RED ACRE and DANISH BALLHEAD were damaged less and harbored fewer maggots than VIKING GOLDEN ACRE. MARKET TOPPER and JERSEY WAKEFIELD were less damaged but harbored as many maggots as VIKING GOLDEN ACRE. Resistance in RED ACRE and DANISH BALLHEAD was due chiefly to low attractiveness, whereas, JERSEY WAKEFIELD and MARKET TOPPER possessed tolerance. Radcliffe and Chapman (1965a,b, 1966) have studied seasonal shifts and relative resistance of several commercial varieties of cabbage to the imported cabbage worm, cabbage looper, diamond backed moth larvae, Plutella maculipennis (Curtis), and the cabbage aphid, Brevicoryne brasAicae (L.). Color or intensity of irradiated light from the leaf surface was important to host selection by aphids. Red was least preferred but was most favorable to aphid increase once they were infested. Nonpreference was the primary mechanism of resistance to the imported cabbage worm and the cabbage looper. Again red cabbages were less susceptible for oviposition by the imported cabbage worm, but the worms established better than on green varieties. Seasonal shifts in damage were noted. Those varieties or plants not damaged by early generation insects were damaged by later generation insects. Data suggested that the shifts result from prior insect injury rather than from physiological changes in the plant. Myzus persicae (Sulzer) , green peach aphid, responded more to changes in nitrogen and potassium levels of Brussels sprouts grown in nutrient solution than did the cabbage aphid (Van Emden, 1966). In New Zealand more than 90,000 acres of rape, resistant to the cabbage aphid, are grown annually. The two resistant types are APHID RESISTANT and RANGI (Lammerink, 1968). A new biotype of the cabbage aphid has been found to attack formerly resistant lines. The old biotype is designated NZI and the new one NZII (Lammerink, 1968).
RESISTANCE OF PLANTS TO INSECTS
235
C. SOLANACEAE The twospotted spider mite, Tetranychus urticae Koch is a pest of tomatoes, Lycopersicon esculentum. Gilbert et al. (1967) observed 15 tomato lines under a heavy natural infestation in Hawaii and concluded that the varieties and hybrids involving lines from Hawaii were more tolerant to defoliation than the United States lines from the Mainland. A reduction in fecundity was noted on TECUMSEH as compared with KOKOMO. Five-week-old plants and mature plants gave similar results (Stoner and Stringfellow, 1967). Many of the wild Lycopersicon species have a dense covering of glandular hairs. These hairs secrete a material which is sticky and entraps many small insects, such as spider mites, aphids, and white flies. Resistance to the potato aphid, Macrosiphum euphorbiae (Thomas), in Solanium penellii was associated with the heavy vesture of glandular hairs. However, L . peruvianum, which has a sparse vesture of glandular hairs, was also resistant to the aphid, probably owing to physiological factors (Gentile and Stoner, 1968). Glandular hairs plus other factors were implicated by Stoner et a1. (1968) in the resistance to oviposition by T . cinnabarinus, carmine spider mite, among several tomato varieties. In a further study of resistance to the twospotted and carmine spider mites in Solanum and Lycopersicon, Gentile et al. (1968) found resistance to both mite species in accessions of S. pennellii, L. hirsutum and L. hirsutum f. glabaratum C . H. Mull. The density of the hairs was stable in S. pennellii but varied in L . hirsutum. In potatoes the wild species S. polyadinum, S . tarijense, and S . berthaultii contain glandular hairs in abundance. Gibson (1971) found that when an aphid mechanically ruptures the cell wall of these glandular hairs, a clear water-soluble exudate is discharged which on contact with oxygen changes into a black, insoluble material which is precipitated on the aphid’s limbs. Stoner (1970) found that he could select tomato plants in a F, population, which were resistant to the common spider mite by visually selecting plants with the greatest concentration of glandular hairs on the leaves. Selection with the naked eye was as effective as hair counts. On plants with a high concentration of hairs the females oviposited 6-50% fewer eggs than on plants with few hairs. Young healthy plants from L. hirsutum and L. hirsutum f. glabaratum had a strong repelling effect on adults but not larvae of the tobacco flea beetle, Epitrix hirtipennis (Melsheimer) . However, when 75% ethanol was used to remove the glandular exudate from the leaves the beetles were not repelled. Senescent unwashed leaves also did not repel beetles (Gentile and Stoner, 1968).
236
F. G. MAXWELL, J. N. JENKINS, AND W. L. PARROTT
Sanford and Peel (1970) studied the tobacco flea beetle on potatoes. An examination of selected terraploid stocks from the USDA potato breeding program led them to conclude that the additive genetic variation in reaction to tuber attack by the beetle is large enough to permit an increase in resistance within this group by individual phenotypic selection. Several lines of cultivated tomato L. esculentum Mill. have genes for adult nonpreference or larval antibiosis or both to the leaf miner Liriomyza brassicae (Riley). Virtual immunity was found in accessions of L. hirsutum and L . hirsuturn f. glubaratum (Webb et al., 1971). The wild species of Solanum have been examined quite extensively by Radcliffe and Lauer (1966, 1970) for resistance to the potato aphid and the green peach aphid, Myzus persicae. They present detailed tables of the resistance scores of over 400 accessions to the two aphids at Grand Rapids, Minnesota. Resistance to both aphids was found primarily in the potato species indigenous to Mexico. Resistance to the two species of aphids was generally associated. Species resistant to the green peach aphid include S . bulbocastaniini Dun., S . michoacanunz (Bitt) Rydb., and S . stenophyllidium Bitt. Species resistant to the potato aphid include S . bulbocasfanum, S. hjertingii Hawkes, S . polytrichon Rybd., and S. stoloniferum Schlechtd and Bche. The wild species of Solanum display all degrees of ploidy. The cultivated potato, S. tuberosum, is a tetraploid. Radcliffe and Lauer (1970) feel that many of the crosses suggested by their work present no particular obstacle to the plant breeder. If aphid resistance can be incorporated in S . tuberosum parents, future development of resistant varieties may be feasible. In a study with the Colorado potato beetle, Leptinotavsa decemlineata (Say), Bongers (1965) dealt with the question of learning by larvae as it affects adult preference. He concluded from his study with 4 species of Solanaceae that it appeared unlikely that the preference for food of sexually mature beetles can be conditioned by any experience on the part of the larvae or young adults. For example, they found temperature was important in that adults at low temperatures oviposited on potato but at high temperatures on bittersweet, S . dulcamara. Bongers believes that food and oviposition responses are based on two different mechanisms. Studies conducted by Hibbs et al. (1964) showed that sugar concentration was of importance to the potato leafhopper Empoasca fabae. Tolerant varieties had a higher sugar content. Dahlman and Hibbs (1967) studied the response of the potato leafhopper to substances in the Solanaceae which are biologically active to the Colorado potato beetle. They concluded that some of the same components of Solanum plants are potentially active in the ecological interaction of potatoes and these two insects. The tobacco hornworm, Manduca sexta (Johannson) , is almost entirely
RESISTANCE OF PLANTS TO INSECTS
237
restricted to plants of the family Solanaceae. The biological factors responsible for feeding, oviposition, host selection and discrimination are very well refined in this insect. These factors have been studied in detail by Yamamoto and Fraenkel (1960a,b) and Yamamoto et al. (1969). D.
CONVOLVULACEAE
Resistance to five insects of sweet potatoes, Ipomoea batatas, has been studied by Cuthbert and Davis ( 1970, 1971). Resistance to the major insect pests of sweet potatoes is readily available. The commercial variety CENTENNIAL is resistant to the sweet potato flea beetle, Chaetocnema confinis Crotch, and the varieties NEMAGOLD and NUGGET are resistant to the wireworm Conoderus falli Lane; Diabrotica; and the Systena complex. Resistance to the wireworm, banded cucumber beetle, Diabrotica balteata Le Conte, the spotted cucumber beetle, D. undecimpunctata howardii Barber, and the elongate flea beetle, Systena elongata F., appear to be controlled by common resistance factors.
E. COMPOSITAE Dunn (1960) has studied the lettuce root aphid, Pemphigus bursarius (L.), on cultivated lettuce Lactuca spp. He found resistance in the form of antibiosis in several varieties.
F. LILIACEAE Most of the work with onions, Allium spp., has been with the onion maggot Hylemya antiqua (Meigen). The A . fistulosum L. varieties NEBUKA and HISHIKO were significantly more resistant to onion maggot than 44 other varieties involving several Allium species (Perron et al., 1958). Later Perron et al. (1960) were able to show that the resistance in these two varieties was due to their being much less attractive to the onion maggot flies for oviposition. Perron and Jasmin (1963) found that these two resistant varieties did not show antibiosis to the onion maggot. G . CUCURBITACEAE
Hall and Painter (1968) published in an Experiment Station Bulletin the results of 6 years’ work evaluating 387 plant introductions of Cucurbita pep0 L., C . maxima Duch., C. moshata Duch., and C . okeechobeenis for resistance to the squash bug, Anasa tristis DeGeer, spotted cucumber beetle, Diabrotica undecimpunctata howardii Barber,
238
F. G. MAXWELL, J. N. JENKINS, AND W. L. PARROTT
and striped cucumber beetle, Acalymma trivittata (Mannerheim) . They developed techniques for evaluating resistance including rating scales and field plot techniques to minimize the effects of insect migration. This was of especial importance with these insects. Sources of resistance to the 3 species of insects were found. A long list of the resistant lines in all 4 cucurbit species is given in their bulletin. However, no resistant line had suitable horticultural qualities coupled with resistance, so that it could offer immediate promise. Brett and Sullivan (1970) also published an Experiment Station Bulletin in which they rated the resistance of current commercial varieties of cucurbits to pickleworm, Diaphana nitidalis (Stoll) , striped cucumber beetle, spotted cucumber beetle, serpentine leaf miner, Liromyza brassicae Riley, and Mexican bean beetle, Epilachna varivestis Mulsant. They found resistance to pickleworm in some varieties of each of squash, cucumbers, pumpkins, cantaloupes, and watermelons. A number of varieties of squash were found resistant to striped cucumber beetles. In studies with the spotted cucumber beetle they found that resistance in the seedling stage did not necessarily mean resistance in the mature plant stage. They found resistance in the seedling stage in varieties of squash and cucumbers and resistance to foliage feeding in varieties of squash, cucumber, pumpkins, cantaloupe, and watermelons. Some varieties of cucurbits were almost immune to attack by the serpentine leaf miner and other varieties may be severely damaged. Greenhouse studies showed that butternut squash were more resistant to the squash bug than BLACK ZUCCHINI. Nonpreference and antibiosis were both involved in the resistance (Novero et al., 1962). Resistance to the squash bug in C. pepo is a genetically determined character governed by at least 3 genes, partial dominance and additive gene action both being important (Benepal and Hall, 1967). Immunity to squash vine borer, Melittia cucurbitae Han., was found in the following cucurbitacae: butternut pumpkin, honey cream watermelon, Iroquois muskmelon, zucca melon, and red-seeded citron, and national pickling and straight4 cucumbers (Miller, 1956). Resistance to the spotted cucumber beetle under no choice situations was found in muskmelon varieties HEARTS OF GOLD and PMR 450. The insects starved rather than feed when caged on these two varieties. Both varieties were resistant to the striped cucumber beetle only when the insects were given a choice of plants on which to feed. Two varieties of squash, E. G. BUSH SCALLOPED and ROYAL ACORN, were resistant to the two beetles only under choice situations (Wiseman et al., 1959). Inheritance of resistance to the striped cucumber beetle in the C . pepo resistant varieties ROYAL ACRE, EARLY GOLDEN, BUSH SCALLOP, and the susceptible variety, BLACK ZUCCHINI was studied by Nath and Hall (1963). Resistance
RESISTANCE OF PLANTS TO INSECTS
239
was partially dominant and additive factors were also important. Resistance was influenced by the environment. Resistance is inherited to such a degree as to be useful. Resistance to the pickleworm has been reported in some of the Cucurbitaceae. In field experiments Dilbeck and Canerday (1968) found that the cultivars of C. moshata and C . maxima were more resistant than those of C. pepo. No high degree of resistance was found in cantaloupes to the pickleworm; however, some varieties were less susceptible than others (Canerday, 1967). Concentrations of D-glucose above 1 % in squash confers resistance to the pickleworm. Also galacturonic acid is important in resistance. It was found only in the most resistant variety BUTTERNUT 23 (Brett etal., 1961, 1965). A class of tetracycline triterpenoids called cucurbitacins found in the Cucurbitaceae are specific feeding attractants for cucumber beetles (Chambliss and Jones, 1966a). Cucumbers with the genotype bi bi completely lack cucurbitacins and are called nonbitter. These plants are resistant to cucumber beetle but are susceptible to the twospotted spider mite; whereas Bi Bi plants contain the bitters principal and are resistant to mites but susceptible to cucumber beetles (Dacosta and Jones, 1971; Kooistra, 197 1 ) . Kooistra also found that susceptibility to mites involved more than just the absence of the bitters principal since four of his susceptible lines of cucumbers did contain the bitters principal. In watermelon the main bitters principal has been identified as elaterinide. Lack of bitterness in watermelon fruits is due to a single recessive suppressor gene suBi active in the presence of the Bi gene for plant bitterness (Chambliss et al., 1968).
H. LEGUMINOSAE One of the most important insects feeding on cultivated peas Pisum sativum L., is the pea aphid, Acyrthosiphon pisum Harr. This insect is also a pest of many other legumes and small grains and has been discussed there also. Nutritional differences between resistant and susceptible varieties of peas have been found. The susceptible varieties PERFECTION, DAISY, and LINCOLN contained more nitrogen and less sugar than the resistant varieties LAURIER,CHAMPION OF ENGLAND, and MELTING SUGAR,when examined at various stages of plant growth (Maltais and Auclair, 1957). In the same 6 varieties of peas, the susceptible varieties generally contained a higher concentration of free and total amino acids (Auclair et al., 1957). Aphids fed at a higher rate on susceptible plants with a greater proportion of the material ingested being excreted and a lesser proportion assimilated than on resistant varieties. This might indicate that aphids were in a semistarved condition on resistant plants and thus utilized more and consumed
240
F . G. MAXWELL, J . N. JENKINS, AND W. L. PARROTT
less on resistant Varieties (Auclair, 1959). Auclair and Cartier (1960) suggest that reduced growth on resistant varieties is not the result of toxic substances in host plant since growth and reproduction of aphids on resistant host plants of peas was similar to semistarved aphids. Seven clones of pea aphid were collected on alfalfa in Southern New Mexico and reared continuously for 7 months on peas and alfalfa. No conditioning toward peas occurred ( Auclair, 1966). Pea seedlings were more susceptible than older plants whereas alfalfa seedlings were more resistant. Three biotypes of pea aphid are recognized in southern Quebec (Cartier, 1959). The biotype R , has been selected as the standard culture biotype for use in southern Quebec for testing peas for resistance (Cartier, 1960). In an examination of 13 varieties of peas in the field and in the greenhouse, Cartier (1963) found that yellow green plants were preferred over green plants. Plant height had two opposite effects: (1) a barrier effect contributing to a higher initial infestation in tall plants due to the interception of migrating winged forms and ( 2 ) an expose effect that reduced ensuing populations due to longer internodes and less dense foliage exposing the aphids to an adverse environment. Cartier suggests the following in breeding peas for resistance to pea aphid: (1 ) use a mean greenhouse temperature of 65OF, ( 2 ) use a moderately virulent strain of pea aphid, ( 3 ) use solid seeded square plots at the density of commercial plantings in field plots, do not use rows; and ( 4 ) use cage tests with induced populations rather than natural infestations. The Mexican bean beetle, Epilachna varivestis Mulsant, feeds selectively on certain species of Phaseolus to the exclusion of the representatives of this and other genera of beans. Wolfenbarger and Sleesman (1961b) did not find any resistant P. vulguris lines. Lima bean, P . lunatus, lines varied in their response from intermediate to susceptible. Earliness was correlated in their study with susceptibility. Visual rating of damage was comparable to larval counts in rating resistance. Sucrose, glucose, and fructose have been found to elicit arrestant and feeding responses. A short range attractant has also been found in beans for the larvae (Augustine et al., 1964). Sixty varieties of snapbeans and lima beans were tested for resistance to the Mexican bean beetle. Snapbean varieties resistant were IDAHO REFUGEE, WADE, LOGAN, SUPER GREEN, BLACK VALENTINE, and REFUGEE U S . No. 5. Resistant lima bean varieties were BABY FORDHOOK BUSH LIMA, TRIUMPH, BURPEES BUSH LIMA, EVERGREEN, and HENDERSON BUSH. Antibiosis (smaller insects) and reduction in oviposition were noted on the resistant varieties (Campbell and Brett, 1966). Potato leafhopper, Empoasca fubue (Harris), is a pest of beans. Wolfen-
RESISTANCE OF PLANTS TO INSECTS
24 1
berger and Sleesman ( 1961a,c,d) found that pubescence was associated with resistance, but other factors are also important. They suggest that common beans that are tall, are mosaic resistant, have pink or striped seed, flower between 50 and 59 days, and mature in 105-114 days are more resistant than beans not possessing these characteristics. Hopperburn and number of nymphs present are not necessarily equivalent. For example, Dolichos lablab L., Bonavist bean, is resistant to hopperburn but susceptible to nymphal infestation; whereas cowpeas, Vigna sinensis (Torner) (Sari), are susceptible to hopperburn and nymphal infestation but are rated as tolerant to leafhopper injury. Wolfenbarger and Sleesman ( 1961a) found resistance to nymphs in the common bean, P. vulgaris, PI 151015 and PI 173024. Intermediate resistance was also found in 28 additional lines of common beans. Ten lines exhibited tolerance (i.e., no hopperburn in presence of nymphal infestation). The resistance of large-seeded FORDHOOK found by Wolfenbarger and Sleesman was substantiated in work by Eckenrode and Ditman (1963). While the leafhopper infestation was lower on THAXTER and EARLY THOROUGHGREEN, the yield reductions were greater on FORDHOOK. This is assumed by Eckenrode and Ditman (1963) to result from a difference in response to the toxic salivary secretions injected into the plants by feeding leafhoppers. Except for a slight cupping of the leaves on FORDHOOK, there was no visible injury to the plant. Guevara (1958) reports that he has selected a few varieties of beans resistant to Apion pod weevil, Apion godman. Sme of these are already in commercial production in Mexico. The tarnished plant bug, Lygus linealaris, is a pest on beans. The preference among bean varieties shown by the adults for oviposition generally were not the same as those shown by first-instar nymphs for feeding and resting. Thus, oviposition and viability of eggs and nymphs were at least partially favored by a different set of conditions. It appears that genetically different races of the bug exist on different wild hosts. In oviposition tests more eggs were deposited on FORDHOOK 242, a large lima bean, than on small lima beans or other beans. The first instar nymphs preferred to rest on CALIFORNIA PINK much more than on other varieties including FORDHOOK 242 (Taksdal, 1963).
V.
Forest Trees
The value of forest and forest products is increasing, and the supply of high quality wood is diminishing. These factors are only two of the many contributing to the need for controlling forest insects. Numerous examples
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already mentioned in this chapter demonstrate the advantages of having crop plants resistant to insect attack; therefore, insect-resistant trees, like field crops, offer an efficient, economical, and permanent method of controlling forest insects. Several international agencies have recognized the crucial need for close cooperation among the various disciplines. As a result, Gerhold (1969) reported that over 50 programs in 19 countries are involved in various phases of research to develop trees resistant to insects. Smith (1960) reported that Pinus jeflreyi Grev. and Balf. crossed with P. coulteri D. Dan produced progenies resistant to the pine reproduction weevil, Cylindrocopturus furnissi Buchanan. Also a 3-way hybrid of these two and P. jeflreyi gave resistance which indicated that resistance was inherited as a dominant character. Hall (1959) tested the performance of these hybrids under field conditions and found they had both weevil resistance of Coulter and lumber quality of Jeffrey pine. Results of this study stimulated the U.S. Forest Service to embark on a large-scale pollination program to produce hybrids for planting in California. A study by Vit6 and Wood (1961) indicated that the incidence of fatal attack to ponderosa pine, Pinus ponderosa, by the bark beetle, Dendroctonus monticolue Hopkins, was associated with low oleoresin exudation pressure. Wright and Gabriel (1959) examined 10 introduced wliite pine species in the northeastern United States to explore the possibilities of developing strains of pine resistant to the white pine weevil, Pissodes strobi (Peck). They concluded that the western white pine, Pinus monticoln Douglas, showed the most promise as a source of resistance. Western white pine was also reported by Soles et al. (1970) to be resistant to attack by the white pine weevil. The authors suggested that the resistance mechanisms either inhibit the weevil from traveling to the tree or induce them to leave. Wright et al. (1967) evaluated 108 natural stands of Scotch pine, Pinus sylvestris L. They found that under field conditions, larvae of the European pine sawfly, Neodiprion sertifer (Geoffroy), were one-half to one instar later in development when they fed on the resistant variety URALENSIS as compared to other varieties. A voluminous amount of material has been compiled by Gerhold et ul. (1964) which includes the proceedings of a joint meeting between N.A.T.O. and N.F.S. Research workers representing various geographic regions of the world were in attendance. The proceedings include ( 1 ) research related to forest trees resistant to insects, (2) basic knowledge on the mechanisms of resistance and ( 3 ) implementations of present knowledge and approaches for developing new breeding methods for producing forest trees resistant to insect attack. Roth (1970) has made a current literature review of the more important
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insects and diseases of forest trees. He summarized that most notable progress which has been accomplished in this area and emphasized the need for more interdisciplinary research in host plant resistance.
VI.
Miscellaneous
A.
SOLANACEAE Tobacco
There are few sources of resistance in tobacco varieties to associated insects, but several studies have been made to determine the mechanism of resistance occurring in tobacco. Thurston ( 1961) tested tobacco introductions and wild Nicotiana species in the laboratory and in the field for resistance to the green peach aphid, Myzus persicae. Selections and crosses of resistant tobacco were less susceptible than any of the standard burley tobacco varieties. Ky 61, TI 566B, and TI 567 were more susceptible than the other tobacco varieties tested. Nicotiana gossei, N . repanda, and N . trigonophylla were highly resistant, but crosses of N . gossei with N . tabacum did not show the high resistance of the N . gossei parent. Burk and Stewart (1969) tested 45 Nicotiana species and 12 related varieties and polyploid or horticultural strains. The highly resistant species were related phylogenetically, indicating that the mechanism of resistance may have a common origin. A possible relationship between polyploidy and resistance also was noted. An explanation for the ability of the green peach aphid, M . persicae (Sulz.), to adapt to a plant capable of nicotine biosynthesis has not been resolved experimentally. The work reported by Guthrie et al. (1962) offers chemical, radiometric, and histological evidence which suggests that the aphid is able to subsist on tobacco because it feeds in the phloem and avoids the nicotine-containing xylem. Abernathy and Thurston (1969) suggested that toxicity of resistant varieties of tobacco, Nicotiana tabacum L., and N . benthamiana Domin, to Myzus persicae (Sulz.) increased as the plants matured. This paralleled an increase in the amount of the exudates on certain trichomes of these plants. Thurston and Weber (1962) reported that resistance in Nicotiana species to the green peach aphid appeared to result from the production of a toxic material produced by the aerial parts of the plant. The symptoms of such poisoning resemble those of nicotine poisoning, but resistance does not appear to be correlated with the amount of nicotine in the leaf. Later Thurston et al. (1966) found alkaloids were secreted by
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trichomes of 7 Nicotiana species tested. Nicotine, the major alkaloid constituent, was identified in the secretion from all species and anabasine, and probably nornicotine, from t h o species. Aphids were killed by contact with these secretions, and resistance to Myziis persicae, results from this mortality. M y z u ~persicae showed a reduced relative growth rate on plants given soil drenches of 1% CCC ( a plant growth retardant), and a greater reduction when CCC was applied as a foliar spray. Growth rate of aphids as a conveniont measure of their performance was used in relation to plant resistance (Van Emden, 1969). A very limited amount of work has been done on the tobacco budworm, H . virercens (F.). However, it has been reported that some varieties of flue-cured tobacco suffered significantly less damage than others due to the feeding of the tobacco budworm. When exposed to the same natural populations of budworms, SPEIGHTS G-36 and COKER 80F consistently showed less damage than COKER 139 and BELL 29. This resistance appears to be related to the disappearance of the budworms from the plant, not to failure of the adults to select these varieties for oviposition (Girardeau, 1968). Several aspects of resistance in tobacco to the tobacco hornworm have been evaluated. Parr and Thurston (1968) found that Nicotiana bentharniana Domin, N . repanda Willd., N . stocktonii Brandeg., N . tiesophila Johnst., N . gossei Domin, Petunia inflata Lindl., and P. violacea Lind. were highly toxic to larvae of Munduca sexta (Johannson). The toxicity in the Nicotiana species appears to result from the secretion of alkaloids by the trichomes of these plants. A stimulant was isolated from tomato and related plants of the family Solanaceae. Preliminary characterizations indicate the material to be a glycosidic substance; however, its complete chemical identity has not been elucidated as yet. In the presence of this substance the insect attacks filter paper, lettuce leaves, or artificial agar diets, but only when certain nutrients, particularly sugars, are also present (Yamamoto and Fraenkel, 1960b). Oviposition and feeding of the tobacco hornworm are almost entirely restricted to plants of the family Solanaceae. Oviposition appears to be initiated by olfactory stimuli widely distributed in this plant family, and consequently it is largely suppressed in the absence of host plants or after excision of the antennae. Both in the field and laboratory, the moths preferred tomato foliage over other solanaceous plants for oviposition. Larval feeding appears to be governed by gustatory stimuli common to plants of this family. On certain members, such as Nicandra and Petunia, feeding and growth are limited by the presence of repellents or toxin (Yamamoto and Fraenkel, 1 9 6 0 ~ ) A . generalized scheme concerning the oviposition
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behavior of Manduca sexta (Johan.) moths was constructed from observation and large cage experiments. There are two phases in the orientation to plants, the approach and the landing. The approach appears nondiscriminatory and the moths utilize visual clues during this phase. The landing is discriminatory and is based on fixed or learned responses to olfactory stimuli. After landing, contact chemostimulation elicits deposition of eggs (Yamamoto et al., 1969). McFadden (1968) demonstrated that movement of tobacco hornworm larvae between plants does occur, but only under abnormally crowded conditions. Jones and Thurston (1970) found that larvae of Manduca sexta (Johan.) reared on burley tobacco grown in the greenhouse consumed almost twice as much leaf area as larvae reared on burley tobacco grown in the field, but the dry weight of the field-grown tobacco was greater than that of the greenhouse-grown tobacco. Larvae that fed on field-grown dark tobacco consumed the smallest leaf area, but the dry weights of the field-grown burley and dark tobacco consumed were very close, 6.054 and 6.468 g, respectively. Stewart and Baker (1970) reported that the larval period was 2 days longer when Manduca sexta (Johan.) larvae were reared on tobacco leaves than when they were reared on artificial diet; however, larvae consumed about one-third more tobacco leaves (by weight) than artificial diet. An integrated control approach for reducing populations of tobacco pests has been described by Gentry et al. (1969). Rabb et al. (1964) described cultural practices to aid in the control of the tobacco hornworm and other pests. Other pests associated with tobacco are Hylemya spp. flies (Kring, 1968), cabbage looper (Elsey and Rabb, 1967), southern potato wireworm (Creighton et al., 1963), and cigarette beetle, Lasioderma serricorne (Yamamoto and Fraenkel, 1960a). B.
CHENOPODIACEAE Sugar Beet
There is a limited amount of research on resistance to sugar beet insects. The two leaf spot and spider mite resistant varieties, GW 674 and GW 359, inhibited the development of the sugar beet root aphid, Pemphigus betae. GW 674 is a selection of GW 359, but has higher sugar content and greater leaf-spot resistance. Nine other varieties were susceptible to root aphid infestation (Harper, 1964). The results of Russell (1966a,b) and Lowe and Russell (1969) in glasshouse experiments have confirmed that inbred lines of sugar beet differ in each of three types of resistance to Myzus persicae Sulz. and Aphis fabae Scop., namely, resis-
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F. G. MAXWELL, J . N. JENKINS, AND W . L. PARROTT
tance to settling, resistance to multiplication, and tolerance. Resistance to multiplication was not invariably associated with resistance to settling, although plants of 'some lines showed both forms of resistance. Plants that were resistant to settling of alatae were not always resistant to apterae of the same species, and there was not a close relationship between resistance to M . persicae and to A . fabae. The mechanisms involved in resistance to aphids in sugar beet are not fully understood. Apterous M. yersicue, which had been reared on sugar beet, settled more readily on beet seedlings than did aphids previously reared on Brassica pekinensis. This effect was greatly reduced when the beet seedlings were covered with tumblers immediately after infestation. Apterous A . fabae, transferred from Viciu faba, settled almost as well on beet seedlings as aphids transferred from sugar beet (Russell, 1966a,b). The distributions of M . persicae (Sulz.) and Aphis fabae Scop. were related to the ages of the leaves of sugar beet plants and spindle bushes when tested in pots in the greenhouse and under field conditions. The ages of leaves were estimated on an arbitrary scale of 10 types of leaf distinguished by degree of unfurling and color. The diverse types of distribution recorded were all reducible to a common general pattern: growing and senescing leaves were more susceptible to colonization than maturing, mature and dying leaves (Kennedy et al., 1950). Ibbotson and Kennedy (1950) using infestations of apterous Aphis fabae Scop. on potted sugar beets, found that leaves were very suitable when young, but became unsuitable as they matured. They became suitable again just after maturity and then unsuitable as they senesced. But among leaves at any given stage, those which were growing or senescing rapidly were more suitable than those changing slowly, unless the rate of senescence was very high. In detached leaf experiments, Macias and Mink (1969) found that apterous M. persicae (Sulzer) preferred yellows-infected leaves to symptomless, mosaic-infected, or curly top-infected leaves. Twice as many alate aphids were found on yellows-infected whole plants as on healthy or mosaic-infected plants 24 hours after release, but no marked differences were found in numbers of alate aphids on healthy, mosaic-infected, and yellows-infected whole plants 48-96 hours after release.
VII.
Problems Associated with Breeding for Resistance to Insects
There are a number of problems that will occur at various stages in the development of any host plant resistance program. Usually one of the primary limiting factors is proper financing over a long period of time. Considerable work, as evidenced by this and earlier reviews, has been done
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over the years on a piecemeal type of approach. Many times the effectiveness that the research would have had was lost because of the inability to follow through with a sustained program after the initial investigation. State experiment stations have, in general, been financially unable or else unwilling to fund the long-term research necessary for the development of resistant varieties. The USDA has not, until recent years, recognized the full potential of this avenue of insect control. Lack of proper funding at the state and federal level still hinders the rate at which progress can be achieved. Administrators must be convinced that long-term, well organized, multidisciplinary team research in host plant resistance offers an excellent opportunity for solving major insect problems. The importance of emphasizing biological approaches has been brought home strikingly with increasing awareness of insecticide resistance, residues in food and fiber crops, and general insecticide contamination of our environment. The importance of a multidisciplinary team approach has not been recognized sufficiently in the past. Efforts by entomologists and plant breeders have often been thwarted by fragmented or isolated endeavors without proper coordination and cooperation. A close-knit team is absolutely essential to progress and success in a host plant resistance program. Not only does the entomologist and the plant breeder need to have extremely close working relations, but they must also be willing to bring in plant pathologists, biochemists, plant physiologists and other disciplines at various stages of the project to help solve complex problems which will undoubtedly arise. Also, host plant resistance needs to be the primary project for the entomologist and plant breeder, not a secondary one. Fortunately, in recent years, the USDA has recognized the need for the team approach and has stationed entomologists and plant breeders at their new laboratories where host plant resistance programs are being undertaken. Outstanding examples of the success and productivity of such teams are the host plant resistance groups at the Boll Weevil Research Laboratory at State College, Mississippi, and the Corn Borer Laboratory at Ankeny, Iowa. The entomological problems connected with breeding plants for resistance to insects differ from other insect control in several ways. First, a very intimate knowledge of the biology (life table) and feeding habits of the insect is absolutely essential. Often, knowledge is inadequate, necessitating detailed studies before launching into the program. Also insect variability is a problem that must be closely monitored. Biotypic differences within insect species may cause different behavior in laboratory-reared colonies than in field-selected populations. Natural variability in growth, fecundity, and other parameters of insect development must be carefully determined and accounted for in tests for resistance. Second, a population
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PARROTT
of insects must be built up using field or laboratory cultures rather than being destroyed. This is often a limiting factor on how fast a program can progress because results that can be obtained on the initial aspects of resistance studies usually are proportional to the number of plants and strains that can be analyzed. Third, methods must be designed to examine large numbers of plants for insect infestation or damage, frequently in a very short period of time. Quantity, in other words, may be more important than a high degree of accuracy in the initial screening programs. Seedling screening techniques for mass selection should be developed wherever possible as a valuable time saving tool. Careful consideration must be given to correlation of resistance in the seedling stage to resistance in the mature plants, however. The success in developing synthetic resistant varicties of alfalfa in a short period of time to the spotted alfalfa aphid and pea aphid can be attributed largely to seedling rating techniques couplcd with simple recurrent selection. Special problems in technique development often occur in working with annual fruiting crops such as cotton where fruiting bodies are usually the primary target of insects. Tropical nurseries are often necessary to speed crossing and seed increase of selected materials. Plants selected initially in a program must usually be selfed and increased to a family to provide sufficient material for reliable replicated tests to confirm the presence of resistance. This procedurc is timc consuming and expensive, but at the present time there are no methods to circumvent this procedure. If chemical causes of resistance can be ascertained early in the program, a precise chemical testing procedure could be very useful at this stage. The relevance of the biochemical approach to real-life situations must be adequately demonstrated. Unfortunately, resistance is most often found in off types or exotic wild species, and considerable crossing and selection is required to move the resistant genes into a desirable agronomic background. This involves the time-consuming process of discarding all the undesirable characters. For this reason it is extremely important that plant pathologists and agronomists be involved early in the program to help decide the characters that are to be retained or disposed of. In some cases certain concessions may have to be made in levels of insect or disease resistance and/or agronomic qualities in order to provide the best overall variety for a particular geographical area. Another problem frequently encountered is that a line or variety developed for resistance to one pest may in fact be more susceptible to another insect that had previously been of noneconomic significance. Excellent recent examples include the glanded and glandless condition (gossypol glands) in cotton. Removal of the glands through breeding caused greater
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susceptibility to bollworms, H . zea and H . virescens (Lukefahr et al., 1966; Oliver et al., 1970, 1971). In addition, a number of leaf-feeding insects not previously known to cause damge to cotton became serious pests (Maxwell et al., 1965). The frego character in cotton imparts high field resistance to the boll weevil (Lincoln and Waddle, 1966; Jenkins et al., 1969; Jenkins and Parrot, 1971a,b), but unfortunately causes increased susceptibility to the tarnished plant bug, Lygus lineolaris (Jones, 1971; Lincoln et at., 1971). The glabrous condition in cotton which contributes resistance to bollworms causes greater susceptibility to thrips, leafhoppers, aphids, fleahoppers, and certain other pests. These brief examples are sufficient to point out that a morphological or chemical resistant mechanism, while contributing resistance to one pest may directly or indirectly (linked factors) create greater susceptibility to another pest. Plants found resistant to a pest should be tested early in a breeding program to test susceptibility to other economic or potentially economic insects and diseases. Such procedures may necessitate changes in approaches, but would possibly prevent loss of many years’ work and expenditures of large amounts of money. In some cases it may be economically unfeasible to proceed with a program if there is a high probability of creating an equal or worse problem with another insect.
VIII.
Utilization of Resistant Varieties
In some cases where resistance is at a relatively high level, it may be sufficient in itself to affect control (Painter, 1968). In areas where resistant varieties are planted over large areas the effect on the population of the insect resisted has been observed to be specific, persistent, and cumulative. The result is often a drastic reduction to noneconomic levels of the insect species. Holmes and Peterson (1957), Painter (1958a, 1968), Dahms ( 1969), Chiang ( 1968), Pathak ( 1970), Luginbill ( 1969), and Luginbill and Knipling (1969) have provided statistical information on the impact of resistant varieties on certain insect populations wherever they have been employed as the principal method of control. It is very difficult to obtain good qualitative data on insect populations as affected by cultivation of resistant varieties. The best data available concern Hessian fly on wheat in California and Kansas, where infestation levels dropped from 100% to less than 1% after resistant varieties were released. The greatest use of resistant varieties in the future will undoubtedly be as one component part of a pest management system. In this type of management system the value of low levels of resistance is magnified because
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the resistance works as just one of possibly many suppressant factors integrated to prevent the target species from gaining an economic threshold Ievel. In addition the resistant crop may in fact enhance natural biological control organisms. If selective insecticides are applied, resistant varieties may indirectly increase their effectiveness. Varieties containing high or moderate resistance when used in combination with other suppressant measures such as pheromone trapping may be sufficient to reduce an insect species to such a low level as to make feasible the sterile-male technique. Frego cotton, resistant to boll weevil, is being considered as one of several suppressant measures to reduce boll weevil populations to a level such that eradication by the sterile-male technique may become feasible. The Hessian fly has been reduced to such low levels in many areas by resistant varieties that elimination by sterile males is a distinct possibility. Once a pest has been greatly suppressed or eradicated from an area, resistant varieties may serve over the years as the major deterrent factor to reestablishment of a species.
IX.
Summary
Examination of the literature pertaining to resistance to insects in plants reveals that there have been numerous outstanding successes in breeding for resistance. The value of these resistant crops cannot be fairly estimated except to say that it would range in the high millions of dollars annually. Second, the literature points out strikingly the great diversity within crop varieties and species screened for insect-resistant mechanisms. Only limited screening for a very restricted number of insects has been attempted, hence a virtual gold mine of potential for future exploitation lies before us. The increasing problems being encountered with insecticide resistance in insects, loss of insecticides by legislation, increasing concern of the general public over contamination of the environment are forces which will necessitate greater emphasis of this method of insect control. Even low levels of resistance to insects in crops will be extremely important as we combine cultural, biological, and selective factors into more sophisticated pest management systems for future insect control. Plant breeders, entomologists, plant pathologists, physiologists, and biochemists and workers in related disciplines have a great challenge before them to work effectively together in close, cooperative team efforts to provide the leadership, research competence, and impetus to move host plant resistance to insects to its full national and international potential.
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ACKNOWLEDGMENTS The authors wish to thank Dr. David G. Holder and Dr. Michael F. Schuster, Research Associates, Department of Entomology, Mississippi State University, for their help and constructive criticism in the preparation of this manuscript. REFERENCES Abdel-Malek, S. H., Heyne, E. G., and Painter, R. H. 1966. J . Econ. Entomol. 59, 707-710. Abernathy, C. O., and Thurston, R. 1969. J . Econ. Entomol. 62, 1356-1359. Adkisson, P. L., Bailey, C. F., and Niles, G. A. 1962. Tex. Agr. Exp. Sta., Misc. Publ. MP-606. Agarwal, R. A. 1959. Indian Sugarcane 1. 8, 12. Agarwal, R. A. 1965a. Indian Sugarcane 1. 9, 108-110. Agarwal, R. A. 1965b. Indian Sugarcane J . 15, 541-549. Agarwal, R. A., and Kandaswamy, P. A. 1959. Proc. Int. SOC.Sugar-Cane Technol. 10, 829-835. Akeson, W. R., Haskins, F. A., and Gorz, H. J. 1969. Science 163,293. Allen, R. E., Heyne, E. G., Jones, E. T., and Johnston, C. 0. 1959. Kans. Agr. Exp. Sta., Tech. Bull. 104. Annappan, R. S., Venkataraman, N., and Kamalanathan, S. 1965. Madras Agr. J . 52, 412-414. Anonymous. 1970. Agric. Res., 1916. Anonymous. 1959. Annu. Rep. Rockefeller Found., New York. Anonymous. 1965. Annu. Rep. Rockefeller Found., New York. Anonymous. 1966. Tew., Agr. Exp. Sta. L-689. Areson, W. R., Manglitz, G. R., Gorz, H. J., and Haskins, F. A. 1967. 1. Econ. Entomol. 60, 1082-1084. Arridge, H. 0. 1954. Univ. Nac. Eva Peron, Rev. Fac. Agron. (Tercera Epoca) 30, 85-101. Arridge, H . O., and Re, R. R. 1963. Univ. Nac. La Plata, Rev. Fac. Agron. (Tercera E P O C ~39, ) 35-50. Athwal, D. S., Pathak, M. D., Bacalangco, E. H., and Pura, C. D. 1971. Crop Sci. 11, 747-750. Atkins, R. E., Pesho, G. F., and Dicke, F. F. 1963. Iowa State J . Sci. 37, 447-452. Auclair, J. L. 1959. Entomol. Exp. Appl. 2, 279-286. Auclair, I. L. 1963. Ann. Entomol. SOC.Amer. 8, 439-490. Auclair, J. L. 1966. Ann. Entomol. SOC.Amer. 59, 780-786. Auclair, I. L. 1967. J. Insect Physiol. 13, 431-446. Auclair, J. L., and Cartier, J. J. 1960. Entomol. Exp. Appl. 3, 316-326. Auclair, J. L., and Raulston, J. R. 1966. Ann. Entomol. SOC.Amer. 59, 1011-1016. Auclair, J. L., Maltais, J. B., and Cartier, J. J. 1957. Can. Entomol. 89,457-464. Augustine, M. J., Fish, P. W., Davidson, R. H., Laysides, J. B., and Climy, R. W. 1964. Ann. Entomol. SOC.Amer. 57, 127-134. Bailey, J. C., Maxwell, F. G., and Jenkins, J. N. 1967a. J. Econ. Entomol. 60, 1275-1279. Bailey, J. C., Maxwell, F. G., and Jenkins, I. N. 1967b. J . Econ. Entomol. 60, 1279.
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Ueckert, D. N. 1968. Aitn. Entorirol. SOC.Anier. 61, 1539-1544. Van. 7.. K., and Guan, C. K. 1959. hlnlnysirrn Agr. I. 42, 307-210. Van den Burgh, R. S., Norwood, B. L., Blickenstaff, C. C . , and Hanson, C. H. 1966. 1. Econ. Enfoniol. 59, 1193-1 198. Vanderzant, E. S. 1967. 1. Econ. Eniontol. 60, 813-816. Van Emden, H. F. 1966. Eittonio/. Exp. A p p l . 9, 444-460. Van Emden, H. F. 1969. Entonrol. Exp. Appl. 12, 125. Varis, A. L. 1958. I. Sci. A g r . Soc. Fin. 30, 271-275. Virtanen, A. I. 1961. Sottn?. Kenristilelrti B 34, 29-3 1. VitC, J. P., and Wood, D. L. 1961. Contrib. Boyce Thompson Znst. 21, 67-78. Wahlroos, O., and Virtanen, A. I. 1959. Actu Ckrrn. Scand. 13, 1906-1908. Walker, J . K., Jr., and Niles, G. A. 1971. Tcx., Agr. Exp. Stn., Bull. 1109. Wallace, H. A. H., and Sinha, R. N. 1961. Can. I. Plant Sci. 41, 871. Wallace, L. E., and McNeal, F. H. 1966. U S . , Dep. Agr., Tech. Bull, 1350, 1-50. Walter, E. V. 1957. I . Ecorr. Entorno/. 50, 105-106. Walter, E. V. 1962. Proc. A m e r . SOC.Hort. Sci. 80, 485-487. Walter, E. V. 1965. I. Econ. Entornol. 58, 1076-1078. Wann, E. V., and Hills, W. A. 1966. Proc. Arner. Soc. Horf. Sci. 89, 491. Wannarnaker, W. K. 1957. 1. Econ. Erriornol. 50, 418-423. Webb, R. E., Stoner, A. K., and Gentile, A. G. 1971. Proc. I. A m e r . Soc. Hort. Sci. 96, h5. Webster, J. A., Sorensen, E. L., and Painter, R. H. 1968. Crop Sci. 8, 15-17. Wene, G . P., and Sheets, L. W. 1966a. I. Ecort. Entomol. 59, 1538-1539. Wene, G. P., and Sheets, L. W. 1966b. Ariz., Agr. Exp. Sta., Tech. Bull. 166. Wessling, W. H. 1958a. J. Econ. Entomol. 51, 508-512. Wessling, W. H. 1958b. 1. Ecorr. Enrornol. 51, 502-506. Widstrom, N. W. 1967. J. Ecort. Enton~01.60, 791-794. Widstrom, N. W., and Davis, 1. B. 1967. C r o p . Sci. 7 , 50-52. Widstrom, N. W., and Hamm, J. J. 1969. C r o p Sci. 9, 216. Widstrom, N. W., Wiser, W. J., and Bauman, L. F. 1970a. C r o p Sci. 10, 674. Widstrom, N. W., McMillian, W. W., and Wiseman, B. R. 1970b. I . Econ. Erztornol. 63, 803. Williams, R. N.. and Schuster, h l . F. 1970. Prsqui. Agropcc. Brtrzil 5, 215-218. Wilson, F. D. 1971a. C r o p Sci. 11, 419. Wilson, F. D. 197lb. C r o p Sci. 11, 268. Wilson, M. C., and Davis, R. L. 1958. 1. Econ. Entorno[. 51, 219-222. Wilson, M. C., and Shade, R. E. 1966. A n n . Entomol. Soc. Ainer. 59, 170-173. Winter, J. D. 1929. Minn., Agr. Exp. Stn., Bull. 61. Wiseman, B. R., Hall, C. V., and Painter, R. H. 1959. Proc. A m e r . Soc. Hort. Sci. 74, 546-551. Wiseman, B. R., Painter, R. H., and Wasson, C. E. 1966. J. Econ. Entomol. 59, 141-142. Wiseman, B. R., Painter, R. H., and Wasson, C. E. 1967a. I . Econ. Entomol. 60, 1738. Wiseman, B. R., Wasson, C. E., and Painter, R. H. 1967b. Agron. 1. 59, 279. Wolfenbarger, D., and Sleesman, J. P. 1961a. 1. Econ. Entornol. 54, 705-707. Wolfenbarger, D., and Sleesman, J. P. 1961b. 1. Econ. E n t o i d . 54, 1018-1022. Wolfenbarger, D., and Sleesman, J. P. 1961c. I . Econ. Entomol. 54, 1077-1079. Wolfenbarger, D., and Sleesman, J. P. 1961d. J. Econ. Entomol. 54, 846-849. Wolfenbarger, D., and Sleesman, J. P. 1963. J . Econ. Entomol. 56, 895-897.
RESISTANCE OF PLANTS TO INSECTS
265
Wood, E. A., Jr. 1971. J . Econ. Entomol. 64, 183-185. Wood, E. A., Jr., and Curtis, B. C. 1967. J. Econ. Entomol. 60, 1084. Wood, E. A., Jr. 1961a. J. Econ. Entomol. 54, 1171-1173. Wood, E. A., Jr. 1961b. 1. Econ. Entomol. 56, 303-305. Woolcott, G. N. 1927. Bull. Entomol. Res. 18, 79-82. Wressell, H.B. 1960. Forage Notes 6, 13. Wright, J. W., and Gabriel, W. J. 1959. Northeast. Forest Exp. Sta., Pap. 115, 35. Wright, J. W., Wilson, L. F., and Randall, W. K. 1967. For. Sci. 13, 175. Yamamoto, R. T., and Fraenkel, G. 1960a. J . Econ. Entomol. 53, 381-383. Yamamoto, R. T., and Fraenkel, G. 1960b. Ann. Entomol. SOC.Amer. 53, 499-503. Yamamoto, R. T., and Fraenkel, G. 1960c. Ann. Entomol. Soc. Amer. 53, 503-507. Yamamoto, R. T., Jenkins, R. Y., and McClusky, R. K. 1969. Entomol. Exp. Appl. 12, 504-508. York, J. O., and Whitcomb, W. H. 1963. Arkansas Farm Res. 12, 1963. York, J. O., and Whitcomb, W. H. 1966. Arkansas Farm Res. 15, 2. Yoshida, S., Onishi, Y., and Kitagishi, K. 1959. Soil Plant Food ( T o k y o ) 5, 23-27. Yushima, T., and Tomisawa, J. 1957. Jap. J. Appl. Entomol. Zool. 1, 180-185.
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TRACE METALS IN SOILS, PLANTS, AND ANIMALS Donald J. Lisk Pesticide Residue laboratory, N e w York Stale College of Agriculture and l i f e Sciences, Cornell University, Itham, N e w York
I. Introduction .................................................... 11. The Soil-Plant Complex ......................................... A. . Sources of Metals in B. Associations and Beh C. The Plant Factor . . . ....................... D. Specific Metals . . . . 111. Aquatic Systems . . . . . . . A. Water . . . . . . . . . . . . B. Aquatic Organisms ........................... ........ IV. Continuing Research ............................................ References .....................................................
I.
267 268
275
302 309 3 11
Introduction
Research dealing with the existence and association of essential metals in soils and their mobility, reactions, and effects in biological systems has been extensive. The recent discovery of relatively high levels of mercury in fish in North America has now focused intense concern on numerous other potentially toxic metals such as lead, cadmium, arsenic, selenium, chromium, nickel, beryllium, antimony, barium, zirconium, and others which may be ubiquitous in ecosystems. This, in turn, has stimulated a spiraling number of survey and other investigations of their effects. It is thus perhaps unfortunate that a review such as this could not instead be prepared five years hence when the expected mountain of new data will have accrued. One can thus merely attempt to collect and assimilate those data now at hand and speculate from their chemistry and by anaIogy to what is known of similar essential metals, the possible action and behavior of certain of them in the environment. Much is known of the gross symptomology and pathology resulting from overwhelming doses of metals in biological systems. Instead the emphasis here will be on possible effects of long-term or lifetime exposure of living 267
268
DONALD J . LISK
systems to trace or ultratrace concentrations of toxic metal contaminants. The term “toxic metals” may in reality be inappropriate, since toxicity is typically dose-dependent and gathering evidence points toward true essentiality for certain previously unsuspected metals. Therefore, where possible, both aspects of the lethality-essentiality controversy will be prcscnted. Except for meteoric debris and volcanic ash, all metals derive ultimately from the soil. The largest part of the total body burden of most metals in biological species is usually from the diet. It is appropriate, therefore, to begin by discussing aspects of the sources, chemistry, and translocation of metals in soils and plants. II. The Soil-Plant Complex
A.
SOURCES OF METALSIN SOILS
Metals in soils include those from the following sources: 1. Metals contained natively in rocks and minerals from which the soil was forrncd 2. Metals added as impurities in fertilizers and lime, as constituents of pesticides and manure or as contaminants of sewage sludge 3. Metals in debris from industrial and mining wastes, fossil fuel combustion products, wind-eroded soil particles, atomic testing, pollen, sea spray, and meteoric and volcanic material which settles or rains out 4. Metals in soil particles displaced through water erosion or metals dissolved or suspended in the water itself. Toxic metals derived from parent soil materials usually constitute by far the major group in soils. Metals from this source might best be dubbed “geochemical pollutants.” The trace metal content of various rocks has been published (Johannsen, 1932; Lindgren, 1923; Oertel and Prescott, 1944). Phytotoxic effects from repeated annual applications of pesticides and fcrtilizers containing toxic metals are evident in many old orchard soils after tree removal and planting of new trees or cultivation for growing other crops (Benson, 1968). Owing to the huge problem of its disposal, the use of digested sewage sludge on agricultural soils has been periodically proposed and variously studied (Carlson and Menzies, 1971; Coffman, 1970; Sciaroni and Lunt, 1957; Toth and Kelly, 1956; Anderson, 1955; Barrow, 1955; Lunt, 1953; Bear and Prince, 1947). Sludge as a soil conditioner promotes desirable physical properties in soils such as friability and improved drainage. However, it typically contains many potentially effective functional groups for chelating, complexing or fixation of relatively high lcvcls of many toxic
TRACE METALS IN SOILS; PLANTS, AND ANIMALS
269
metals from industrial or other sources of pollution which even slow its own microbiological disinfection (Barth et al., 1965 ; Rudgal, 1946). Its use in sanitary land fills has resulted in overwhelming concentrations of iron and chlorides in leachates from them (Carlson and Menzies, 1971). Sludge containing toxic metals and added to soil has resulted in substantial plant absorption of the elements and phytotoxicity depending on plant variety and soil pH. The absorption of metals from sludge by plants is probably promoted by the presence of organic complexing agents which solubilize them (Bender et al., 1970; Jenkins et al., 1964a,b; Jenkins and Cooper, 1964). Sludge may also cause partial fixation of toxic metals added to soil (Nosbers, 1968). Phytotoxicity and drastically reduced seed germination have also been reported (Lunt, 1959). It is noteworthy that the toxicity of metals in sewage to anaerobic bacteria can be obviated by metal precipitation using a sulfide treatment of sewage (Lawrence and McCarty, 1965). Sludge in sufficient amounts increases soil acidity, and liming is recommended (Lunt, 1953). The use of sludge would seem to offer most benefit in reclaiming mine-stripped land for reforestation (Sopper, 1971) or establishing grass on golf courses or in parks (Carlson and Menzies, 1971). Pollution of soil by atmospheric debris is complex and multifarious. Even meteoric debris, which may at first be considered an unlikely source of serious metal contamination, has been estimated to enter the earth’s atmosphere in amounts up to 1000 tons per day (“Chemistry and the Atmosphere,” 1966). Metals in fossil fuels released during combustion contribute a sizable fraction, and data have been published on the content of various metals in coal (Abernethy et al., 1969; Abernethy and Gibson, 1969; Kessler et al., 1971; Goldschmidt, 1935) and petroleum (Shah et al., 1970a,b). Concentrations of metals in the atmosphere of American cities have been determined (“Air Quality Data for 1967,” 1971; Tabor and Warren, 1958; Schroeder, 1968b; Brown and Vossen, 1970). Two further excellent series of publications have been prepared that deal with metals as air pollutants. One series covers aspects of several metals and their compounds including arsenic, barium, beryllium, cadmium, chromium, mercury, nickel, selenium, vanadium, radionuclides and also certain essential elements [“Air Pollution Aspects of (Name of Metal) and its Compounds,” 1969l.The other publication concerns the effects of metals as air pollutants on biological systems and includes barium, chromium, nickel, vanadium, lead, cadmium, mercury, zinc, and manganese (“Air Quality Monographs,” 1969, 1970). Table I lists typical concentrations of toxic metals in fertilizers, sewage sludges, coal, petroleum, and city air. The general pollution of an area near a superphosphate plant by arsenic has been shown (Lindberg, 1964). Nondetectable levels of
N 4
0
TABLE I Typical Concentrations of Some Trace Metals in Source Materials Causing Mc-tal Pollution o f Soil arid Air
Metal
Limestones“ (PPrll)
20 yc Superphusphate*--8 (ppd
tl
0
Sewage
sludge"'
(%I
co>llJ-‘ (PPiIl)
1’c-t. roleo 111 (ppnl)
Krlmn ail”-n (ra/1113)
z
*r
0
4 Antimony Arsenic Barium Beryllium Bismuth Cadmium Cerium Cesium Chromium Gallium Germanium Lead Lithium iMercury Nickel
0.2 1
I20
0-100
NI)’
2,2-1199 0- 100
0 ,009 0.052
-
0.04 12 0.5 11 4 0.2 9 5 0.04 20
7.3-170
NI) ND NI)
-
90
66-243
-
0.140
-
ND
7-92
0.169
0.04-1.6
ND ND
7-34
0.5-5 2-25 20-3000 0.1-1000 0.000-0.0002 0.2-0.5
0.001
30-107
0.05-1 . I 750-1O0On
0 . 0 015-0.0 18
1.3 5-60 5.5 0.0017-0.0048 2-20 0.5-25 0.07-33 10-50
0.05-0.06 0 . 00.5 0-1.5 0.0001- ,0006 0 . 0 0 1 - , 003 0-0.017
-
__
-
0-0.048
-
0.1-9.8
-
0.05
0 . w2-30 49-345
-
0.001-0.064
Niobium Rubidium Scandium Selenium Silver Strontium Tellurium Thallium Tin Titanium Vanadium Zirconium
11.5-44,5 5 30 0-1.5 15-20 2 5 .9 - 3 6 .6 1 9 .5 - 2 2 .6 0.9 3.2-4.1 43-270 2.3-180 50
0.3 3 1 0.08 0.05 610 1.7 0.5 400 20 19 ~~
0 .0 1 - 0 .0 9 0.047 0.001-0.009 0 .0 0 1 - 0 .0 0 9
1-10 500-2000 10-50 7-250
0.01-0.09 0.01-0.09
0.03-1.4 0.0002
-
-
0.05 -
-
0-0.05 0-0.13 0-0.315 0.004
~~
Generalized from Bowen (1966). Generalized from Schroeder and Balassa (1963). Generalized from Schroeder (1970b). d Generalized from U.S.D.A. and T.V.A. (1964). Generalized from Schroeder and Balassa (1965). f Generalized from Schroeder el al. (I967a). 0 Generalized from Schroeder et al. (1964). Ir Generalized from Martin (1971). ’ Generalized from Lewin (1968). i Generalized from Abernethy d al. (1969). a
ND
1-5 15 3 4-7 0.5-2 0 .0 7 - 0 .1 5 0.5-2
Generalized from Kessler et al. (1971). Generalized from Goldschmidt (1935). Generalized from Shah d al. (1970a). Generalized from Shah et al. (1970b). Generalized from “Air Quality Data for 1967” (1971). p Generalized from Brown and Vossen (1970). Generalized from Tabor and Warren (1958). Not detectable. Diesel fuel additive.
‘d
r
P
5m 5u
272
DONALD J . LlSK
specific metals in sludge probably reflects a simple lack of them in the effluents entering the sewage plants which were sampled. The U.S. Geological Survey has long performed yeoman’s service in determining the presence and magnitude of lesser known elements in surficial materials as in the excellent publication by Shacklette et al. (1971 b ) . Much, however, is unknown concerning the toxic metal content of most soils. These levels, even if known presently, are doubtless changing owing to continued pollution and erosion. A method called “geochemical reconnaissance” (Webb et al., 1968a,b; Thornton et al., 1969) is therefore worthy of description. Metals in stream sediments in specific areas are determined so as to judge the content and mobility of them in minerals and soils of the region. The principle is that under typical conditions weathering products including metals will be funneled down surface drainage systems either dissolved or in the active stream sediment. These sediments may thus reflect deficiency or toxicity levels of a large area which could otherwise be assessed only by multiple sample analysis of soils and minerals throughout the region. Agreement between stream sediment analysis and analysis of individual mineral and soil samples is qualitatively good.
B.
ASSOCIATIONS AND BEHAVIOR OF METALSI N
SOIL
Our knowledge of the chemistry of toxic metals in soils is very incomplete and largely speculative. In review, the associations of metals in soils include those ( I ) in minerals as part of their regular structure and as interstitial impurities; ( 2 ) as simple and complex inorganic precipitates; ( 3 ) as simple and complex inorganic ions; ( 4 ) incorporated in organic matter and microorganisms; (5 ) as soluble or insoluble organic complexes or chelates; ( 6 ) adsorbed as ions on charged surfaces of clays, precipitates, and organic matter; and ( 7 ) in various complex combinations of the above associations. Without experimentation one can only weakly attempt to predict the behavior of toxic metals in soils by matching their physicochemical properties in pure systems with their possible reactions in soils which these properties pertinently influence. Thus the standard free energies of formation of metal oxides, silicates, sulfides, sulfates, and carbonates will indicate their general distribution in fused iron, silicate slag, and so on, according to the principles of Goldschmidt (1954). Kee and Bloomfield (1961a) have pointed out the order of coprecipitation of metals with ferric oxide and their order of sorption by it as determined by the content of particular metals in ironstone and lateritic material (Lindgren, 1923; Oertel and Prescott, 1944). The presence of metallic ions in clays will depend on the
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
273
chemical and physical structure and purity of the clay, the valence and ionic radius (or hydrated ionic radius) of the metal in question (GoldSchmidt, 1954), its electronegativity (Ringwood, 1955) and the free energy of formation of the compound or isomorphic mixture (Rankama and Sahama, 1950) as it pertains to isomorphous substitution or interstitial diffusion. Ionic potential (charge/radius) is an extremely useful concept for predicting the affinity of cations for charged surfaces (Kunin, 1958) and the tendency of elements to exist as soluble cations, hydrolyzates, or soluble complex anions (Mason, 1952). Published data are useful on the order of affinity of various ions for cationic or anionic exchange sites on clays (Hasler, 1943) or on ion exchange resins, (Kunin, 1958; Samuelson, 1953), which would indicate their order of exchangeability on carboxylic or hydroxyl sites in organic matter and specific ion effects, such as chemisorption. The affinity of metal ions for exchange sites would also be useful in predicting their flocculating effects in soil clay, but the concentrations of toxic metals are probably too low in most instances to appreciably influence flocculation. Sodium ions from detergents, however, are probably responsible for puddling soils around leach lines in home septic systems by dispersion of clay particles. The tendency of metals to form hydrolytic polynuclear complexes or insoluble hydroxides, phosphates, carbonates, or sulfates (or metalloids to precipitate with calcium, magnesium, iron, aluminum, or manganese) and their phase stability at given pH values and oxidation-reduction potentials would be informative. Whether, based on their oxidation-reduction potentials and pH, they would undergo reduction in submerged or poorly drained soils and the resultant solubility of the reduced metal phosphate, etc., is essential knowledge. Conversely the insolubility of metal sulfides under reduced conditions would importantly determine the availability of toxic metals to plants and to intoxicating reactions such as methylation. Whether methylation of metals is a reaction of concern in the environment will depend on the electronegativity of the metal for reaction with the eIectropositive methyl radical (Rochow et al., 1957), the activation energy of the methylated derivative and the hydrolysis constant 6f the formed derivative (Jernelov, 1970). The valence, ionic radius, and electronic structure of metals will influence the ease of formation and intricacy of complex ions. The tendency of metals to form chelates and their published order of stability is necessary predictive knowledge which may indicate their availability in soil solution and the facility of plant absorption. The electronegativity and sulfide insolubility of metals will determine their affinity for amino, imino, and sulfhydryl groups in soil organic matter (or as functional groups in enzymes), and thus perhaps to some extent their toxicity to soil microorganisms. [The order of increasing toxicity of metals to bacteria has been shown to be Li, Sr, Ba, Cr3+,
274
DONALD J. LISK
Be, Pb'". TI1-, Zr, N P , Cd, Hg2-, Ag (Porter, 1946).] The complexation of metal ions in soil organic matter has been studied by Beckwith (1 955 ). Other physicochemical approaches (Vanselow, 1932; Gapon, 1933; Rao et al., 1968; Bolt and Page, 1965) for describing and predicting the behavior of toxic metals in soils are just beginning. Beyond this, one might only hope to gain insight by analogy, such as by judiciously comparing the known soil reactions of zinc with possible similar ones for cadmium, phosphorus reactions with arsenic, and so on. It is therefore possible to assemble many of thc above physicochemical data from literature sources for particular metals. Data such as this might then aid to predict the effects of additions of lime, fertilizers, sulfur, organic matter, or moisture on the availability or phytotoxicity of the metal to plants and their possible locations and associations in the soil profile. It must be ernphasizcd, however, that a multitude of factors dictate against accurate prediction. For instance, continued extensive pollution by specific metals, the many different compounds of a given metal pollutant as it reaches soil, the host of soil types and competing soil reactions, let alone the modifying cffects of microbiological activity and plant growth, would complicate predictions. Mitchcll ( 1955) has cflectively summarized knowledge of the factors affecting the movement and location of trace metals in soils including many of the above-mentioned as well as Icaching, soil sorting, fractional crystallization, mineral stability, and biological enrichment. Table I1 lists availablc physicochemical data pertinent to the above discussion. It is known that elements such as arsenic, mercury, and selenium tend to be more toxic and show more tendency to undergo biological methylation. While methyl derivatives of most metals may be synthetically prepared, the lower the clectronegativity of the metal, thc greater is the activation energy required for the methylation reaction, and the easier hydrolysis of the formed derivative occurs (Rochow et a/., 1957). In any equivalent metal series, the lower the ionic potential (the larger the metal ionic radius), the more tightly the metal will be bound to ion exchange sites. Also higher ionic potentials usually indicate a higher degree of hydration of the metal in solution. As Mason (1952) pointed out, metals with lower ionic potentials, such as barium, cesium, lithium, rubidium, and strontium, tend to form soluble cations in solution; those with intermediate potentials, such as beryllium, gallium, niobium, scandium, titanium, and zirconium, form hydrolyzates; and those with the highest values, such as selenium and vanadium, form soluble complex anions. Ionic potential can be used to predict precipitation reactions. Thus ferrous or manganous ions with respective potentials of 2.70 and 2.50 would be stable soluble ions in solution, whereas precipitation of iron must be preceded by oxidation to the ferric state with an ionic potential of 4.69 and manganese is precipi-
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
275
tated in the hydrated quadrivalent state having a potential of 6.67. From the solubility product data, it is evident that much opportunity exists for precipitation of toxic metals in highly insoluble forms. The degree of stability of metal chelates is indicated by the magnitude of the stability constant (the logarithm of the equilibrium constant). Those listed in Table I1 are for EDTA (ethylenediamine-N,N,N’,N’-tetraacetic acid). Metals forming more stable chelates tend to be more toxic since they may more readily traverse biological membranes in this form. The rare earth metals form stable chelates with EDTA with stability constants in the range of 15 to 25. Data such as the oxidation-reduction potentials of metals recalculated for a typical submerged soil pH of 7 could also have been tabulated in the hope of predicting what ion species might exist in the potential range of 0.3 to -0.7 V for submerged soils. A lack of knowledge of the complex nature of existing solid phases, their solubilities, and the modifying effects of organic complexing agents, however, makes such predictions very risky. This subject has been dealt with in the literature (Garrels and Christ, 1965; Stumm, 1967). C.
THE PLANTFACTOR
Agronomists have long debated to what extent the soil- or the plant dictates ion uptake by roots. The nature of the plant including its species, size, growth rate, extent and depth of rooting, transpiration rate, and nutritional requirements may effect its efficiency for metal absorption from soils. The mechanism of ion uptake by roots is speculative and may involve direct ion absorption of soluble ions in soil solution as well as exudation of organic complexing anions by roots or bacteria to render fixed metals soluble for absorption. Plant root washings have been shown capable of dissolving manganese dioxide (Bromfield, 1958a,b). Physical contact between the root and adsorbed or precipitated ions may result in direct passage of the ions into the root. This process of contact exchange was proposed and described by Jenny and Overstreet (1939) and has been supported by theoretical considerations (Overstreet, 1945). Certain plants, such as corn, have been dubbed “coarse feeders” since they absorb large quantities of soil nutrients. Trees are especially effective in absorbing many trace metals (including many rare earths) from great depths and translocating them to the leaves as determined by their transpiration rate. Falling leaves then effect a recycling of these metals to the upper soil humus layer. Other plants may accumulate inordinately high levels of specific metals such as cobalt or selenium and conversely other specific metals are largely excluded by plants. Finally one ion species may interfere with root absorption of a different one in the same solution. Since the behavior of
N
-1
h
TABLE I1 Physicochemical and Related Data for Metals
Metal
ElectronegaIonic tiritya potential6
Antimony Antirnony Arsenic Arsenic. Barium Barium Beryllium 3erylliuni Bismu t 11 Risuiuth Cadmium Cesium Chromium Chrorniuni
1 . 8892 2 k? . 2200 0.97 0 97 1 . 47 47 1.67 1 67 1 I .46 0 . 8866 1 . 5566
s.95(S)d
Gallium Gslliu in Germanium Lead Lithium Mercury Sickel
1.82 1.82 2.o.2 1.55 0.97
4.81(3) 7.55(1) 1.67(4)
1.44
1.75
1.35(3) 1.49~2) 5.72(2) 3.12(5) 2.16(2) 0.60(1) 4.77(3)
1.47(1) 1.82(4) 2.90(?)
Solubility product constant ( K S P ) ~ c032-
OH-
HP0d2-
Pod3-
S210-93,a)
10-"(3)
.4s04"
SeOP
10-18 ( r Z I ( 3 ) )
lo-' (Ca) lo-' (Mg) 10-31 (Fc(3)) (h€n(.2))
(Ca) 10-20 (Mg) 10-2" (Fe(3)) 10-29 (Mn(2))
1O - y , ) 10-23(3)
10-32
-.
SOo2-
-
-
Stability constant (log
-
1.4(3) 16.6(?)
10-23(3) -. .
"0 Y(3) 20.3(3)
8
z 2 b 4
Niobium Palladium Rubidium Scandium Selenium Silver Strontium Tellurium Thallium
1.23 1.35 0.89 1.20 2.48 1.42 0.99 2.01 1.44
7.25(5) 1 .67(2) 0.88(1) 4.10(3) 8.00(4)
-
-
0.80(1) i.79(2) 5.72(4) 0.68(1)
10-"(1) 10-9(-)
7.3(1) 8.6(2)
Tin Titanium Vanadium
1.72 1.32 1.45
5.64(4)
-
Zirconium
1.22
5.88(4)
8.48(5)
5.06(4)
18 4 2 )
-
-
-
-
-
22.5(3)
17.3(3) 12.7(2), 25.9(3), 18.1(5) 10-18"(4)
29.9(4)
4 0 P
n
m
E 4
P
2
v,
p
t: Y
c
Generalized from Little and Jones (1960). Generalized from Weast (1969). Generalized from Sillen and Martell (1964). Charge.
cd
r Z
*
"2 P
2
P
t: td
4 4
278
DONALD J. LlSK
toxic metals in soils and the mechanisms of ion uptake by plants are largely speculative, it is most practical to examine the data that have been experimentally gathered concerning metals in soil-plant systems. Aspects of their presence in man and animal systems will also be considered.
D. SPECIFICMETALS 1. Mercury
Mercury is presently of most concern and will therefore be dealt with first. There are many known occupational and industrial sources of mercury contamination for humans including mining (West and Lim, 1968), milling (Rentos and Seligman, 1968), hat manufacture (Goldwater and Nicolau, 1966), dental (Hoover and Goldwater, 1966; Joselow et al., 1968; Cook and Yates, 1969; Gronka et al., 1970), painting (Goldberg and Shapiro, 1957; Taylor, 1965), hospital (Williams et al., 1968), and laboratory exposure, and others, in which high concentrations of the metal have been reported in tissues, especially liver and kidney, and excreta of those involved. The industrial hazards of mercury have been described (Duffield, 1968). Surveys of mercury in organs and tissues of humans unexposed to high concentrations have shown appreciable levels (Joselow et al., 1967; Suzuki et al., 1970). The concentration of total mercury in human hair serves as an approximate indicator of exposure. The use of organic mercury compounds as preservatives in hair preparations can confound the results of hair analysis, and exposure to humans from this source is unstudied. Studies of the distribution in blood (Cember et al., 1968) and elimination (Cember, 1969) of mercury in rats have been made. Administration of mercuric salts markedly decrcases the respiratory excretion of volatile selenium compounds in rats (Parizek et al., 1969d). Ingested inorganic mercury compounds are largely excreted, and the chances of recovery from reasonable overexposure are good. Aryl mercury compounds may be degraded in the liver and kidney and excreted as inorganic mercury. Alkyl compounds are most toxic, only slowly excreted [half-life equals approximately 79 days (Miettinen et al., 1971)], and may penetrate the blood-brain barrier (Glomski and Brody, 1971) to cause permanent derangement. The most disastrous consequences of mercury were those at Minamata Bay in Japan where mercury as methylmercury in fish reached high levels owing to industrial contamination of the aquatic environment. Increasing incidence of mental retardation, deafness, ataxia, and other symptoms, which has become known as the “Minamata” syndrome (Berglund and Berlin, 1969b) then followed in infants,
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adults, and animals. Localization of mercury in certain regions of the brain, such as the cerebellum, correlates with clinically demonstrable cerebellar malfunction (Glomski and Brody, 1971 ). Methylmercury is believed to attach to sulfur-containing amino acids (Burstein and Sperling, 1970; Porter, 1970) and thereby alter protein function. Chromosome breakage in human lymphocytes from consuming fish containing methylmercury has been reported (Skerfving et al., 1970). The risk to humans and other mammals from methylmercury (Berglund and Berlin, 1969a,b) and precautionary measures (Klein and Herman, 1971 ) have been evaluated. Residues of mercury have been reported in various wildlife mammals in Sweden (Borg et al., 1969; Johnels and Westermark, 1969), such as fox and deer, levels in carnivores being generally higher than in herbivores. Mercury in birds has been studied considerably. Probably the main environmental sources of mercury for birds are mercurial fungicide-treated seeds and mercury-containing fish. Numerous reports of residues of mercury in a variety of avian wildlife species in Canada (Fimreite et al., 1970; Fimreite, 1970), Sweden (Borg et al., 1966, 1969), Norway (Wanntorp et al., 1967; Holt, 1969), and Finland (Henriksson et al., 1966) have been published. Mercury concentrations are typically proportional to the position of the species in the food chain. Mercury accumulates in liver, kidneys, and eggs of birds (Platonow and Funnell, 1971; Stoewsand et al., 1971) and most other animals. It is excreted in the feathers of birds (Henriksson et al., 1966; Berg et al., 1966; Stoewsand et al., 1971), and as a residue in human hair may serve to indicate body burden. It is interesting to note that the strain of chickens can profoundly affect the extent of deposition of mercury in their liver or kidneys (Miller et al., 1959a,b, 1967, 1970). Methylation of mercury and storage of methylmercury has been reported in chickens (Kiwimae et al., 1969), but not in Japanese quail (Stoewsand et d.,1971). A continuing decrease in eggshell weight of birds of prey for the past 70 years has been reported and may be due to progressively higher general toxicant levels in the environment (Ratcliffe, 1967). Eggshell thinning at low (1-8 ppm as mercury) dietary levels of mercuric chloride has been shown in Japanese quail (Stoewsand et al., 1971). Complete absence of shells in 25% of the eggs from chickens fed 18 mg of methylmercury per kilogram has been reported (Tejning, 1971). Depending on the dietary level of methylmercury, up to 15% shell-less eggs have also been reported in ring-neck pheasants (Fimreite, 1971 ) . Less effective reproduction in Wood pigeons poisoned by mercury has been indicated (Ljunggren, 1968). Poor hatchability of pheasant eggs containing up to 2 ppm of mercury was found (Borg, 1966). Mercury in soil may result from that contained in parent materials, ap-
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plications of mercurial fungicides, mining, fallout from the combustion of fossil fuels, and other sources (Stahl, 1969). In an extensive analytical survey of over 900 soil samples taken about 80 kilometers apart throughout the United States, Shacklette et al. (1971a) reported a range of 0.01-3.4 and 0.01-4.6 ppm of mercury for the eastern and western sections, respectively. Mercury occurs worldwide in belts that correspond to the earth’s mobile belts and zones of dislocation (Jonasson and Boyle, 1971). Mercury is sometimes associated with gold deposits, and its presence in air above may be sensitively measured to locate gold (McCarthy et al., 1969). The amount of mercury released worldwide per year by wcathering processes and by combustion of coal has been estimated at 230 and 3000 metric tons, respectively (Joensuu, 1971). Mercury in soils located over deposits of cinnabar (mercuric sulfide) may reach 40,000 ppb (Shacklette, 1965). Mercury does not appear to be concentrated to a great extent in plants, which usually contain less than 0.5 ppm (fresh weight) (Shacklette, 1970). Ranges of mercury in many plants and foods have been published and are usually far below 0.5 ppm (Wallace et al., 1971; Nelson et al., 1971 ). Mercury may attain high concentrations in vegetation near sources of mercury, such as mines (Byrne and Kosta, 1970). Droplets of metallic mercury have been found in the seed capsules of Holosteum umbellatum (jagged chickweed) growing on soils rich in mercury (Rankama and Sahama, 1950). Mercury added to soil as mercuric chloride has been shown to become rapidly fixed in the organic matter and clay fractions (“International Conference on Environmental Mercury Contamination,” 1970). It is tightly bound in organic soils (Hasler, 1943). Mercuric chloride may be reduced in soils to metallic mercury, and the process is enhanced by the presence of sewage (Zimmerman and Crocker, 1933). Vapor damage from this source has been reported with roses grown in mercuric chloride-treated soil. Inhibition of at least certain soil bacteria by mercury compounds has not been reported but would seem pIausibIe. Sewage sludge can be expected to be high in mercury owing to industrial pollution and mercury contamination at the treatment plant from broken mercury seaIs in pumps (Knapp, 1970). Addition of sulfur to soil to precipitate mercury as highly insoluble mercuric sulfide has been suggested as a possible remedial measure (Lagerwerff, 1967). Methylation of mercury in submerged soils will be discussed later. Many mercury-containing fungicides including phenylmercury and methylmercury compounds used in agriculture are often a major contributor to mercury residues in plants, and ultimately food. Kimura and Miller ( 1964) have shown the presence of vapors of the parent compounds and of metallic mercury when phenyl-, methyl-, or ethylmercury com-
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28 1
pounds degrade in soil. Final residues in food will depend on many factors, including fungicide stability, number and rate of applications, formulation, interval between application and harvest, rainfall, temperature, light intensity, growth dilution and crop variety. The diffusion of methyl-, ethyl-, or phenylmercury fungicides through the seed coat of treated seeds has been shown (Lindstrom, 1959). Grain grown from seed treated with methyl- or ethylmercury compounds has been shown to contain higher mercury residues (Saha et al., 1970). Translocation of phenylmercuric acetate into the fruit of foliar-treated apple trees has been reported (Ross and Stewart, 1962). Data on the translocation and residues of mercury in many varieties of crops as a result of fungicide applications has been amply gathered and reported by Smart (1968). In conclusion it should be emphasized that mercury is typically lost through volatility and adsorption during analysis. Results reported can thus be seriously low when obtained by methods other than direct neutron activation, closed oxygen flask sample combustion, or meticulously conducted wet ashing procedures accompanied by daily recovery studies at the level of mercury expected. Mercury in the environment has been extensively reviewed (“Mercury in the Environment,” 1970; Goldwater, 1971; Hammond, 1971; Knapp, 1970; “Effects of Mercury on Man and the Environment,” 1970; Borg et al., 1969; Backstrom, 1969; “The Mercury Problem,” 1967; Lofroth, 1969; “Hazards of Mercury,” 1971; Wallace et al., 1971; D’Itri, 1971).
2. Lead Lead has been a continuing problem to man since perhaps the fall of Rome (Gilfillan, 1965). The sources of lead include engine exhausts, water pipes, food containers, insecticides, certain pottery, smoking, and many others. Lead has been frequently reported and studied in humans (Patterson, 1965; Schroeder and Tipton, 1968; Strehlow and Kneip, 1969), being higher in persons living near freeways (Thomas et al., 1967) and workers in printing shops (Hernberg et al., 1969), but not in those handling gasoline containing tetraalkylleads (de Treville et al., 1962; Kehoe et al., 1963a,b). Lead concentrates in bones and the total amount there may represent about 90% of the entire body burden (Schroeder and Tipton, 1968). Among bones it has been found at highest concentration in teeth (Strehlow and Kneip, 1969). Lead increases in various body parts with age, being higher in Americans than Africans or Middle or Far Easterners (Schroeder and Tipton, 1968). Little accumulates in brain, and lead was not found to displace low concentrations of other essential trace metals in soft tissues, such as chromium, manganese, cobalt, copper, or molybdenum (Schroeder and Tipton, 1968). Lead inhibits the conversion of 6-aminolevulinic acid to porphobilinogen by 6-aminolevulinic acid dehy-
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drase, and lead exposure is normally assessed by analysis of urine for the acid (Davis et al., 1968; Hankin et al., 1970) or of erythrocytes for the enzyme (Hernberg et al., 1970). Of most concern is that the average lead level in blood of Americans is about 0.25 ppm while the threshold for acute lead intoxication is in the range of 0.5 to 0.8 ppm (Patterson, 1965). Whereas ingested lead is largely excreted, inhaled lead is effectively absorbed and is believed to account for about 30% of the totaI body burden. Much effort is underway by the petroleum industry to produce low-lead gasolines i“Gasoline : Antipollution Forces Bring Marked Changes to Petroleum Refining Industry,” 1970), but certain of these high aromatic fuels emit considerably more carcinogenic polynuclear hydrocarbons (“Aromatics in Gasoline,” 1971) . Lead from storage battery plates, paint, putty, and gasoline engine oil is a common cause of poisoning in domesticated animals, such as cattle (Christian and Tryphonas, 1971 ). It has been found at relatively high levels in wild frogs near freeways (Schroeder and Tipton, 1968). Weight loss and poor coats were the only toxic symptoms noted in rats fed 5 ppm of lead nitrate (Schroeder et al., 1970b). Environmental lead contamination in birds is believed to be due mainly to consuming lead shot and air-borne lead from gasoline exhaust which can result in contamination of consumed fish (“Lead in the Sea,” 1971). Substantial concentrations of lead in avian wildlife have been reported (Bagley and Locke, 1967; Bagley et al., 1967; Locke et al., 1969). In feeding studies where a single large dose of a lead salt is administered to birds, toxicity may not result (Salisbury et al., 1958). Conversely, consumed lead shot which is retained in the gizzard will result in continuous lead absorption for prolonged periods and resultant lead toxicity (Salisbury et al., 1958). Efforts have been made to find substitute materials for lead in shot form and nickel, tin, Teflon-coated steel (Grandy et al., 1968), and copper shot (Locke et al., 1967) did not produce toxic symptoms in mallard ducks. The use of these materials will depend on their ballistic qualities in shot when compared to lead. The sources of lead in soils include mineral, combustion of fossil fuels, mining and smelting operations, pesticides, fertilizer impurities, and others mentioned above. Major mineral forms of lead include the sulfide, carbonate, and sulfate (Rankama and Sahama, 1950). The total lead content of soils in this country has been reported to range between 10 and 700 ppm (Shacklette et af., 1971b) with an average of about 16 ppm in the earth’s lithosphere (Swaine, 1955 ). Lead is biologically enriched in the upper layers of soils (Mitchell, 1955) and is much higher in soils near highways (Page and Ganje, 1970). Typical lead contents of many food plants and trees are in the range of 1 ppm (Mitchell and Reith, 1966; Warren and Delavault, 1962), and in fungi, algae, and plankton may be severalfold
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higher (Bowen, 1966). Lead in coal has been shown to range from 2 to 20 ppm (Kessler et al., 1971). Lead concentrations in air of American cities has been reported ranging up to 16 pg per cubic meter (“Air Quality Data for 1967,” 1971). Much higher lead concentrations in plants have resulted through atmospheric deposition by their proximity to mining (Alloway and Davies, 1971) and smelting (Hammond and Aronson, 1964) operations and highways (Chow, 1970). Lead from gasoline combustion appears to be the major source of lead in the Los Angeles basin since the isotopic ratios of lead 206 to either lead 204, 207, or 208 in gasoline closely parallels those found in snow and ocean sediments of the area (Chow and Johnstone, 1965). A general and dramatic 400% increase in the concentration of lead in Greenland snow from 1750 to the present has been determined (Patterson and Salvia, 1968). Similar historical increases in the concentration of lead in mosses have been reported (Ruhling and Tyler, 1968). The soil chemistry of lead is little understood. Plants tend to exclude lead in the process of ion uptake from soils. Lad-210 and polonium-210 resulting in that order from the radioactive decay of radon which emanates as a gas from the soil are naturally occurring isotopes. Lead-210 and polonium-210 are found in tobacco, and concentrations of these metals in the bones of human smokers are considerably higher than in nonsmokers (Holtzman and Ilcewicz, 1966; Little and McGandy, 1968). High concentrations of lead in soil may, however, greatly restrict plant growth (Lag et al., 1969). Lead reaching the soil from air-borne gasoline combustion products is not believed to be readily available to plants (Page et al., 1972). Lead in soil is mobilized upon flooding the soil or anaerobically incubating it with plant material (Kee and Bloomfield, 1962). The lead content of soils varies inversely with pH. Similarly the absorption of lead by radishes was reduced by increasing the soil pH from 5.9 to 7.2 (Lagenverff, 1971). Lead appears to only weakly induce iron chlorosis in plants (Hewitt, 1953). Various other growth-inhibiting and -promoting effects of lead in plants have been reported (Brewer, 1955). For further details on the modes of accumulation of lead by plants from air and soil, the reader is referred to several excellent papers (Lagerwerff, 1967, 1972; Allaway, 1968). The general environmental hazards of lead have been reviewed (Schroeder and Tipton, 1968 ; Bryce-Smith, 1971; Smith, 1969; Haley, 1969; Schroeder and Balassa, 1961). 3 . Cadmium
After mercury and lead, probably cadmium is considered next in importance as an environmental pollutant. Its sources are many (Dunphy, 1967) and include smelting and plating operations, lithography, engraving,
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soldering, and welding. In feeding studies with rats, Schroeder has linked cadmium with the production of hypertension (Schroeder and Vinton, 1962; Schroeder, 1964; Schroeder et al., 1966, 1968c) and was able to reverse the syndrome with zinc chelates (Schroeder et al., 1968b; 1970d; Schroeder and Buckman, 1967) and other metal binding agents (Schroeder ef al., 1955). Cadmium levels in the air of 28 American cities has been closely correlated with the incidence of death from hypertension and arteriosclerotic heart disease (Carroll, 1966). Cadmium concentrations were reported to be higher in kidney tissue of subjects dying of hypertensive complication (Schroeder, 1965), but no such correlation has also been reported (Morgan, 1969). Renal cadmium levels in humans have been shown to progressively increase up to about age 50 and then decrease (Schroeder, 1967). Available evidence linking cadmium with hypertensive diseases provides one of the best examples of a valid correlation between environmental trace levels of a toxic metal and a resultant biochemical lesion. Sufficient zinc has been shown to protect the body against toxic effects of cadmium (Schroeder et a[., 1967b; Mason and Young, 1967; Gunn et al., 1961; Parizek et al., 1969b; Parizek, 1957; Powell et al., 1964). Zinc is higher in the blood of Americans in the northeast (Kubota et al., 1968) perhaps owing to air pollution and resultant deposition of zinc-containing dust on plants. Other maladies caused by cadmium poisoning are proteinuria (Piscator, 1962), kidney damage (Axelsson and Piscator, 1966), anosmia, and early testicular atrophy (Vigliani, 1969). Cadmium markedly increases retention of selenium in rats (Ganther and Baumann, 1962; Parizek et al., 1969d). The toxic effects (Flick et al., 1971 ) and other environmental aspects of cadmium (Schroeder, 1971) have been recently reviewed. Cadmium has been determined in various wildlife animals (Schroeder et al., 1967b). Cadmium is found in nature in very small amounts associated with lead, copper, and particularly zinc ores (Athanassiadis, 1969), probably owing to its chemical similarity. It has been suggested as a possible pathfinding element in geochemical prospecting for zinc deposits (Warren and Delavaut, 1956). The ratio of zinc to cadmium in igenous rocks is about 900 to 1 (Rankama and Sahama, 1950). Other sources of cadmium include dusts and fumes from mining and refining of metals, miscellaneous industrial sources (Athanassiadis, 1969), as an impurity in fertilizers (Schroeder and Balassa, 1963), gasoline, oil, coal, tirewear, and many organic waste products (LagerwerR, 1972), and cigarette smoke. Cadmium levels in coal may range from 0.2 to 0.5 ppm (Kessler et al., 1971). Concentrations of cadmium in air of American cities ranging up to 0.1 pg/m3 have been reported (“Air Quality Data for 1967,” 1971; Carroll, 1966; Schroeder, 1970a; Tabor and Warren, 1958). Air polluton by cad-
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mium is particularly serious in Japan. Cadmium levels in water may reflect contamination from metal pipes (Schroeder et al., 1967b). Plants have been reported to contain cadmium concentrations up to 0.1 ppm on a fresh weight basis (Bowen, 1966; Schroeder et al., 1967b), certain fungi containing about 10 times this level. Many plants absorb cadmium from the soil rather easily. The ratio of cadmium concentration in plants to that in the corresponding soil has been reported to be about 10 to 1 (Hodgson, 1970). Increased accumulation of cadmium by plants fertilized with superphosphate which contains cadmium as an impurity has been reported (Schroeder and Balassa, 1963) . Absorption of cadmium by radishes was diminished by raising soil pH from 5.9 to 7.2 (Lagerwerff, 1972). Lagerwerff also showed that direct aerial contamination by cadmium of radish tops when radishes were grown near a highway accounted for more than 40% of their total cadmium content. Cadmium reaching the soil by aerial contamination appeared largely unavailable to radishes. A recent survey of institutional diets showed concentrations of cadmium ranging from 0.027 to 0.062 ppm (Murthy et al., 1971). Allaway (1968) has suggested possible measures to reduce cadmium absorption by plants, such as soil fertilization with zinc- or cadmium-free superphosphate fertilizer. Since cadmium is strongly fixed in organic soils (Hasler, 1943) , addition of organic matter might also be beneficial. 4. Arsenic
Arsenic, probably more so than mercury, is naturally present throughout the environment at trace concentrations in rocks, ores, soils, plants, and animals. Biologically, most arsenic occurs in the pentavalent form whereas the more toxic trivalent arsenic is largely the production form (Schroeder and Balassa, 1966a). Its production is almost entirely as a by-product in the smelting of lead, copper, and gold. The major sources of arsenic pollution from human activities are metal smelting, combustion of fossil fuels, and the use of arsenic-containing pesticides, herbicides, and drugs. The arsenic content of coal (Kessler et al., 1971; Abernethy et al., 1969) and petroleum (Shah et al., 1970b) has been reported to range from about 2 to 20 ppm and from 0.05 to 1.1 ppm, respectively. Arsenic is highly concentrated in coal ashes (Rankama and Sahama, 1950). Arsenic present in cigarette tobacco smoke, probably as the carcinogen triphenylarsine (Holland and Acevedo, 1966), showed a steady decline in concentration between 1961 and 1965. Concentrations of arsenic in air throughout the nation have been reported and are roughly proportional to the proximity to industrial or agricultural sources of arsenic pollution (Sullivan, 1969a). Arsenic levels in humans have been related to known geochemical arsenic levels in specific areas (Heydorn, 1970). Arsenic has been accused of
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being carcinogenic (Vallee et al., 1960), but rats receiving small amounts of arsenites have been found to have a significantly lower incidence of spontaneous tumors (Kanisawa and Schroeder, 1967). Arsenic counteracts the toxic effects of selenium in rats (Levander and Argrett, 1969). Dietary arsenic markedly decreases the respiratory loss of volatile selenium compounds in rats (Ganther and Baumann, 1962; Olson et al., 1963). Arsenic in the diet of chickens decreases the deposition of selenium in eggs and counteracts the decrease in egg production, viability, and body weight brought on by dietary selenium (Thaper et a!., 1969; Krista et al., 1961 ) , There is speculation presently that arsenic may be an essential element (Kanisawa and Schroeder, 1967). The native arsenic content of soils has been reported to range up to about 40 (Williams, 1940) to 70 ppm (Mitchell, 1955), with levels up to 121 (Miles, 1968) and 183 ppm (Greaves, 1934) in orchard soils. The soil chemistry of arsenic has been studied by Woolson et al. (1971). As reported by them, arsenic resembles phosphorus and is fixed in soil in the iron, aluminum, and calcium fractions, iron and aluminum being most effective owing to the lower solubility product of these metals with arsenatc as compared to calcium. Organic matter also affects arsenic fixation. Heavier soils fix arsenic more effectively than lighter ones. Arsenic may also leach down in soils to depths of 6 feet. The fate of cacodylic acid (dimethylarsinic acid), an herbicide of major use in soils, has also been studied by Kearney and Woolson (1971a,b). Under aerobic conditions the compound is microbiologically converted to carbon dioxide and arsenate by carbon to arsenic bond cleavage. Arsenate so liberated would than be fixed in soils by the processes described. Cacodylic acid is also directly fixed by iron, aluminum, and calcium soil fractions. Anaerobically cacodylic acid is converted to arsines, dimethylarsine being perhaps the major product. The alkylarsines are strongly fixed in soil by iron oxides. Oxidation of dimethylarsine to dimethylarsinoxide, and finally again to cacodylic acid, is known to occur in air and may therefore indicate the possibility of an anaerobic degrading and aerobic synthesizing cycle for the herbicide (Kearney and Woolson, 1971a,b). Arsenic in land and marine plants has been reported at levels of about 0.2 and 30 ppm, respectively (Bowen, 1966). Schroeder and Balassa (1966a) reported levels of arsenic ranging from negligible to 3.6 ppm in a variety of plants. The ratio of arsenic concentration in plants to that in soils has been listed as about 0.12 (Hodgson, 1970). Plants growing on soils where arsenic has accumulated have been shown to contain up to 14 ppm (Bear, 1957). Phytotoxicity from soil arsenic accumulation is commonly manifested on old orchard soils (Vandecaveye et al., 1936). As the soil concentration ranges from 25 to over 100 ppm, effects on
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plants may range from negligible to greatly retarded growth of fruit tree seedlings (Benson, 1968). Decreased seed germination and reduced seedling viability of many plants may result. Rice on flooded soil is extremely sensitive to small concentrations of arsenic (Reed and Sturgis, 1936). This may be due in part to its increased availability in the presence of reduced soluble ferrous arsenite as compared to insoluble ferric arsenate compounds and a resultant herbicidal effect. As enumerated by Woolson et al. (1971), many factors affect the toxicity of arsenic to plants including soil fertility, plant vigor, available iron, aluminum, calcium, organic matter, phosphorus, soil density, plant variety, and the nature of the arsenic compound. Addition to soil of lime, organic matter, iron, aluminum, or zinc compounds or chelates (Thompson and Batjer, 1950; Batjer and Benson, 1958) or uncontaminated heavy soil would aid in overcoming arsenic phytotoxicity. Substituting resistant plants might also be efficacious unless they are arsenic accumulators. At low concentrations some plants are stimulated in growth by arsenic. Phosphate fertilization may accentuate arsenic phytotoxicity by competing for sites of fixation. Proving this experimentally may be difficult, however, since phosphate fertilizer may contain arsenic impurities (Schroeder and Balassa, 1966a). Additions of organic arsenicals to soil will be less available and phytotoxic than soluble inorganic forms. The use of organic arsenicals for parasite control, growth stimulation and as antibiotics in farm aniamls is closely regulated (Frost et al., 1961). With rapidly diminishing use of arsenic compounds as pesticides and herbicides, no health hazard appears evident at the presently known arsenic concentrations in the environment. The environmental aspects of arsenic have been reviewed (Schroeder and Balassa, 1966a).
5. Selenium When considering man, one cannot discuss the known toxic effects of metals without also including the possible essential role or beneficial effects of some lesser understood ones. In 1957, selenium emerged as an essential element which prevented dietary necrosis in rats receiving diets deficient in vitamin E (Schwarz and Foltz, 1957). It has since been shown to be involved in the prevention in animals of a variety of nutritional deficiency diseases, including liver necrosis, white muscle disease, hair and feather loss, and so on. Selenium deficiency occurs only rarely in man. Therefore evidence is sparse linking selenium deficiency in humans to disease. Children with kwashiorkor in Guatemala showed low levels of blood selenium (Burk et al., 1967). A survey of selenium in human blood in 210 subjects from 19 locations in the United States revealed levels geographically related to known selenium concentrations in crops in those regions (Allaway et al., 1968). Selenium concentrates in the human kidney, but the body
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burden does not appear to increase with age (Schroeder et af., 1970a). The toxicity of excess selenium to animals has been reviewed (Rosenfeld and Beath, 1964). Sclcnium has been variously purported to be both a promoter of cancer and an inhibitor (Shamberger, 1970). Traces of selenium have been reported to increase the incidence of dental caries (Hadjimarkos, 1965, 1969). Selenium is markedly protective against the toxic effects of cadmium or mercury (Parizek and Ostadalova, 1967; Parizek et al., 1969a; Sikov and Mahlum, 1969). Dimethylselenide is a metabolite of selenium in animals (Hofmeister, 1894; McConnell and Portman, 1952). Parizek (1970) has speculated that perhaps dimethylselenide could result in the transmethylation of inorganic mercury to toxic methylmercury compounds in animals. Mercury and cadmium greatly decrease the respiratory excretion of volatile selenium compounds in rats (Parizek et al., 1969d). A very recent study further elucidates the interactions each of zinc, cadmium, tellurium, and arsenic with selenium in rats (?vkConnell and Carpenter, 1971 ). The biosynthesis of selenomethionine in Escherichia coli has been shown (Tuve and Williams, 1961). Urinary excretion of trimethylselenonium ion by rats that ingested selenite has been reported (Byard, 1969; Palmer et al., 1969). Although an essential element, the narrow range between deficiency and toxicity requires that selenium be considered under the heading of toxic elements. Selenium is widely distributed at a concentration of about 0.09 ppm in the earth’s crust (Goldschmidt, 1954). It is largely concentrated in sulfide minerals. The geochemistry and mineral associations of selenium have been amply studied (Anderson et al., 1961; Searight et al., 1946; Wells, 1967; Searight and Moxon, 1945; Hawley and Nichol, 1959; Coleman and Delevaux, 1957; Lakin and Trites, 19561. Selenium is used in many products which probably do not cause environmental contamination. Its use in printing and xerography may result in paper contamination, the combustion of which could contribute to air pollution. Selenium in coal and oil have been found at concentrations up to 5 (Kessler et al., 1971 ) and 1.4 ppm (Shah et al., 1970b) in coal and petroleum, respectively. It is also present as an impurity in fertilizers (Wells, 1966). Concentrations of selenium in the air over New England cities have been reported to average 0.002 pg/m” (Hashimoto and Winchester, 1967). Sulfur dioxide, of which perhaps 0.1% could consist of selenium dioxide (Schroeder et al., 1970a), has been determined at concentrations in city air up to 658 pg/m3 (“Air Quality Data for 1967,” 1971 ). High levels of selenium occur in large areas of the western United States and several other countries (Lakin, 1961; Byers, 1935). The soil chemistry of selenium is poorly understood, and many anomalies exist between the magnitude of plant absorption and the corresponding soil content of
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the element. Selenium in acid soils appears to be fixed as ferric selenite in the zones of accumulated iron and aluminum compounds and is thus largely unavailable to plants (Cary et al., 1967; Cary and Allaway, 1969). On arid alkaline soils, soluble calcium selenate is available. Water-soluble organic selenium compounds are also present. Selenium may also be rendered unavailable by reduction of selenate to selenite, selenium metal, and finally selenides. Subsequent reoxidation of these latter products is then possible. The proportions of selenium in the various oxidation states theoretically depend on redox potential, soil pH, other ions which may fix selenium, such as iron and microbiological effects (Geering et al., 1968). Soil colloids have a negligible effect on the availability of selenates (Gile et al., 1938) but reduce that of selenites (Gile and Lakin, 1941). Selenates are most available to plants followed by selenites, and, last, selenium metal (Bisbjerg and Gissel-Nielsen, 1969; Gissel-Nielsen and Bisbjerg, 1970). The associated cation in selenite salts including sodium, barium, or ferric does not appear to be a factor in the extent of plant absorption of selenium (Watkinson and Davies, 1967a). Surveys of selenium in plants from various locations have been reported (Kubota et al., 1967; Byers, 1935). Plants may be classified according to their extent of absorption of selenium. Grasses, clovers, and garden vegetables have a relatively low tolerance to selenium and may absorb 5 ppm. Cereals and onions may absorb up to 30 ppm without signs of phytotoxicity. Certain other species including the vetch Astragalus and the woody aster Xylorrhiza accumulate tremendous concentrations (up to 15,000 ppm has been reported) and thus serve as plants indicative of seleniferous soils. These plants upon death and decomposition therefore serve to contribute large amounts of eventually available organic selenium compounds to upper soil layers. Successive cropping of selenium fertilized soil results in progressively diminished plant absorption and effects soil depletion (Allaway et al., 1967). Selenium is rapidly absorbed and translocated following foliar applications of sodium selenite ( Watkinson and Davies, 1967b). Factors affecting absorption of selenium by plants include plant species (Ehlig et al., 1968), form of selenium, soil composition, growth stage, and other factors determining plant vigor. Diminished absorption of selenium by plants may be effected by the addition to soils of sulfate fertilizers (Gissel-Nielsen, 1971; Gile et al., 1938; Gile and Lakin, 1941 ) or barium salts (Ravikovitch and Margolin, 1959). Patterns of absorption and translocation of selenium in plants may vary considerably depending on the plant species, method of application, and other factors. This subject has been reviewed (Anderson et al., 1961). Selenium in plants has been found as selenite, selenate, selenoamino acids, and other compounds that may be soluble peptides of selenoamino acids
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DONALD J. LlSK
(Allaway et al., 1967). Volatile but yet unidentified selenium compounds are released by both selenium-accumulator and nonaccumulator plants (Lewis et al., 1966). Loss of selenium during the drying of crops at elevated temperature has been reported (Gissel-Nielsen, 1969). Many of the above aspects of selenium in soils and plants have been effectively reviewed (Anderson er al., 1961; Rosenfeld and Beath, 1964; Allaway et al., 1967). Allaway (1968) has reviewed the considerations involved in properly controlling and adjusting the selenium levels in foods to assure the presence of essential levels in the human and animal diet. Our present understanding of the chemistry of selenium in soils and its mode of passage into plants and animals has been comprehensively reviewed (Allaway et al., 1967). Aspects of selenium in agriculture have been compiled (Anderson et al., 1961 ).
6 . Chromium Chromium has received much recent publicity as evidence accumulates linking a deficiency of it in humans to a condition of abnormal glucose metabolism resembling diabetes. It now appears to be essential for glucose metabolism and the proper remedial action of insulin. Trivalent chromium lowers serum levels of both glucose and cholesterol in rats (Schroeder, 1969) and may indirectly be a beneficial factor in preventing atherosclerosis (Schroeder et al., 1970c) and as an anticholesterogenic agent (Schroeder, 1968a). It may also be antagonistic to lead toxicity (Schroeder et a/., 1970b). An eye lesion consisting of corneal opacity developed in rats on a chromium-deficient diet (Roginski and Mertz, 1967). Chromate is absorbed by body organs much less readily than chromite, although neither crossed the placenta of rats (Visek et al., 1953). The rate of absorption of chromate and chromite by body organs and their membrane permeability have been correlated with their colloidal behavior, complex formation, and protein and red cell binding (Visek et al., 1953). The concentration of chromium diminishes progressively with age in Americans, but not in several foreigners studied. This may be due to our extensive use of refined sugar and other foods from which chromium . use has been removed during preparation (Schroeder et al., 1 9 7 0 ~ )The of brown or raw sugar resulted in considerably lowered serum glucose and cholesterol levels in rats as compared to white, refined sugar (Schroeder, 1969). The distribution and retention of chromium in animals and man has been studied (Baetjer et a!., 1959). The whole topic of the biological role of chromium (Mertz, 1966, 1969) and its environmental implications (Schoeder, 1970b) has been effectively reviewed. Chromium is widespread in the environment, small amounts usually being present in most rocks and soils as chromic oxide. Total chromium
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
29 1
in igneous and sedimentary rocks may range from 11 to 100 ppm (Bowen, 1966). As a mineral it is mostly found as chromite (FeO-Cr,O,). Uses of chromium in plating, tanning, paints, corrosion inhibitors, and fungicides (Sullivan, 1969b) all contribute to its dissemination in the environment. Coal (Kessler et af., 1971) and petroleum (Shah et al., 1970b) have been found to contain 5-20 and 0.0015-0.018 ppm of chromium, respectively. Combustion of these and wood (Schroeder, 1970b) contribute to air contamination. Chromium in urban air of the United States has been shown to range up to 0.1 pg/m3 (“Air Quality Data for 1967,” 1971). American soils may contain from 1 to 1500 ppm of chromium (Shacklette et af., 1971b). Serpentine soils are commonly higher in chromium (Cannon, 1970). The soil chemistry of chromium is largely speculative. Chromium is relatively unavailable in soil as very insoluble oxides. It also substitutes (as Cr3+) for A13+ in the [ N O , ] groups of aluminosilicates to become part of the mineral structure (Rankama and Sahama, 1950). Addition of chelated chromium to soils to promote plant absorption would probably result in only a brief effect with rapid formation instead of the correspondingly more stable iron chelates (Allaway, 1968). Chromates would not be stable in soils except perhaps in an alkaline oxidizing environment (Allaway, 1968). Chromic ion does not exist in solution, but rather complexes with water and other anions under acid conditions. In an alkaline solution it forms polynucleate hydroxyl compounds (olation) (Schroeder, 1970b). Under some circumstances hexavalent chromium is reduced to the trivalent form by organic matter (McKee and Wolf, 1971). Chromium in red oak leaves and corn oil has been reported present at about 40% as the tri-, and 60% as the hexa-, valent element (Schroeder et al., 1962b). Soil chromium is sometimes mobilized when soils are flooded and then drained or incubated with organic matter (Kee and Bloomfield, 1962), presumably by production of soluble organic complexing agents, but no increase in the availability of chromium to plants on poorly drained soils has also been reported (Mitchell et af., 1957). Regarding the unavailability of chromium it should be noted that trivalent chromium is unique among many cations in that it is so tenaciously held by certain synthetic cation exchange resins that it can be removed only by ashing the resin. Its unavailability in soil may therefore in part be due to similar strong attraction to negatively charged sites on clays or in organic matter, a specific ion effect. Plants absorb only a small proportion (0.01-1 ppm) of the chromium in soils. The ratio of total chromium in plants to that in soil is reported by Hodgson (1970) as 0.02. Traces of chromium have been reported in many plants (Robinson et al., 1917). Chromium additions to soil normally result in only very small increases in plant absorption of the element (Alla-
292
DONALD J. LISK
way, 1968). It is noteworthy, however, that foliar application of river water containing chromium-5 1 as a contaminant resulted in appreciable absorption of the isotope in several plants (Perkins et al., 1960). Also irrigation of vegetables with sewage waste waters containing chromium resulted in increased plant absorption of chromium by a factor of 3-10 over that of controls (Sedova, 1958). Perhaps efficient plant absorption of continuously provided chelated chromium was operative here. Mosses, lichens, ferns, and grasses growing on alkaline soils may contain 5-50 times thc concentration of chromium (Schroeder et al., 1962b) found in most other plants. Both plant growth stimulation and phytotoxicity by chromium have been reported and reviewed by Pratt (1966). Hexavalent chromium is usually more toxic. Inhibition of nitrification by tri- or hexavalent chromium addition to soil was also observed (Sedova, 1958). The chromium content of a wide variety of foods has been reported by Schroeder (1970b). Levels in most are in the range of 0.1 ppm, except in fruit and fish, which contain about 0.01 ppm, and condiments, which contain about 2.7 ppm. Total intake of chromium by consumption of institutional diets has been found to be in the range of 0.36-0.89 mg per day (Murthy et al., 1971). Some chromium may be transferred to food from stainless steel cooking utensils (McKee and Wolf, 1971). Conversely, as noted above, chromium is lost in the refining of sugar (Schrocder et al., 1 9 7 0 ~ ) .
7. Other Metals Several other metals have been studied regarding their toxicity or possible essentiality. These include tin, vanadium, and nickel. A comprehensive study has been made of the concentrations of tin in numerous plants, foods, human tissues, and other items (Schroeder et af., 1964). It was concluded that the metal was probably not essential, but rather a general environmental contaminant mainly from the storage of food in tin containers. Convcrsely, it is suggested as a possible essential element owing to its growth-promoting effects in rats (Schwarz et af., 1970). Vanadium may be useful in controlling blood cholesterol levels (Hudson, 1964; Lewis, 1959; Anonymous, 1960; Wright et al., 1960; Azarnoff et al., 1961; Valberg and Holt, 1964) and catalyzing tissue oxidations (Bernheim and Bernheim, 1938, 1939). Diets low in vanadium resulted in poor feather dcvelopment in chickens (Hopkins and Mohr, 1971). Excess vanadium depressed the cystine level in rat hair (Mountain et al., 1953) and human fingernails (Mountain et al., 1955). Vanadium in water has been reported to protect human teeth against dental caries (Tank and Storvick, 1960). Animal sources of dietary vanadium may be deficient since it is stored largely in bones. The levels of vanadium in humans in the United States is very low (Allaway et al., 1968). Environmental con-
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
293
siderations of vanadium have been published (Schroeder et al., 1963b; Bertrand, 1950). When deficient in the diet of chickens nickel has been reported to result in the production of thickened legs, enlarged hocks, and orange-colored skin (Nielsen and Higgs, 1971). Nickel may be involved in the development of the integumentary color or its absence in birds and may therefore be essential in their diet (Schroeder et al., 1962a). Nickel in tobacco has been implicated as a possible carcinogen in cigarette smoke (Sunderman and Sunderman, 1961 ). The production of nickel carbonyl as an air pollutant from the incomplete combustion of coal is particularly toxic. The environmental aspects of nickel have been reviewed (Schroeder and Balassa, 1962; Schroeder, 1970c; Mastromatteo, 1967). Small doses of lithium have been found to be very effective in the control of certain forms of mania in humans (Cade, 1949; Malenfant, 1970; Pfizer, Inc., 1971; Muniz et al., 1971). Its possible essential role is unknown. It has also produced teratogenesis in rats (Wright et al., 1971 ) . Beryllium is another metal which is extremely hazardous. Workers may be exposed during ore refining, alloying, milling, and machining the metal (Chamberlin, 1959; Cholak et al., 1967) unless proper measures are taken (Donaldson, 1965; Hyatt et al., 1959; Viles, 1959). A variety of chronic lung ailments may result from inhalation, but the severity may depend on the chemical compound of the metal (Spencer et al., 1968). Particles of beryllium in an external wound prevent healing and must first be excised out. Beryllium is no longer used in rocket propellants or fluorescent lights owing to its toxicity. The absorption and excretion of ingested beryllium has been studied in rats (Reeves, 1965). Very little published information is available on general environmental occurrence of the metal. Possible hazards to man from long-term exposure to barium are uninvestigated. Its presently increasing use as an additive to diesel fuel may therefore be of concern. Unusual metals may enter man’s environment in various ways. Thus antimony and barium are used in the primers of gun shells and will be present in the gases emitted when guns are discharged. Zirconium silicate is a toothpaste additive. Schroeder ( 1970d) has reviewed the environmental aspects of barium. Metal radionuclides are a continuing source of human apprehension. They result mainly from nuclear fallout, as by-products of nuclear reactors and power stations, and those present natively in the environment. Global air movements are predominantly toward northern rather than southern hemispheres and a continual deposition of metal radionuclides and other toxicants in arctic species is to be expected. Thus, interesting studies of the concentrations in the food chain and ultimately in the consuming natives of cesium-137 in the Canadian north (Bird, 1968) and cesium-137 and strontium-90 in Alaska (Hanson, 1968) have been published. I
294
DONALD J. LISK
Cesium-137 was particularly accumulated by lichens, passing then to foraging caribou and reindeer, and finally to natives consuming these animals. In this same food chain strontium-90 tended to become immobilized by storage in the bones of the animals. Lead-210 and polonium-210 have been found in human and other food chain organisms (Hill, 1965; Baratta et al., 1969). A clear, informative account of the whole subject of nuclear fallout has been prepared (Comar, 1966). Schroeder, again, has extensively examined other metals, such as germanium (Schroeder and Balassa, 1967), niobium (Schroeder and Balassa, 1965), tellurium (Schroeder et al., 1967a), titanium (Schroeder et al., 1963a), and zirconium (Schroeder and Balassa, 1966b) including analysis of many plants, soils, foods, and human tissues to hopefully develop occurrence patterns from which one might speculate regarding their essentiality. Although these elements are ubiquitous, further investigation will be required before their true role, if any, can be understood. A recent survey of several metals in institutional diets consistently revealed surprisingly high levels of antimony, but the health significance of this is unknown (Murthy et al., 1971). Antimony present in enamel or tin food containers may contaminate food (Murthy et al., 1971). Schroeder has studied other metals, including antimony, arsenic, cadmium, chromium, gallium, germanium, indium, lead, nickel, niobium, palladium, rhodium, scandium, selenium, tellurium, tin, titanium, vanadium, yttrium, and zirconium in feeding studies with rats and mice to determine their effects on growth, survival, life-span, teratogenic, carcinogenic, and other toxic effects (Kanisawa and Schroeder, 1967; Schroeder et al., 1968a,d, 1970b; Schroeder and Mitchener, 1971a,b,c). Schroeder has also reviewed the environmental effects of trace metals in several other notable publications (Schroeder, 196813, 1970a,c,e; Schroeder and Nason, 1971). Schroeder (1968a) studied the effects of several metals in the diets of rats on serum cholesterol levels, and evidence implicated chromium, nickel, and niobium as possibly exerting anticholesterogenic properties. Strontium and lithium may be instrumental, with fluorine, in aiding to reduce tooth decay (Losee and Adkins, 1969). The use of titanium in surgical implants and for bone prosthesis with no side effects is noteworthy. Other multimetal surveys have been conducted of soils and plants to relate their concentrations to cardiovascular mortality (Shacklette et al., 1970) and of heart tissue in relation to age and sex (Wester, 1965), but correlations were obscure. Cardiovascular death rates have been shown to be higher in areas with softer waters (Schroeder, 1966). Analysis of human tissues for many trace metals in Americans and foreigners revealed definite geographical correlations as regards the concentrations of nonessential metals (Tipton et al., 1965).
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
295
An exhaustive study (Anderson and Stewart, 1969, 1970) involving 24 elements in pheasants has been published. Interesting correlations were observed between the concentrations of these elements (including metals) in soil and grit in three (rated good, fair, and poor) pheasant ranges, the number of pheasants occupying the ranges, and tissue levels of these elements in the birds. Calcium, magnesium, potassium, chromium, cobalt, and molybdenum were suspected as influencing the distribution of pheasants. Numerous other possibly important, observations were made concerning the concentration of certain metals in particular tissues or bones, the comparative levels of these elements in corresponding human tissues and the possibly essentiality of certain previously unsuspected metals such as barium and strontium for pheasants. Owing to the scarcity of reliable information concerning the soil chemistry and plant absorption of other metals, it is also best to attempt to summarize this material in one section. Of the remaining metals the behavior of nickel and vanadium in soils has been studied to some extent. Nickel appears to be fixed and less available to plants at soil pH values above or below 6.5 to 7.0 (Crooke, 1956; Painter et al., 1953; Filipovic et al., 1961; Pratt et al., 1964). Nickel and vanadium are mobilized in soil following flooding and aeration and are more available in poorly drained soils (Mitchell et al., 1957; Kee and Bloomfield, 1962; Swaine and Mitchell, 1960; Kee and Bloomfield, 1961a). Oxides of the metals are solubilized by anaerobically fermenting plant material (Kee and Bloomfield, 1961b). Poor drainage can promote the solubility of other elements, such as barium (Mitchell et al., 1957), perhaps gallium (Kee and Bloomfield, 1961b), and tin (Swaine and Mitchell, 1960). Gallium has been indicated as essential to the growth of Aspergillus niger (Steinberg, 1938). Nickel interferes with plant absorption of iron, and sufficient iron reduces the phytotoxicity of nickel (Crooke, 1955; Crooke and Knight, 1955; Crooke et al., 1954; Nicholas and Thomas, 1954). In studies with alfalfa, barley, lettuce, and peas, beryllium was found to be concentrated in plant roots with only minor translocation, mostly to leaves (Romney and Childress, 1965) . Beryllium promoted phosphorus absorption by plants but decreased uptake of calcium and magnesium. The metal is strongly absorbed by soils and clays, displacing other adsorbed cations, such as barium, calcium, magnesium, and strontium. Beryllium has a strong tendency to form complexes and colloidal aggregates when the pH is raised to about 5.5. The toxicity of beryllium to plants is reduced as the pH of the soil is raised. Zirconium and niobium are also strongly fixed by soils and accumulate mainly in the roots of carrots (Vlamis and Pearson, 1950). Based on appreciable sorption of these metaIs by carrots
296
DONALD J. LISK
and yet strong soil fixation, these authors postulate a mechanism of absorption based on contact exchange (Jenny and Overstreet, 1939). The exchange of zirconium and niobium adsorbed on clays to a strong acid cation exchange resin (Sengupta, 1949) would support this theory. Vlamis and Pearson (1950) also suggest that exudation of organic acids by plant roots or bacteria may effect solution and absorption of these metals. Rare earth elements have been found in many plants (especially certain nut trees) and soils (Robinson et al., 1917, 1938; Robinson, 1943; Borneman-Starinkevitch et al., 1941 ). Reduced conditions in soils may favor their availability to plants by reduction of metals, such as cerium, which may be precipitated in the quadrivalent state to the soluble trivalent form. Complexation of rare earth ions by organic agents in soils is probably also important in promoting their availability (Robinson et al., 1958). The concentrations of many toxic metals in soils and plants have been published (Shacklette et al., 1971b; Swaine and Mitchell, 1960; Cannon, 1970; Mitchell, 1944). Studies on the absorption of toxic metals by plants and their phytotoxic or growth-stimulating effects have been reviewed (Chapman, 1966; H. C. Brewer, 1948, 1951, 1953, 1955; McKee and Wolf, 1971). Table 111 summarizes the known approximate concentrations of trace metals in soil parent materials, soils, plants, and humans. Bowen (1966) has noted that elements that are concentrated in blood plasma are essential, and many of those which are toxic or nonessential concentrate in red cells. Table IV lists typical concentrations of metals in major food classes. In preparing these data, an attempt was made to avoid data for metals in foods which had been stored in tin or possibly contaminated otherwise by metals in processing. Allaway (1968) published an excellent account of the environmental cycling of trace elements from soils through plants and to man and animals. As pointed out by him, plants may mature quite normally and yet contain insufficient cobalt, chromium, copper, manganese, selenium, and zinc for animal needs. Other plants may grow normally but contain levels of selenium, cadmium, molybdenum, or lead which are toxic to animals. Plants exclude antimony, arsenic, beryllium, chromium, mercury, titanium, vanadium, nickel, zirconium, and others by only minimal absorption of these metals from soil. Lead in soil is taken up more poorly by young than by mature plants. Plants may concentrate chromium in their roots rather than tops. Some metals such as zinc, although present in soybeans in sufficient amounts, may be fixed in a form, such as the phytate, that is nutritionally unavailable to animals. Chromium is more assimilable in yeast than in many other foods, perhaps because it is present as a readily absorbed porphyrin complex. Vanadium is stored largely in the bones of
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
297
TABLE I11 Trace Metal Content of Parent Materials, Soils, Plants, and Humansa
Metal
Igneous rocks (ppm)
Antimony 0.2 Arsenic 1.8 Barium 425 Beryllium 2.8 Bismuth 0.17 Cadmium 0.2 Cerium 60 Cesium 1 Chromium 100 Gallium 15 Gadolinium 5.4 Germanium 5.4 Lead 12.5 Lithium 20 Mercury 0.08 Nickel 75 Niobium 20 Palladium 0.01 Rubidium 90 Scandium 22 Selenium 0.05 Silver 0.07 Strontium 375 Tellurium 0.001 Tin 2 Thallium 0.45 Titanium 5700 Vanadium 135 Zirconium 165
Shales (PP~) 1.5 13 580 3 1 0.3 59 5 90 19 4.3 1.6 20 66 0.4 68 11
140 13 0.6 0.05 300
6 1.4 4600 130 160
Sandstones (ppm) 0.05
1 50
<1 0.3 0.03 92 0.5
35
I2 2.6 0.8 7 15 0.03 2 0.05
60 1 0.05 0.05 20
-
0.5 0.82 1500 20 220
Ratio Soil (plants/ ( P P ~ ) soil) 6 6 500 6
0.06 50
6 100 30
1 10 30 0.3 40 21
100 7 0.2 0.1 300 10.1 10 5 5000 100 300
0.01 0.03 0.11 0.03
5.3 0.5 0.03 0.01 0.04
-
0.25 2.3 1 0.05 0.05 0.11
Human whole blood (mg/l) 0.005 0.49 0.07 <0.0001 (0.01 0.007
0.003 0.03 0.0005
0.4
0.27
0.007 0.04 4.2-6.4'
-
-
0.2 0.001 1.0 1.5 0.09 0.02 0.09
2.7 0.08 0.27 0,024 0.04 1.1" 0.13
-
-
0.001 0.008 0.03
0.03 0.02 0.4c
Human total body burden (mg) 18 7.9 22 0.04
50
1.5 1.7
-
120 2.2
13 10 110
320
13 <1 320 8.2 <17 < 6
Generalized from Aldrich et al. (1951), Bowen (1966), Hilgeman et al. (1970), Schroeder and Balassa (1965, 1966b, 1967), Schroeder et al. (1967a), and Tipton (1972). Red blood cells. Serum.
animals. Other metals, including, copper may be more available to animah in cured hay rather than in fresh grass. Considering metals in soils and plants from a toxicological or nutritional standpoint, one must ask several questions. Is the element in the soil? Is
TABLE IFConcentrations (ppm) of Trace Metals in Major Food Classes0
Metal
Sea fooda
Arsenic Cadmium Chromium Germanium Lead Niobium Nickel Selenium Tellurium Tin Titanium Vanadium Zirconium
1.5-15.3 0.05-3.66 0-0.44 0.12-3.63 0.15-2.50 0-2.9 0.02-1 . 7 0.12-2.02 04.78 0.49-4.30 0.02-0.88 0-5.1 0-1.26
Cereals and grains 0-2.4 0.01-0.57 0-0.52
0-1.05 0-7.49 0.3-3.44 0-6.45 0.09-1.11 0-2.93 0-2. 63 0-0.99 0-6.03 0-6.59
Fruits
Vegetables
Meats
0-0.17 0.01-0.03 0-0.2
0-1 . 3 0.01-0.45 0-3.62 0-1.07 0-1.26
0-1.4 0.19-3.49 0.03-0.27 0.15-0.75 0-0.57 0.12-1.83 0-4.5 0.1!+4.17 0 0.39-3.44 0.01-0.76 0 0-3.81
0-0.85
0.03-0.38 0-1.07 0-0.34 0
0-0.38 0-0.18
0.05-1 . 5
0.05-2.53
0-2.59 0-0.57 0-0.77 0-9.07 0-0.42 0-6.0 0.31-4 .24
Dairy products 0-0.23 0.1-0.56
0.3-1 .51 0-0.79 0.27-2.44 0 - 0 . 03 0.06-1.46 0-2.39 0.19-0.96 0
-
0.8-5.64
Nuts
0.03-0.07
0-1 .93
-
0.49-0.94
-
0-1 .03 0-1.09
0-1.96 1.17-3.13
Generalized from Schroeder and Balassa (1961, 1965, 1966a,b, 1967) and Schroeder d al. (1962a,b, 1963a,b, 1964, 1967a,b, 1970a).
tl 0
s r
0
4
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
299
it available to plants? Where in the plant or animal does it accumulate? How much of the element accumulates? Is it available in an assimilable form? What are the possible effects of plant maturity, curing, or other food processing on its digestibility? It is obvious then that the mode and degree of uptake of metals by plants may be unfavorable from a nutritional standpoint but simultaneously beneficial from a toxicological one and vice versa. Allaway ( 1968) suggested several unique agronomic measures to control the movement of trace elements from soils to plants and animals. In summary, the soil-plant system can therefore be viewed as a dynamic living system of competing biological, chemical, and physical reactions. Organic matter, resistant to oxidation, is inexorably mineralized by soilforming organisms. Metal ions are concomitantly incorporated in organic matter. Cations adsorbed on active surfaces are in continuing equilibrium with those in the contacting soil solution. Plant roots, through the energy of respiration, pump metal ions, singly or chelated, out of this contacting solution. The insolubility of metal precipitates confronts the stability of the soluble complexed or chelated metals. Insoluble oxidized metal precipitates may be rendered soluble by submerged reducing conditions. Fixation of metals by adsorption, precipitation, and fixation cooperate to reduce leaching losses but may overzealously render them unavailable to plant roots. Flocculation is opposed by deflocculation, wetting by drying, expansion of clay lattices and release of ions by collapse and entrapment of ions, and so on. Ill.
Aquatic Systems
A.
WATER
1. Sources, Movement, and Associations of Metals in Water The possible hazards from toxic metals in water are being widely discussed (“Hazards from MetaIs,” 1970). The general sources of pollution of water which would include metal contamination have been accounted by LeGrand (1965). These include domestic and municipal sewage, industrial wastes, organic wastes from food and lumber processing, radioactive effluents, mineral wastes (metal processing, mining and ore extraction, oil production, chemical industries), cooling and irrigation water and intrusion by seawater. Other air-borne sources listed earlier for soil contamination would also apply. LeGrand (1965) has clearly summarized modes of movement of water which may spread contamination. Surface water may flow down poorly constructed, defective, or abandoned wells to pollute water below. Over-
300
DONALD J. LlSK
pumping of water by man can cause underlying salty water to move upward to mix with surface water. Overpumping may also cause the edgewise diffusion of polluted or seawater into undesired aquifers. The soil strata through which water Aows may purify it. Thus clay may remove contaminants whereas passage through coarse material or seams in rocks might have little effect. Mixing of the upper (epilimnion) layers of lake water in cool autumn months with the lower (hypolimnion) layers is another natural phenomenon resulting in a redistribution of surface water contaminants. Pumping wastes into deep abandoned wells and underground nuclear explosions or earthquakes could also conceivably result in the spread of water contamination. The toxicological impiications of metals in water are complex because the associations of them are many. Metals may exist in water as dissolved ions, in organic complexes, adsorbed on clay particles, as suspended precipitates, contained in decaying organisms and other forms. It is evident, therefore, that attempts to interpret water analysis data are often folly, when customarily the suspended solids are filtered out and discarded prior to determining metal concentration. The ion species of a given metal may depend on the proximity of sampling to the source of pollution. Metal ions may chelate or complex with other pollutants. Thus it is believed that certain metals would react with the strong nitrilotriacetate chelating group found in NTA detergents. A useful and comprehensive compilation of literature references on the subject of water pollution and its control has been published (Ingram and Mackenthun, 1963). 2. Metals in Water
a. Mercury. Mercury in water and fish from many sources is presently of greatest concern and has been widely publicized. Industrial pollution such as that from chlor-alkali industries has been a direct source of water pollution. Another direct source, the use of mercury-containing algicides, has been recently outlawed (Anonymous, 1971). Geochemical sources of mercury pollution are widespread, and relatively high water concentrations have been found in acidic hot springs in Japan (Ohta and Terai, 1971). In seawater, mercury may exist as the divalcnt mercuric tetrachloride anion [HgCl+I-2 (“International Conference on Environmental Mercury Contamination,” 1970). Mercury is particularly insidious for several reasons. It is very toxic, it is volatile, it is ubiquitously distributed, and it undergoes methylation and therefore cycling. Thus mercury is oxidized by microorganisms in aquatic bottom muds to mercuric ion and finally to methylmercury and dimethylmercury (Jensen and Jernelov, 1969). The former metabolite is water soluble and is absorbed by fish and stored by them intact probably combined with sulfur-containing compounds. Dimethylmercury is water insolublc and volatile and therefore vaporizes into the
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
301
atmosphere (“International Conference on Environmental Mercury Contamination,” 1970). Under the influence of light or acidic conditions it is converted to methylmercury or mercury metal, respectively, which then again settles or rains out on the soil or water surface to complete the cycle (“International Conference on Environmental Mercury Contamination,” 1970). Its presence in prehistoric animals and plants and fossil fuels attests to its long history as an environmental contaminant. Precipitation of mercury as extremely insoluble mercuric sulfide in bottom muds would greatly limit the availability of mercury for methylation (Jernelov, 1969). If the sulfide is oxidized to soluble sulfate, methylation of mercury could then again proceed (Jernelov, 1969). The methylation of mercury may be mediated by a methyl transferase enzyme perhaps having a cofactor of cobalamine (“International Conference on Environmental Mercury Contamination,” 1970). The methylation of mercury in vitro in the presence of methylcobalamine has been shown (Bertilsson and Neujahr, 1971; Imura et al., 1971). The transfer of methyl from Co3+to Hg2+has been shown to occur by the action of an anaerobic bacterium isolated from canal mud in Holland (Wood et al., 1968). The kinetics and mechanism of this alkyl group transfer has been studied (Schranzer et al., 1971). The mechanism has been studied further by Landner (1971) showing that methylation of mercury by the methanogenic bacterium Neurospora crassa was stimulated by the presence of cysteine and homocysteine. Biological methylation of mercury is possible by many metabolic pathways (Wood, 1971). At this point one might logically wonder if methylation of metals is a common metabolic reaction. It is believed that this reaction would occur only with metals of sufficient electronegativity. Thus the order of electronegativity for the following elements is: S
> Se > Te > As > Hg > Pb = Sn > Cd > Zn
Although methyl derivatives can be prepared synthetically for any of these, the higher activation energy and ease of hydrolysis of those near the right would probably prevent formation and existence of such metabolites in living cells (Jernelov, 1970). The methylation of arsenic by methylcobalamine has been described (McBride and Wolfe, 1971 ). The methylation of sulfur, selenium, tellurium, and arsenic by microorganisms in the environment has been reviewed (Challenger, 1945). b. Other Metals. Lead may become a water pollutant from industrial pollution, gasoline exhaust from motor boats (English et al., 1963) and as a pollutant in rain (Lazrus et al., 1970; Ter Haar et al., 1967) and snow (Tatsumoto and Patterson, 1963). Lead aerosols in marine atmospheres have been analyzed (Chow et al., 1969; Chow and Johnstone, 1965). Contamination of water by chromium (Perlmutter et al., 1963;
302
DONALD J. LlSK
Davids and Lieber, 195 I ) and cadmium (Lieber et al., 1964) has been reported. Arsenic found in detergents and enzyme soaks at levels of 10-70 ppm (Angino et al., 1970) was recently reported. The use of aquatic herbicides containing arsenic or copper sulfate for algal control are sources of metal entry. Calcium and sodium chlorides used, respectively, for settling dust and melting ice on roads may be a serious source of water contamination and sodium chloride is of concern if the resulting water is ingested by persons on salt-free diets (“Of Salts and Safety,” 1971). Seawater reaching fresh waters may contribute traces of virtually all metals. Estimates of the total dissolved metal reserves in ocean water have been made (Spilhaus, 1966). An excellent analytical survey of metals in waters throughout the United States has been compiled (Kopp and Kroner, 1967) and another (“Heavy Metal Survey,” 1970) is underway.
B.
AQUATIC
ORGANISMS
1 . Modes of Absorption and Toxicity of Metals Owing to the importance of fish and aquatic plants such as algae and plankton as a potential source of human food and also as an obstacle in water to recreation, transportation, and sewage treatment, much study has been directed toward their culture and control. Factors affecting the absorption by and mode of toxic action of metals upon aquatic organisms have been clearly summarized (“Water Quality Criteria,” 1968). In brief all metals or elements are taken up against a concentration gradient. The affinity of metals for living matter has been studied and may depend on the magnitude of their valence, atomic weight, and other factors. Thus the order of affinity of divalent metals for brown algae is given (Water Quality Criteria,” 1968) as: Pb
> Mn > Zn > Cu, Cd > Co > Xi
and for plankton as: Zn
> Pb > Cu > 5fn > Co > Xi > Cd
For other metals the order of affinity for brown algae is: Fe
> La > Cr > Ga > Li > .41 > Si
and for plankton is: Fe
> Al > TI > Cr, Si > Gx
Ion uptake may occur by an active metabolic process or by simple diffusion. Most primitive animals, unicellular protozoa, and algae absorb by diffusion. The cell vacuoles of many marine species are able to open inter-
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
303
mittently to exude fluid and thus control cell volume and osmotic pressure. Active ion uptake occurs only in freely respiring cells. It may be limited therefore by factors effecting respiration. A popularly held concept of the mode of active ion uptake is the “carrier hypothesis,” which postulates that ions are transported across membranes as chelates with metabolically produced organic molecules. Ion diffusion is important in replenishing the concentration of ions in close proximity to absorbing membranes. Similarly, the internal bulk diffusion of absorbed ions away from the membrane is rate determining. Metals have been arranged according to their toxicity to eels (Doudoroff and Katz, 1953) as: Hg
> Cu = Zn = Cd > Sn = A1 = Ni
= Fe3+
> Fez+ > Ba > Mn > K
=
Ca = Mg
> Na
to cyprinodont fish (Orizias Zatipes) (Doudoroff and Katz, 1953) as: Hg = Cu
> Au = Pd > Th > Pt > Cd > Ce > Ba > K =Co>Li=Mn>Ca=Sr>Mg=Na
and to Daphniu magna (Warnich and Bell, 1969) as: Cu
> Hg = Ag > Cd > Cr > Zn > Pb = Co > Ni > Ba
Concerning the mode of toxic action of metals the mechanism believed most important is enzyme poisoning. Electronegative metals such as copper and mercury have a strong affinity for reactive enzyme sites such as amino, imino, and sulfhydryl groups. They are also chelated by organic molecules such as prior to penetrating cell membranes. Thus correlations have been attempted between metal toxicity and properties such as their electronegativities, the insolubility of their sulfides and the stability of their chelates. As examples, the order or electronegativities is given (“Water Quality Criteria,” 1968) as: Hg
> Cu > Sn > Pb > Ni > Co > Cd > Fe > Zn > Mn > Mg > Ca > Sr > Ba
The insolubility of the sulfides is: Hg
> Cu > Pb > Cd > Co > Ni > Zn > Fe > Mn > Sn > Mg > Ca
The order of chelate stability is: Hg
> Cu > Ni > Pb > Co > Zn > Cd > Fe > Mn > Mg > Ca
When comparing the order of toxicity of metals by the above properties, variations may be expected since more than one enzyme is usually being poisoned and many more metal atoms may react with the enzyme than simply the specific number required to block the reactive sites (“Water Quality Criteria,” 1968). In this regard mercury, lead, copper, beryllium, cadmium, and silver have been found to inhibit alkaline phosphatase, catalase, xanthine oxidase, and ribonuclease in fish (Jackim et al., 1970).
304
DONALD J. LISK
Other modes of toxic action of metals (“Water Quality Criteria,” 1968) are : 1. Action as antimetabolites, such as arsenate substituting for phosphate. Permanganate, antimonate, selenate, tellurate, tungstate, and beryllium may also act in a similar manner. 2 . Formation of precipitates or chelates with essential metabolites. As examples, aluminum, beryllium, scandium, titanium, yttrium, and zirconium may react with phosphate, barium with sulfate, or iron with ATP. 3. Catalyzing the degradation of essential metabolites, such as the decomposition of ATP in the presence of lanthanum and other lanthanide cations. 4. Reaction with cell membranes and altering their permeability. Thus gold, cadmium, copper, mercury, lead, and uranium may affect the transport of sodium, potassium, chlorine, or organic molecules across membranes or can even rupture thcm. 5. Replacing structurally or electrochemically important elements in cells and then not functioning. Lithium substituting for sodium, cesium for potassium, and strontium for calcium are examples. An example of toxicity by affecting cell membranes is that of heavy metals upon fish. It was first observed by Carpenter (1927, 1930), and is now generally accepted, that, in sufficient concentration, certain heavy metals, such as lead, exert their toxic effect on fish by precipitating or coagulating the normal mucus secreted by the gills and skin. The resultant surface plugging, and thus interference with respiration, secretion of waste products, and salt balance may then be fatal. Carpenter (1927) detected no lead in the bodies of fish killed in solutions of lead nitrate and then externally extracted with acetic acid to remove mucus. The acetic acid washing, however, contained most of the lead to which the fish were initially exposed. A similar mode of action was observed with zinc, copper, cadmium, and mercury. It should be noted, however, that at lower concentrations metals such as zinc (Lloyd, 1960; Mount, 1964) and cadmium (Mount and Stephen, 1967) may exert their toxicity by direct damage to the gill epithelium. Aside from the nature and concentration of metals, their toxicity to fish are affected by many other factors. These include: ( 1 ) valence, ( 2 ) the associated anion, ( 3 ) other cations, (4) pH, (5) purity of test materials, ( 6 ) form of metal in water, ( 7 ) time of exposure, ( 8 ) volume of water, ( 9 ) temperature, ( 10) dissolved oxygen, and ( 11 ) nature and condition of fish. Oshima (1931 ) found hexavalent chromium to be much less toxic to eels than the trivalent metal. Although ferric ion may be more toxic than ferrous (Doudoroff and Katz, 1953), it is also more subject to precipita-
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
305
tion as the insoluble hydroxide (Jones, 1964). At the same molar concentrations, Jones (1935) found the sulfates of heavy metals much less toxic than the chlorides or nitrates. The effect of other metals in solution is important. Thus hardness in water has been shown to decrease the toxicity of zinc to fish (Jones, 1938). Ion antagonism has been indicated in the reduced toxicity of various metals in the presence of calcium (Ellis, 1937; Kruger, 1928). Ion synergism has also been noted (Bandt, 1946). Whereas the toxic effect of zinc and cadmium or nickel and cobalt as the sulfates were additive, the combined toxicity of nickel and zinc, copper and zi.nc, or copper and cadmium was much greater than additive. The effect of pH will manifest itself insofar as it renders metals more soluble or insoluble. The presence of toxic metal impurities in the metal being investigated, such as zinc in manganese (Oshima, 1931), can lead to sources of error. Several of the above factors have been correlated with the rate of absorption of trace metals by mollusks (Pringle et ul., 1968). As pointed out earlier, metals may exist in water in several forms. Whereas low concentrations of single heavy metal ions may simply coagulate external mucus which subsequently sloughs off, other more complex ions such as methylmercury are cumulatively stored by fish (Johnels and Westermark, 1969; Bache et al., 1971). Although metals may be present in various colloidal and adsorbed forms, they may still exert their toxicity by redissolving in the higher carbon dioxide concentrations adjacent to gill membranes of fish. Obviously a decrease in the time of constant exposure or intermittent exposure would reduce metal toxicity. Raising water temperature or volume and lowering dissolved oxygen similarly enhances metal toxicity. Finally the nature and condition of the fish are important factors. The species, age, and general condition of fish will affect their foraging, metabolic, and excretory capabilities. The prior exposure of fish to a toxic metal may serve to acclimatize them to subsequent exposures to the same metal. This has been noted for zinc (Goodman, 1951 ;Affleck, 1952) and copper (Paul, 1952). Fish also exhibit avoidance reactions to specific metals, and their ability to detect and avoid them has been shown to be in the order: lead, mercury, zinc, and copper (Jones, 1964). Many of the above factors influencing metal toxicity have been comprehensively reviewed (Doudoroff and Katz, 1953). The reader is referred to the literature (“Water Quality Criteria,” 1968) for a general discussion of many aspects of the pollution of the aquatic system by metals and other contaminants. 2. Metals in Aquatic Organisms a. General Considerations. Since nutritionally and recreationally fish constitute a most important segment of the aquatic ecosystem, toxic metals
306
DONALD J. LISK
in fish are obviously of great concern. Surprisingly few extensive surveys of metals in fish have been conducted. Newer sensitive instrumental methods of analysis are beginning to be applied to this problem. Activation analysis has been recently used to analyze determinable levels of 25 metals in Great Lakes fish (Lucas and Edgington, 1970). In a recent paper Bryan (1971) has reviewed the physiological behavior of heavy metals in fish. Heavy metals such as zinc may be absorbed across the entire body surface of fish as well as the gills. They may attach to proteins during this passage and may be tightly protein-bound in the blood. Absorption from the digestivc tract also is important since consumed marine plants contain virtually all trace metals in amounts that reflect metal concentrations in the contacting water (Young and Langille, 1958). (In this regard current experimentation with raising plankton ultimately for foraging fish in water to which sewage sludge is added could result in high residues of metals in fish.) Excretion of metals may occur in urine or feces or by diffusion back across surface membranes. Major storage of metals is in renal organs. Lucas and Edgington (1970) have also shown major storage of heavy metals in fish liver. It is possible that proteins such as metallothionein (Margoshes and Vallee, 1957) may be involved in heavy metal binding during storage in fish organs. Other toxic effects of metals in fish include prolongation of egg hatching (Shaw and Brown, 1971), decrease in extractable muscle protein (Castell et al., 1970), and other morphological and behavioral effects (Bryan, 1971 ) . 6. Mercury. Much publicity has appeared recently about relatively high levels of mercury in fish (“Mercury Stirs More Pollution Concern,” 1970; “Ontario Halts Fishing in Mercury-tinged Lakes,” 1970; “And Now, Mercury,” 1970; “Mercury-Major New Environmental Problem,” 1970; “Mercury Menace Prompts Firm to Offer Test Data,” 1970; Klein and Goldberg, 1970). Of greatest concern is the finding (Westoo, 1966) that it is present largely as methylmercury compounds. Fish do not typically exhibit toxic effects during methylmercury storage, apparently because brain accumulation is not pronounced in fish. Total mercury in fish concentrates progressively with age, and the percentage of the total present as methylmercury is much higher in older fish (Johnels and Westermark, 1969; Bache et al., 1971). Mercury is methylated in the presence of decaying fish (Jensen and Jernelov, 1969) but not by certain algae, even though these algae contain the cobalamine methyl transferase enzyme system (“International Conference on Environmental Mercury Contamination,” 1970). Mercuric ion is “absorbed” by goldfish with extreme rapidity but appears to be initially associated with the external mucus of the fish (McKone et al., 1971 ). Organomercurial fungicides reduce photosynthesis by plankton (Harriss et al., 1970). The subject of mercury in aquatic sys-
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
307
tems has been reviewed (Berglund et al., 1971; Fimreite, 1970; Ackefors, 1971; Westoo and Rydalv, 1969, 1971). c. Lead. A survey of about 400 fish of various species from 50 waters in New York State was conducted (Pakkala et al., 1972b). Concentrations of total lead ranged from about 0.3 to 1.5 ppm. No correlation was noted between lead concentration and size, species, age, or sex of fish but fish from certain waters showed predominantly higher lead levels. d . Cadmium. Cadmium was similarly surveyed in the above New York fish (Lovett et al., 1972). Fish from waters in the central and western regions of the state usually contained less than 20 ppb, but those taken from waters in the Adirondack region were quite consistently higher, ranging up to 150 ppb. The predominance of lead, copper, and zinc mines and deposits (with which cadmium is typically associated) in this area may have contributed ta these higher concentrations in fish. Certain fish such as whitefish and goldfish appeared to accumulate cadmium, perhaps by their particular foraging habits. Cadmium concentration in lake trout was not related to the age of the fish. e. Arsenic. Arsenic was also analyzed in the above collected fish from New York waters and was found to range from about 0.03 to 0.5 ppm (Pakkala et al., 1972a). Larger fish generally contained higher arsenic concentrations. Fish from notably polluted waters such as the Hudson and St. Lawrence rivers were also higher. Arsenic was not found to be cumulative in lake trout of increasing age. Kearney and Woolson (1971a) found no biological magnification in aquatic food chains in studies with radiolabeled organic arsenicals. Arsenic is higher as a residue in shellfish (Vallee et al., 1960). Arsenic may be present in shrimp at high concentrations and as trimethylarsine (Goldwater, 1971). The form of arsenic stored in shrimp was found to be rapidly excreted in the urine of rats consuming the shrimp as compared to storage of arsenic fed to them as As20, (Coulson et al., 1935 ) . Cacodylic acid (dimethylarsinic acid), a widely used weed and brush killer probably is reduced in streams to a volatile arsine, but the latter compound may again be oxidized to cacodyl oxide and cacodylic acid to effect a complete cycling (Kearney and Woolson, 1971b). f. Selenium. Selenium tends to reach relatively high levels in fish and fish products sometimes approaching 2 ppm (Arthur, 1971). In the New York fish survey selenium ranged from 0.1 to 1 ppm. Concentrations were not related to size or age of fish but certain species such as whitefish were quite consistently higher as well as fish from specific waters (Gutenmann et ul., 1972). g. Chromium. The accumulation and excretion of chromium by rainbow trout has been studied (Knoll and Fromm, 1960). Absorption of hex-
DONALD J. LlSK
308
avalent chromium is probably by simple diffusion across the gill membrane. It accumulates in the stomach, kidney, and spleen. The degree of absorption of hexavalent chromium is directly related to the water concentration and length of exposure (Fromm and Stokes, 1962). The metal does not interfere with gill respiration (Fromm and Schiffman, 1958) but can induce various blood changes (Schiffman and Fromm, 1959). h. Other Metals. Table V summarizes typical levels of metals found in fresh and salt water as well as aquatic organisms. Concentration ranges in fresh water fish were based on analysis of 50 to 500 fish from about 50 waters in New York collected in 1969. Analyses were made by spark
T.4BLE V Trare Metal Content of River and Seawater, Marine Algae and Fisha ~
River water
Fresh water fish
Element
(PPt))
(PP"')
Antimony Arsenic Barium Berylliuni Bismuth Cadniiurn Cerium Cesium Chromiiiiri Galliuni Germanium Lead Lithium Merr u ry Nickel Niobiurn Rubidium Sca nd iuin Selenium Silver Strontium Thalliuni Tin Titanium Vanadiuni Zirroniurii
0.13 0 4 51 1
0 03-0 5 0 05-0 25
80
-
0 20 0 18 <1
-
-
-
0 O?-0 15
-
-_ --
5
0 5-2
1 1 0 08 10
0 1-1
-
-
<20 80 0 02 0 04 8 6 1 2 6
__
0 03-3 8
-
0 1-1 0 01-0 1
0 05-19 5
0 1-a 7
-
~~
-
0 33 3 30 0 0006 0 017 0 11 0 4 0 5 0 05 0 03 0 07 0 03 180 0 03 5 4 0 01 120 < O 004 0 09 0 5 8100 0 01 3 1 2 0 02 ~~
a
Marine plants (dry wt.) (PP4
Seawater (PPb)
30 93
-
0 .4
0.07 2.4 1
-
6.7 5.4 0.03 17
7.4
Generalized from Bowen (1966) and Tong and Lisk (1974).
0.2 0.3
-
0.04? 3
-
0.2 0.15? 0.3? 0.5
0 3? 1
-
-
-
0.84 0.27 570
0.5-4
17 34 3.5 20
3" 0.2 0.14
-
~~
Marine fish (dry wt.) (wm)
11
-
-
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
309
source mass spectrometry, colorimetric, fluorescence, and atomic absorption methods. For further information on the toxicity, accumulation, and effects of metals in aquatic systems one can refer to the excellent and comprehensive compilation prepared by McKee and Wolf ( 1971).
IV.
Continuing Research
Undoubtedly only pollution abatement and judicious recycling of used products will provide the final solution to our present environmental dilemma. Efforts will continue, however, to learn the location, association, and magnitude of pollutants whether the sources of them are human or geochemical. Trace metal research is difficult. The chemistry of metals in biological systems is complex and only partly unraveled. Analytical method development, particularly for metabolites, holds the key to success in many instances. Many difficulties remain, such as adsorption or vaporization losses during ashing and analysis, microbiological contamination of solutions, and possible inadvertent removal of hitherto unknown essential elements during purification of animal diets. Much progress has been made in the development of sensitive selective analytical methods and instrumentation. In addition to past methods, such as emission spectroscopy and radioisotopic techniques, many newer methods are being developed. Neutron activation analysis and atomic absorption spectrophotometry have been useful additions for analysis of metals. Most recently flameless (Hatch and Ott, 1968) and hot tube atomic absorption (Woodriff and Stone, 1968; Donega and Burgess, 1970; Kahn, 1970), spark source mass spectrometry (Tong et al., 1969; Evans and Morrison, 1968; Yurachek et al., 1969), metal chelation gas chromatography (Karayannis and Corwin, 1970; Pommier and Guiochon, 1970; Hansen et al., 1971), microwave-powered emission gas chromatography (Bache and Lisk, 1971), and anodic stripping voltammetry (Bender et al., 1970; Allen et al., 1970) have been added. Toxicological or nutritional feeding experiments involving metals and laboratory animals may now be conducted much more precisely than in the past. The use of “clean room” techniques and diets containing synthesized amino acids, recrystallized vitamins, purified fats, and highly purified minerals drastically reduce background contamination and greatly facilitate interpretation of experimental results, especially concerning metal essentiality. Finally much research is underway to develop and improve antidotal schemes for humans exposed to toxic metals. Many treatments including ascorbic acid (Samitz et al., 1968; Samitz and Katz, 1965), glutamic acid
310
DONALD J . LlSK
(Winter ef al., 1968), and estrogen (Gunn et al., 1965) are available. Use of the aurintricarboxylic acid lake reaction with beryllium (Schubert and Rosenthal. 1959), EDTA (Spencer and Rosoff, 1965 ) and various thiol compounds (Gunn er al., 1966) for heavy metal intoxication remain as effective measures. Recent research appears most promising such as the use of spironolactone ( a very nontoxic, hormonally inactive steroid containing a thioacetate group) which protects rat kidneys from the calcification and necrosis caused by mercuric chloride (Selye, 1970) and the use of sulfhydryl- or thiol-containing resins for removing methylmercury compounds from animals (“Cholesterol Flush,” 1971 ; “Chemicals Affect Brain in Diverse Ways,” 1971 ). Other sulfur compounds that may be effectively used for mercury or methylmercury poisonings include N-acetyl-DL-penicillamine (Kark et a / . , 1971 ) and possibly thioglycolic acid (Takahashi and Hirayama, 1971). The use of metal binding agents in medicine has been reviewed (Seven and Johnson, 1960; Schroeder, 1956). Other investigations dealing with the counteraction of one metal on the toxicity of another, such as zinc to alleviate cadmium toxicity and selenium to diminish toxic effects of mercury, may lead to other effective therapeutic measures (Parizek et al., 1968, 1969a,c,d; Levander and Argrett, 1969; Parizek and Ostadalova, 1967; McConnell and Carpenter, 1971 ). In summary, a further understanding of the true role and action mechanisms of trace metals throughout the environment could prove immeasurably beneficial to mankind. These benefits could include the efficaceous use of sludge, fertilizers, and lime for further promotion of crop quality and yield by improving the mineral nutrition of plants and obviating phytotoxicity as well as superior methods of land reclamation and reuse. In aquatic systems proper management of metals in solution could improve the prospects for successful hydroponics, fish farming, weed and algal control, perhaps without herbicides and averting and correcting water pollution. Finally the nutritional and toxicological outlook for animals and man would brighten with final confirmation of those elements which are truly essential, means of obviating those which are toxic, and closely controlling the concentrations of those which can be both essential and lethal.
ACKNOWLEDGMENTS The author is indebted to W. H. Allaway, J. H. Baker, J. Kubota, M. Peech, F. N. Ponnamperuma, 0. Sardi, and R. G . Young for their many helpful suggestions and for providing source material used in preparing the manuscript; to H. A. Schroeder for kindly supplying published reprints of his superb and extensive research; and to A. Wykes for providing the exhaustive computerized literature scan. Finally, without the patience and perseverance of Nancy R. Goodman in
TRACE METALS IN SOILS, PLANTS, AND ANIMALS
311
literature searching, assembling, and typing of the manuscript and proofreading the finished copy, completion of the paper would not have been possible.
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D. S Frear. R H. Hodgson. R H Shimabukuro. and G G Still Agricultural Research Service.
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I. Introduction .................................................. I1 Benzoic Acids ................................................ A Introduction ........................... B . Benzoic Acid .......................... C . Chlorinated Benzoic Acids .................................. D Chloramben .............................................. E. Dicamba ................................................. F Dichlobenil and Related Compounds .......................... G Substituted Benzamides .................................... H Ioxynil and Bromoxynil .................................... I Discussion and Summary . . . . . . . . . . . ..... I11 Dinitroanilines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . ............ B. Metabolism ............................................... C Selectivity ................................................ D. Interactions .............................................. E. Discussion and Summary .................................... IV Triazines ..................................................... A Introduction .............................................. B. Metabolic Pathways ........................................ C . Enzyme Reactions ......................... ............ D . Selectivity ................................................ E Discussion and Summary ................................... V Heterocyclics ................................................. A Picloram ................................................. B Pyrazon .................................................. VI Diphenylethers ................................................ A . Introduction .............................................. B Metabolism ............................................... C Selectivity ................................................ D. Discussion ................................................ VII Substituted Ureas .............................................. A Introduction .............................................. B Metabolism ............................................... C Enzymes ................................................. D Selectivity ................................................ E Interactions ............................................... F Summary .................................................
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.................... .............................................. B. Propachlor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Propanil . .. .. ............ X. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .................... ................ .......
I.
3 63 363 367 368 368 368 369 371 372
Introduction
The past few years have witnessed an increasing number of reports and reviews on the behavior of pesticides in plants (Kearney and Kaufman, 1969; Casida and Lykken, 1969; Menzie, 1969; Sijpesteijn, 1969; Plimmer, 1970). This increased research activity reflects a growing concern and interest in the fate of pesticides in our envirionment. A complete survey of the rapid progress in this broad subject area is not feasible. Instead, the authors have chosen to limit the present review to a discussion of the most recent reports on the metabolism and fate of herbicides in higher plants. For convenience, each class of herbicide included in this review is discussed separately. Particular attention has been given to (1) the role of metabolism in herbicide selectivity, ( 2 ) the need for additional information on the identification and significance of many soluble and insoluble polar metabolites and residues in plants, and ( 3 ) the need for basic biochemical studies on plant enzyme systems responsible for pesticide metabolism. Except for a relatively few references required to provide background information and to place current information in proper context, most of the cited references are selected from the literature of the past three years.
II.
A.
Benroic Acids
INTRODUCTION
Benzoic acid and its substituted analogs constitute a heterogeneous group of compounds which are used principally as phytocidal agents (Plimmer, 1970). The substituted benzoic acid, chloramben,l and its analogs recently had sales exceeding $25 million per year for use as herbicides Chemical designations of pesticides mentioned in the text appear in Table I.
BEHAVIOR OF HERBICIDES IN PLANTS
329
(Neumeyer et al., 1969). Methoxy and cyano derivatives, such as dicamba and dichlobenil, represent other examples of benzoic acid derivatives which find important herbicidal use. In addition, amide derivatives of benzoic acid recently have shown promise as herbicides (Beynon and Wright, 1968; Viste et al., 1970). The chemistry and uses of most of the diverse compounds discussed in this section are outlined elsewhere (Melnikov, 1971; Plimmer, 1970; Weed Science Society of America, 1970). Several comprehensive reviews have appeared treating various aspects of the metabolism, fate, and physiological effects of substituted benzoic acids in plants and soils (Hilton et al., 1963; Swanson, 1965; Moreland, 1967; Casida and Lykken, 1969; Menzie, 1969; Swanson, 1969; Frear and Shimabukuro, 1971; Helling et al., 1971). Several of the compounds have been shown to inhibit the gibberellin-induced synthesis of a-amylase in seeds and seed parts (Ashton et al., 1968; Devlin and Cunningham, 1970; Jones and Foy, 1971), and to influence the carboxylic acid cycle substrate oxidation in mitochondria (Foy and Penner, 1965). The influence of these and other classes of herbicides on chloroplast electron transport and ATP levels has been reviewed (Moreland, 1969; Gruenhagen and Moreland, 1971). In the present discussion, emphasis is placed on selected aspects and recent developments in the metabolism of representative derivatives of benzoic acid. B.
BENZOICACID
The degradation of benzoic acid in plants has not been followed in great detail; however, the formation of products resulting from ring hydroxylation and complexing with glucose, amino acids, and other naturally occurring plant products has been noted and reviewed (Swanson, 1969). Ring cleavage has not been reported.
C. CHLORINATED BENZOICACIDS The principal chlorinated benzoic acid which is used as an herbicide is 2,3,6-TBA. Its development, use pattern and movement, morphogenic effects, and degradation in plants have been described (Swanson, 1969). A report by Mason (1959) indicated that the principal route of metabolism involved the formation of protein complexes. Numerous studies have recorded the persistence and apparent lack of appreciable 2,3,6-TBA degradation in soil (Swanson, 1969). Reports of dechlorination, without the identification of metabolites, have been discussed by Swanson (1969) and Menzie (1969). The cometabolism of 2,3,6-TBA by a species of Brevibacterium has also been reported (Horvath, 1971 ) . This bacterium
330
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T.IBLE I Clieuiiral Designations of Pesticides Mentioned in Test Common o r tradc iiaiiie .tlachlor Atrazirie Barlbari I3en e fiI i Rroniclvynil Carbaryl
Chloranihen Chlorhrotiiuron Clilorferi\ iiiplios Chiorprophain Chlort hiatiiid 2.1-D Dicsiiitia Dicliloheiiil I)ichloriilate Dicryl IXuroii D yf oiia te T)ipropa liii r)isuifotorl Fensulfottiion Fluorortifen CS 13528 Tovynil
Tpazinr Linuron Malathion Monuron Nitralin Kit rofeii Phenniecliphn 111 Phorate Picloraiii Pronietryne Pronarnide Propachior I'ropanil Propha in Prynachlor Pyrazon Simazine Swep Taridev 2,S,G-TBA Turhutol 2,3,5-TIBh Trifluraliii I S B 3581
Cheiiiical iiaiiie 2-Cliloro-.?',6'-d~ethyI-S-(riiethov~1iiethyl )arctaiiilide ?-Chloro-1-(ethylaiiiino)-G-(isopropylariiiiio)-.~-triazii~e &Chloro--?-butynyl m-clilorocarbnnilate S-Butyl-S-ettiyl-ol,a.a-triAuoro-.?,8-dinit ro-p-toluiche S,~-Dibroiiio--l-liydro.\-yl~ettzonit rile I-Kaphthyl iiiethylcarbaiiintc Y-.~ri~irio-~,,j-dicliIoroberizoic acid ~-(~-~~ro~i~o-~-chloroph~riyl)-~-metho~y-l-iiietliylurea :!-Cliloro-l-(~,~-tlielilornplien~l)~inyl rlietliyl phosphate Isopropyl m-eliloroearl,anilate 3,G-Dichlorot~iio~~enzamide .L,1-Dicliloropheiio\gar.etic acid
3,fi-DichIolo-o-a~iisicacid 2,6-Dirliloro~~en7oriitri~e
3,S-DicIilorol)enzyl iiietliylcnrl~aiiiate ~',~'-Dichloro-~-~iietliylacrylanilide Y-(~,~-Dichloroplieiiyl)-~,l-dinretliylurea 0-Ethyl-S-phenyl-ettiyl phosphoiiodithioate 2 ,G-Dinitro-.~',.~-di-n-propyI-p-toli~idine O,O-Ethyl 8-2(et Iiylt Iiio)rt hyl phosphoroditfiioa te O,O-IXethyI O-I'-(iiiethylsulfiriyl~phenyl phospliorothioate p-Xitrophenyl ol,ol,a-trifluoro-:!-nitl.o-p-tolyl ether -?-Cliloro-4-dhylaniirio-6-ferf-butylani~no-~--tr~az~1ie 4-IIydroxy-Y,5-diiodobenzonitrilr 2-Chloro-4-(diet hyla 1iiino)-6-(isoprop~Iairii1io)-s-tr~azine Y-(Y,1-DicIilorophen).l)-~-111et hovy-1-iiiethylurea O,O-Diniet hyl S-his(carboethovy)ethyl phosphorodithioate Y-(y-CtilorophcnyI)-I,l-dinieth~lurea ~-(Jlet)iylsulfonyI)--L,G-diiiitro-~~',.~'-dipropylaniline 2 ,1-Dichlorophe1iyl p-uitroplieiiyl ether Methyl m-hydroxyrnrl)ariilate m-nietIiyIcarl)nnilate C~,U-I~icthyl-S-(ethyltliioniet hyl) ~iliospliorodithioate S-.~niino-S,,j,G-trieliloropicolinicacid -?,l-Bis(isopropyIanii1io)-6-1~ietliyltliio-.~-triazi1ie .Y-(1,l-Diriiet tiyl-.?-propynyl)-S,j-dichlornl~ctizaiiiide 2-Chloro-S-isopropylacetanilide 3',1'-Dichloropropionanilide Isopropyl ca rba nila t e ?-Chloro-S-( I-met hyl-'2-propynyl)aeetanilide j - . ~ I n i n o - ~ - c l i l o r o - ~ - p l i e i(2lf)-pyridaziiione iyl-~ 2-ChIc.r0-$,6-bis(cthylaniino)-s-triazine Methyl Y,S-dicIilorocarba~~ilate m-(Y,Y-~iinethylureido)phe~iyl tert-\,utylc,?rhaiiiate 2,S,G-Tricliloro henzoic acid 2,G-Di-terf-hutyl-p-tolyl methylcarbamate .?,3,5-Triiodobenzoie acid a,a,a-Trifluoro-2,6-dinitro-.~,.~-~lipropyl-~-tnluidine 53,.~3-Dietliyl-2,,l-riinit io-G-trifluoroiiiethyI-l,3-phenylenediamine
BEHAVIOR OF HERBICIDES IN PLANTS
33 1
was incapable of utilizing 2,3,6-TBA as a sole carbon source, but cells grown in the presence of benzoic acid released 1 mole of chloride per mole of 2,3,6-TBA oxidized. The herbicide was degraded to 3,5-dichlorocatechol through the intermediates 2,3,6-trichloro-4-hydroxybenzoate and 2,3,5-trichlorophenol. Benzoic acid derivatives which are halogenated in the 3- and 5-positions are generally resistant to microbial degradation (Horvath, 1971). In soybean (Glycine Max Merr.) , however, Spitznagle et al. (1969) found that 2,3,5-TIBA was dehalogenated. The 2,5- and 3,5-diiodobenzoic acid derivatives were recovered as conjugates. Some of these conjugates were acid labile, and the formation of lipid conjugates was suggested.
D. CHLORAMBEN The discussion of chloramben by Menzie (1969) and Swanson (1969) amply documents the conjugation of chloramben in numerous plant species to form N - ( 3-carboxy-2,5-dichlorophenyl)glucosylamine. Rapid and extensive conjugation, coupled with the evident stability of the conjugate apparently limits the further degradation of chloramben in most species studied. Glucosylation appeared to be a detoxication mechanism and was thought to be the basis for selectivity (Colby, 1965). However, glucosylation by itself does not explain selectivity completely since extensive glucoside formation has been reported in both susceptible and resistant species. The enzymatic nature of glucosylchloramben formation in plant tissue sections was described (Frear et al., 1967). The various tissues tested differed widely in their ability to glucosylate chloramben and tissue differences did not correlate with plant susceptibility to chloramben. The UDPg1ucose:arylamine N-glucosyltransferase catalyzing the glucosylation was partially purified (Frear, 1968a). The activity of the enzyme in plant tissue paralleled the ability of tissue sections to glucosylate chloramben. Several different substituted anilines were found to serve as substrates for the enzyme. Stoller and Wax (1968) found that nearly all the radioactivity from chloramben-14C could be recovered from tissues of resistant and susceptible seedlings as glucosylchloramben, “amiben-X,” and unaltered chloramben. These authors suggested that tolerant plants detoxified a larger portion of the chloramben by glucosylation thereby reducing the accumulation of free chloramben and “amiben-X.” Stoller ( 1968) subsequently showed that “amiben-X’ was relatively nonphytotoxic. It was metabolized to chloramben and glucosylchloramben in plant tissues. A later study (Stoller, 1969) showed that the distribution of chloramben and metabolites in susceptible
332
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and resistant species was comparable if the chloramben was supplied at dosages which were equally effective (ED,,) in inhibiting growth. Stoller concluded that differences in chloramben concentration required to saturate the glucosylating enzyme determined differences in susceptibility by affecting the level of free chloramben in the tissues. While this theory contributes to an understanding of the mechanism of chloramben selectivity, it is noted that absolute tissue concentrations of chloramben were higher in resistant than in susceptible plants, even when both were treated at their ED,,, rates. E.
DICAMBA
Swanson (1969) has reviewed the behavior and fate of dicamba in plants. He noted that the degradation of dicamba is species dependent. No dicamba degradation was observed in purple nutsedge (Cyperus rotundis L. ) based on thin-layer (Magalhaes et af., 1968) and gas-liquid chromatographic (Ray and Wilcox, 1969 ) evidence. Foliar application caused a temporary reduction in respiration rate and a decrease in membrane permeability of nutsedge 5 days after treatment (Magalhaes and Ashton, 1969). Broadhurst et al. (1966) reported that dicamba was rapidly conjugated with little or no decarboxylation in wheat ( Triticum aestivum L . ) and bluegrass ( P o a pratensis L.). Hydrolysis of the conjugates yielded approximately 90% 5-hydroxy-3,6-dichloro-~-anisicacid (5-OH-dicamba), 5 % 3,6-dichorosalicylic acid, and 5 % dicamba. The 5-OH-dicamba was present as a glucose ether while dicamba was present as a glucose ester. Carboxyl-'T dicamba was uniformly distributed in resistant barley (Hordeurn vulgare L.) and wheat, but it was selectively accumulated in the meristematic tissues of Tartary buckwheat [Fagopyrurn tataricum (L.) Gaertn.] and wild mustard (Sinapis arvensis L . ) (Chang and Vanden Born, 1971 ). The major metabolite, SOH-dicamba, was recovered from all species, and the salicylic acid derivative was found in minor amounts in the grasses. The species were ranked in order of decreasing resistance, decreasing rate of metabolism, and increased absorption and translocation of dicamba as follows: wheat, barley, wild mustard, and Tartary buckwheat. It was concluded that selective absorption, translocation, and metabolism all contributed to herbicidal selectivity. Other studies pertinent to the determination of differential phytotoxicity have been reported by Chang and Vanden Born (1971) and by Arnold and Nalewaja (1971). The latter authors showed that dicamba treatments increased the RNA and protein levels in wheat treated at the boot stage and in buckwheat (Polygonutn convolvulus L. ) treated in early vegetative and flowering
333
BEHAVIOR OF HERBICIDES IN PLANTS
stages. They suggested that a DNA-histone-dicamba occurred.
complex had
F. DICHLOBENIL AND RELATED COMPOUNDS The discovery, herbical properties, translocation and metabolism of dichlobenil have been reviewed recently (Swanson, 1969 ). Dichlobenil was converted predominantly to 2,6-dichlorobenzoic acid in bean (Phaseolus vulgaris L. ) , alligatorweed [Alternanthera philoxeroides (Mart.) Griseb.], and certain fungi (Pate and Funderburk, 1966). Recent studies indicate that dichlobenil is not readily metabolized in bean roots (Verloop and Nimmo, 1969). Approximately 90% of the dichlobenil translocated to the shoots was lost by volatilization and the remaining 10% was metabolized. 3-Hydroxy-2,6-dichlorobenzonitrieand 4-hydroxy-2,6-dichlorobenzonitrile were the principal metabolites recovered after 5 days. They were present in a ratio of approximately 4 to 1. These metabolites were shown to be toxic to bean leaves. Only trace amounts of the hydrolysis products, 2,6-dichlorobenzamide and 2,6-dichlorobenzoic acid, were observed. Verloop and Nimmo (1969) found that conjugates of dichlobenil also were formed and that only a portion of these could be extracted. The extractable conjugates were acid hydrolyzable to the hydroxybenzonitriles. The distribution of metabolites in bean after 5 days was approximately 20% free phenols, 60% extractable and hydrolyzable conjugates (presumably glucosides), and 20% insoluble conjugates. It was concluded that dichlobenil was concentrated 3-fold from the nutrient solution and rapidly translocated to the shoots. Approximately 90% was lost from the shoots as unaltered dichlobenil vapor. Of the remaining dichlobenil in shoots, about 90% was hydroxylated, conjugated, and subsequently incorporated into insoluble plant residues which accumulated with time. Wheat ( Triticurn vulgare L. ) metabolized dichlobenil to the hydroxy derivatives more extensively than did rice (Oryza sativa L.) (Verloop and Nimmo, 1970). The herbicide chlorthiamid was reported to be converted to dichlobenil in sterile soil. Chlorthiamid and several 2,6-dichlorobenzaldoximes also were converted to dichlobenil in nonsterile soil (Milborrow, 1963, 1965). Conjugates of chlorthiamid from apple (Malus spp.) and wheat yielded the 3-hydroxy and 4-hydroxy analogs of dichlobenil when hydrolyzed by pglucosidase (Beynon and Wright, 1968). Only a portion of the conjugates was hydrolyzable. They found that the phenolic derivatives of chlorthiamid were formed in wheat, but not in rice. These patterns are similar to those found for dichlobenil (Verloop and Nimmo, 1969, 1970). Wijma et al. (1970) reported that most of the products obtained by the condensation of chlorthiamid with aromatic or aliphatic aldehydes pos-
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sessed high preemergence phytoactivity, presumably due to the generation of dichlobenil in the plant tissues. The phytotoxicity of variously substituted benzonitriles and derivatives has been investigated (Gentner and Danielson, 1970). A seedling bioassay with cquimolar concentrations of dichlobenil and 30 related compounds revealed that the 2,6-dibromo-, 2-bromo-6-chloro-, 2,3,4-trichloro-, and 2,3,5-trichlorobenzonitriles and the 2,6-dichloro-/3-benzddoximeacetate analogs were at least as active as dichlobenil. Other analogs were less active. While these findings support the contention that the variously substituted compounds may be converted differentially to dichlobenil, they do not preclude the possiblity that the active agent may be other than dichlobenil (Gentner and Danielson, 1970).
G.
SUBSTITUTED BENZAMIDES
Alkyl substitution of the amide nitrogen in dichlorobenzamides is required for activity (Koopman and Daams, 1965; Wijma et al., 1970; Swithenbank et al., 1971). Swithenbank et al. (1971) indicated that significant herbicidal activity could be achieved by a combination of chlorination of the phenyl moiety. substitution of a dimethyl propynyl group on the nitrogen, and retention of the carbamyl function. Pronamide, which meets these requirements, has biological activity superior to other analogs tested (Swithenbank et al., 1971; Viste et al., 1970). The benzamides are most effective against grassy weeds (Swithenbank et al., 1971; Viste et al., 1970), and the most active analogs are substituted in the 3 5 , 3,4,5-, and 3-positions. In contrast to the benzamides, the chlorinated benzoic acids are effective primarily against dicotyledonous species, and the most active analogs are substituted in the 2,3,6-, 2,5-, 2,3-, and 2,6-positions (Smith, 1961). Pronamide has shown a high degree of activity for control of grassy weeds, including quackgrass [Agropyron repens (L.) Beauv.] (Viste et a/., 1970), and its degradation in soil and alfalfa (Medicago sativa L.) has been reported (Yih and Swithenbank, 1971a; Yih et al., 1970). Two major metabolites were detected in soil. They were 2-( 3,5-dichlorophenyl)-4,4-dimethyl-5-methyleneoxazoline,formed by cyclization of pronamide and N - ( 1,l-dimethylacetonyl) -3,5-dichlorobenzamide, formed from the inethylene oxazoline metabolite. Neither metabolite possessed as much herbicidal activity as did pronamide. In alfalfa, the radioactivity from carbonyl-l'C pronamide was methanol extractablc, but an increase in the unextracted residue occurred with time (Yih and Swithenbank, 1971a ) . Chromatography of methanol extracts re-
BEHAVIOR OF HERBICIDES IN PLANTS
335
vealed that pronamide was metabolized slowly in alfalfa. Minor amounts of nine metabolites were recovered from alfalfa 17, 50, and 112 days after treatment. One metabolite, p- (3,5-dichlorobenzamido)-p-methylbutyric acid, accumulated in tissue to a level equal to that of pronamide after 112 days. A tentative scheme for the metabolic transformations occurring in soils, plants, and animals has been proposed (Yih and Swithenbank, 1971b). An unidentified metabolite, found in alfalfa but not in soils, was presumed to be a derivative of 3,5-dichlorobenzoic acid (Yih and Swithenbank, 1971a), and may be related to the unextracted residues obtained in alfalfa. Several insecticides inhibited pronamide metabolism in leaf disks of lettuce (Lactuca sativa L.) (Chang et al., 1971a). The most effedtive inhibitors were Dyfonate,* chlorfenvinophos, fensulfothion, and disulfoton. Pronamide did not affect the metabolism of carbaryl in tomato (Lycopersicon esculenturn L.) leaf disks, nor did it influence the metabolism of Dyfonate or malathion (Chang et al., 1971b). An anatomical study indicated the growth-inhibiting properties of pronamide on roots and rhizomes of quackgrass (Peterson and Smith, 1971; L. W. Smith et al., 1971). Vascular disruption, precocious differentiation, and nuclear enlargement were noted. In addition, L. W. Smith el al. (1971) showed that nodes at rhizome apices exhibited increased DNA, RNA, protein, and cellulase levels.
H.
IOXYNIL AND
BROMOXYNIL
The development and selectivity of ioxynil and bromoxynil has been reviewed by Swanson (1969). The possible conversion of ioxynil in soils and plants to the corresponding benzoic acid derivative with loss of iodine was also discussed. Spray retention was a major factor in the selectivity of ioxynil in barley (Hordeurn distichon L.), pea (Pisurn sativurn L.), and white mustard (Sinapis alba L.) . Another factor in selectivity may be enhanced translocation in susceptible mustard compared to barley and pea (Davies et al., 1967, 1968a). Schafer and Chilcote (1970b) reported the metabolism of ioxynil to 4hydroxy-3,5-diiodobenzamideand 4-hydroxy-3,5-diiodobenzoic'acid. Small amounts of the amide derivative of ioxynil were detected in pea and white mustard 4 days after treatment (Davies et al., 1968b). The amide derivative
' Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
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was found in greater quantities in resistant barley together with the benzoic acid derivative and unknown compounds. It was also noted that higher concentrations of ioxynil were required to inhibit respiratory metabolism in leaf segments of barley than in pea or white mustard (Davies et aZ., 1968b). Heywood et a/. (1964) reported that the recovery of recognizable ioxynil derivatives from plants decreased with time. This may have resulted from instability of the amide or benzoic acid derivatives, or possibly from deiodination (Zaki et al., 1967). Degradation accompanied by reincorporation or complexing of I*C from ring-labeled ioxynil could explain the reported occurrence of radioactivity in starch, gluten, and glucose fractions of wheat grains (Hart et al., 1964) and would not necessitate invoking ring cleavage. Schafer and Chilcote (1970a) showed that a combination of differential spray retention and translocation accounted for approximately one-third of the 100-fold difference in susceptibility between resistant wheat ( T . aestivrrm ) and susceptible coast fiddleneck ( Amsinckia intermedia Fisch. and Mey .) to bromoxynil. Radioactivity from lC-cyano-labeled bromoxynil was more mobile in fiddleneck than in wheat, and higher levels were found in soluble forms (Schafer and Chilcote, 1970b). Wheat contained more insoluble residues and evolved more WO, than did fiddleneck. It is not known whether the insoluble residues represented conjugates of bromoxynil or natural plant constituents derived from the incorporation of "CO,. The liberation of ''C0, indicated that bromoxynil was converted to its benzoic acid derivative followed by decarboxylation. Ioxynil and bromoxynil represent benzoic acid derivatives in which differential spray retention and penetration evidently play a major role in selectivity. The relative importance of differential translocation, metabolism, and sites of action is undetermined at present.
I. DISCUSSION AND
SUMMARY
This diverse group of pesticides exhibits metabolic and formative effects in plants and undergoes biotransformations which differ slightly from compound to compound. Dehalogenation, ring hydroxylation, hydrolysis, and side-chain modifications occur in the variously substituted benzoic acids. However, ring cleavage in plants has not been demonstrated convincingly. There are numerous reports of insoluble residue or conjugate formation. Generally, these reports fail to identify the nature of the conjugates found. With the exception of chloramben, the plant enzymes which catalyze the major metabolic conversions of the benzoic acid herbicides have not been characterized. Conjugates and insoluble residues evidently contain unde-
BEHAVIOR OF HERBICIDES IN PLANTS
337
graded and variously substituted benzene rings which appear to be persistent. Therefore, future investigations should emphasize the characterization of these polar degradation products.
111.
A.
Dinitroanilines
INTRODUCTION
The substituted dinitroanilines represent a relatively new class of selective herbicides. The discovery of the superior herbicidal activity of 2,6dinitroanilines with a dialkyl-substituted amino group and a substituent group in the 4-position of the ring (Alder et al., 1960; Soper et al., 1961) has resulted in the development of several new and important herbicides. Chief among these are trifluralin, benefin, and nitralin. Trifluralin and benefin 'have received the principal research attention, and trifluralin has become one of the 5 pesticides whose sales exceed $25 million per year (Neumeyer et al., 1969). Other trifluralin analogs, such as dipropalin and N3,N3-diethyl-2,4-dinitro-6-trifluoromethyl1,3-~henylenediamine(USB 3584), are in various stages of development. Various aspects of the dinitroaniline herbicides have been reviewed. The relative stability of trifluralin (Casida and Lykken, 1969), and the degradation of trifluralin in animals, plants, and microorganisms has been outlined (Menzie, 1969). The chemistry, metabolism, and mode of action of trifluralin (Probst and Tepe, 1969; Plimmer, 1970; Melnikov, 1971) and the effects of dinitroanilines on plants have been discussed (Frear and Shimabukuro, 1971). The behavior of this and other classes of pesticides in soils has been reviewed comprehensively (Helling et al., 1971).
B. METABOLISM The persistence, metabolism, and fate of trifluralin in soils and plants has been investigated (Probst et al., 1967; Golab et al., 1967). The aboveground portions of a wide variety of plants grown in trifluralin-treated soils did not contain trifluralin or its degradation products, based on residue analyses having a sensitivity of 5 to 10 ppb. Roots from plants grown in treated soil exhibited a residue in the region of soil incorporation. The residues in roots of cotton (Gossypium hirsutum L.) and soybean were distributed in lipid, glycoside, hydrolysis product, protein, and cellular fractions. From 25 to 30% of the radioactivity from 14C-propyl-labeled trifluralin was found in the cellular fraction. Radioactivity from I4C-trifluoromethyl-labeled trifluralin was recovered predominantly from the gly-
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coside fraction. No trifluralin or major degradation products of trifluralin were recovered following hydrolysis of the glucoside fraction. The wide distribution of the radioactivity and lack of recognizable metabolites did not suggest any specific pathways of incorporation. Small amounts of 14C0, were liberated from soils and plants. Residues of trifluralin and its degradation products in carrots (Daucus Carota L.) grown in treated soil were investigated (Golab et al., 1967). Radioactivity was concentrated in the peel and near the phloem-xylem junction. Tissue extracts contained predominantly unchanged trifluralin, with small amounts of metabolites indicating that trifluralin was metabolized by dealkylation to ~,~u,cu,-trifl~oro-2,6-dinitro-N-(n-propyl)-p-toluidine. Trace amounts of a,cu,a-trifluoro-5-nitro-N4-( n-propyl) -toluene-3,4diamine were recovered, suggesting a reduction of one nitro group and dealkylation of one alkyl group. A small amount of the oxidized derivative, 4-(di-n-propylamino) -3,5-dinitrobenzoic acid was also detected. The substituted benzoic acid metabolite was not recovered from soil; therefore, it appears that it was formed in the carrot tissue. Chromatography of extracts from peanut (Arachis hypogaea L.) and sweet potato (Ipornoea batatas L.) grown in trifluralin-"C solutions for 72 hours (Biswas and Hamilton, 1969) indicated the presence of dealkylated and reduced trifluralin metabolites. Infrared studies indicated the possible formation of phenolic and benzoic acid derivatives (Biswas and Hamilton, 1969). Schmidt ( 1970) suggested that trifluralin or its monopropyl derivative formed complexes with an unknown constituent in soybean and that these complexes were dissociated by treatment with acetonitrile. Small amounts of unidentified polar metabolites and unextracted residues were noted. The polar products may be intermediates in the formation of terminal residues from trifluralin degradation (Probst et a[., 1967, Golab et al., 1967). The fatc of benefin in soil, plant, and animal systems has been studied recently (Golab et al., 1970). Peanuts and alfalfa were grown in treated soil and analyzed for "C-labeled benefin and metabolites. After 129 days, small amounts of radioactivity were recovered from peanut plants. Most of the radioactivity was present in roots and hulls. A major portion of the radioactivity was not extractable, and most of the extractable radioactivity was polar material. Only stems, roots and hulls contained significant 2,6amounts of the products; a,a,a-trifluoro-5-nitrotoluene-3,4-diamine, dinitro-a,cu,a-trifluoro-p-cresoland cu,a,cu-trifluorotoluene-3,4,5-triamine.In alfalfa, after 227 days, 65% of the radioactivity was extractable, but almost all of this was as polar products. Small amounts of benefin and traces of the same dealkylated and reduced products found in peanut were recovered. The similarity of recognizable degradation products in soils and in peanut and alfalfa plants suggested the possibility that both benefin and
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its degradation products were absorbed directly from the soil. Formation of the cresol derivative from benefin was attributed to soil type and conditions, since it was also detected in a subsequent experiment when trifluralin was incorporated in the same soil (Golab et al., 1970). Radioactivity from labeled trifluralin and benefin accumulated as polar products in soiIs, rumen fluid, and plants (Probst et al., 1967; Golab et al., 1969, 1970; Williams and Feil, 1971). The sequential steps involved in the degradation of benefin and trifluralin can be divided into two major routes (Golab d al., 1970). The aerobic route involves oxidative dealkylation reactions, and the anaerobic route is characterized by reductive reactions. Neither route is mutually exclusive, and the major metabolites under anaerobic systems, such as flooded soils and rumen, occur as minor metabolites in aerobic systems such as plants and well aerated soils. Polar products are eventually formed by both routes, and it was suggested that aromatic amines formed by reduction or dealkylation are probably precursors of the polar products (Golab et al., 1970). The formation of these products was postulated to be the main pathway for degradation of dinitroanilines to carbon dioxide and water. The enzymes responsible for in vivo metabolism of these substituted dinitroanalines have not been characterized, but crude extracts of peanut and sweet potato leaves were active in degradation of trifluralin (Biswas and Hamilton, 1969).
C.
SELECTIVITY
Dinitroanilines are used principally for selective control of grasses and broadleaf weeds in crops such as cotton, soybean, and certain root and vegetable crops (Weed Science Society of America, 1970). They are generally applied as preemergent soil-incorporated treatments. The seedling stages of susceptible species are most sensitive to dinitroanilines (Bayer et al., 1963). Inhibition of growth and/or cell division in roots has been noted in corn (Zea mays L.), cotton, oats (Avena sativa L. ) , sugar beets (Beta vulgaris L.) , safflower (Carthamus tinctorius L.), and other species (Arle, 1968; Bayer et al., 1967; Feeny, 1966; Hacskaylo and Amato, 1968; Oliver and Frans, 1968; Anderson et al., 1967; Schweizer, 1970; Talbert, 1965). Much of the experimental work with dinitroanilines has been done with trifluralin and the morphogenetic effects noted with trifluralin may be typical of this class of herbicides. The development of the injury syndrome has been documented in resistant and susceptible species, and has been reviewed recently (Schmidt, 1970). Differential susceptibility of tissues within a given species has been noted (Negi and Funderburk, 1968; Rahman and Ashford, 1970; Standifer and
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Thomas, 1965). Inhibition of lateral root formation, accompanied by percyclic and endodermal thickening in otherwise normal primary roots of cotton has been reported (Bayer et al., 1967). Other species exhibit similar abnormalities (Bayer et al., 1967; Hacskaylo and Amato, 1968; Talbert, 1965 ) . Acropetal translocation generally is restricted (Schultz et al., 1968; Standifer and Thomas, 1965), and the radioactivity from trifluralin is retained primarily on the surface of cotton and soybean roots (Strang and Rogers, 1971b). Differential phytotoxicity of trifluralin and nitralin has been noted in several species (Barrentine and Warren, 1970). The relatively greater toxicity of trifluralin to shoots in their study was ascribed to a limited vapor activity of nitralin. Dinitroanilines are known to reduce cell division, decrease nucleic acid and protein synthesis and increase carbohydrate content in plants (Probst and Tepe, 1969). Mitochondria1 oxygen uptake and oxidative phosphorylation are also inhibited (Negi et al., 1968). In addition, trifluralin inhibited dipeptidase activity in cucumber (Cucum’s sativus L.) and proteolytic activity in squash (Cucurbita maxima Duchesne) (Ashton et d., 1968; Ashton and Tsay, 1970). Trifluralin may inhibit cell division in corn and cotton roots by affecting sulfhydryl groups (Shahied and Giddens, 1970). The repression of mitotic activity by trifluralin may be due to destruction of IAA or kinetinlike compounds (Hassawy and Hamilton, 1971b). However, the data above do not explain fully the basis for selectivity of this class of herbicides. Selectivity may be based partly on physiological factors which enable roots of tolerant species to grow rapidly into untreated soil (Probst et al., 1967). The amount of trifluralin absorbed from moist soil per unit weight of germinating seeds was not correlated with species susceptibility (Schmidt, 1970). However, the same investigator found that species susceptibility was correlated with the ratio of trifluralin to water absorbed. Tolerant soybean and okra (Hibiscus esculentus L.) absorbed more water per unit of trifluralin absorbed than did susceptible sorghum (Sorghum vulgare Pers.) and wheat ( T . aestivum). The postulated formation of a trifluralin complex in resistant species also may contribute to selectivity (Schmidt, 1970). Lipid content of seeds likewise may contribute to the selective action of trifluralin (Hilton and Christiansen, 1972). Lipids applied to soil or filter paper containing trifluralin prevented the expression of phytotoxicity to a number of plant species. In addition to the effectiveness of external lipid applications, it was found that the lipid content of seeds was closely correlated with seedling sensitivity to trifluralin. Seeds having a high lipid content thus might be able to trap trifluralin and prevent it from reaching an active site.
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D. INTERACTIONS Synergistic injury responses of corn, beans, and barley ( H . vulgure) to combinations of trifluralin and carbaryl have been reported (Hamill and Penner, 1970). Soybeans were injured less by atrazine residues in soil when treatment was combined with trifluralin (Adams et al., 1970; Espinoza et al., 1968). Antagonism was observed in germination studies of beans, corn, and barley treated with trifluralin in combination with fluorodifen or chloramben (Hamill and Penner, 1970). Trifluralin injury to corn and wheat was reduced by malathion (Smith, 1970). The toxicity of trifluralin to cotton was reduced by applications of malathion, phorate, or disulfoton (Arle, 1968). Phorate reduced the trifluralin-induced lateral root inhibition in cotton (Hassawy and Hamilton, 1971a). The mechanisms involved in these interactions remain generally obscure.
E. DISCUSSION AND
SUMMARY
The dinitroaniline herbicides are effective in selective control of germinating weed species. They are not absorbed, translocated, or metabolized by plants as extensively as are many other classes of pesticides. They are effective in disrupting cell division and perhaps hormonal balance in tissues placed in contact with the herbicide preparations or in treated soil. Various biochemical effects of the dinitroanilines on plant tissues have been noted, but probably do not explain the selective phytotoxicity observed. Differential metabolism may not explain selectivity since little or no metabolism of the compounds occurs in plants. Those metabolic conversions which have been explored indicate that dealkylation and reduction occur, followed by the formation of polar products probably generated from amine intermediates. Ring cleavage has not been demonstrated. Small amounts of 14C0, were evolved from soils and plant tissues treated with 14CF,labeled trifluralin (Probst and Tepe, 1969), but it is not known whether this contributes significantly to the overall degradation pattern. Selectivity is probably based on a combination of factors, including a differential ability of resistant and susceptible species to trap the herbicide in lipoidal fractions, and differential germination and growth characteristics which permit roots of resistant species to grow rapidly into untreated zones of soil. The difficulties of determining the basis for the selective action of the dinitroanilines have been described (Probst and Tepe, 1969). These difficulties emphasize the need for further investigations to characterize more definitively the sites of metabolism, the degree of enzyme involvement in metabolism and the relative contribution of several possible mechanisms
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which may contribute to the final expression of selective phytotoxicity of the dinitroanilines.
IV.
A.
Triazines
INTRODUCTION
The symmetrical triazine herbicides have shown remarkable selectivity for control of weeds in corn, sorghum, sugarcane (Saccharurn oficinarum L.), and other crops. The first report of herbicidal activity in s-triazines appeared in 1955 (Knuesli et al., 1969). The high tolerance of corn to s-triazines led to investigations on the physiological action of these compounds and the basis for resistance in corn. The discussion on mechanism of action and other physiological effects of s-triazines is included in published reviews (Hilton et al., 1963; Moreland, 1967; Frear and Shimabukuro, 1971) . Recent reviews discuss metabolism of s-triazines in the soil environment (The Triazine Herbicides, 1970) and in different biological systems (Knuesli et al., 1969; Shimabukuro et al., 1971b). This short review on s-triazines will be limited primarily to a discussion on the metabolic fate of these compounds in plants.
B. METABOLIC PATHWAYS Simazine and atrazine are two of the few herbicidal compounds which have been investigated extensively with regard to their metabolic fate in plants. Most of our information on s-triazine metabolism is based on studies with the 2-chloro-s-triazines given above. Information on the metabolism of 2-methoxy- and 2-methylmercapto-s-triazines in plants is very limitcd.
I . Hydrolysis Dechlorination of atrazine and simazine and the substitution of a hydroxyl group in the 2-position was the first degradation reaction identified in plants (Fig. 1 ) (Gysin and Knuesli, 1960; Castelfranco et al., 1961; Hamilton and Moreland, 1962; Roth, 1957; Hamilton et al., 1962). This reaction was found to be nonenzymatic, and the active catalyst in corn was isolated and identified as 2,4-dihydroxy-7-methoxy1,4-benzoxazine3-one (benzoxazinone) (Hamilton et al., 1962; Wahlroos and Virtanen, 1959). Relatively high concentrations of benzoxazinone are found throughout the corn plant, the roots containing higher concentrations than other parts of the plant (Klun and Robinson, 1969). Although benzox-
343
BEHAVIOR OF HERBICIDES IN PLANTS
AN
N/
KE
benzoxazinone catalyzed RlN H
H
F . 2
Simazine - Rl and % = C,H, Atrazine - Rl = C,H,; % = - CH(CH,),
FIG.1. Benzoxazinone catalyzed hydrolysis of 2-chloro-s-triazines.
azinone acts as a catalyst in the hydrolysis reaction, relatively high molar ratios of benzoxazinone to 2-chloro-s-triazines are required for effective conversion of the 2-chloro-s-triazines to their hydroxy analogs (Tipton et al., 1971). It is suggested that molecular aggregates of benzoxazinone may be the catalytically active form (Tipton et al., 1971). The hydroxylation of 2-chloro-s-triazines seems to be limited to benzoxazinone-containing species such as resistant corn and Coix lacrymajobi L. and susceptible wheat and rye (Secale cereale L.) (Hamilton, 1964; Shimabukuro, 1967). Resistant sorghum, intermediately susceptible pea and cotton, and susceptible oat, barley, and soybean failed to form the 2-hydroxy derivatives (Hamilton, 1964; Shimabukuro, 1967, 1968; Shimabukuro and Swanson, 1970). None of these plants contain benzoxazinone. 2 . N-Dealkylation The oxidative removal of alkyl side chains in plants was first indicated when 14C0, was recovered from corn, cotton, and soybean treated with 14C-chain-labeledsimazine (Funderburk and Davis, 1963). It was reported that at least 70% of the total radioactivity absorbed by corn plants as 14C-chain-labeledsimazine was expired as 14C0, within 6 days (Miieller and Payot, 1966). These results indicated very clearly that both resistant and susceptible plants are capable of degrading the substituted alkylamine groups of simazine and atrazine. One of the two possible N-dealkylated metabolites of atrazine, 2-chloro-4-amino-6-isopropylamino-s-triazine (IV) (Fig. 2 ) was first isolated and identified from atrazine-treated pea plants (Shimabukuro et al., 1966). The concentration of (IV) was at least twice that of atrazine after 48 hours. Significant amounts of (IV) and 2-chloro-4-amino-6-ethylaminos-triazine (111) were formed in atrazine-treated sorghum. Lesser amounts of the same metabolites were detected in soybean, wheat, and corn (Shimabukuro, 1967). N-dealkylation, as illustrated in the upper half of Fig. 2, may occur to some extent in most higher plants. This is in contrast to the hydrolysis reaction which is limited to a few selected plant species.
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Atrazine (1)
CH,CH,N H
c
t.
OH
OH
NH,
t
-
NCH(CHd2 H
CHsCH2N H
H2N
OH
NCH(CH,), H
(vm) FIG.2. Metabolism of atrazine by N-dealkylation and hydrolysis in plants.
N-dealkylation of (111) and (IV) gave 2-chloro-4,6-diamino-s-triazine (V ) in sorghum (Shimabukuro, 1969). A slightly more polar unknown metabolite (IX) was also detected in sorghum. Dechlorination had occurred in (IX),but the substituent in the 2-position was not the hydroxyl group (Shimabukuro, 1969). The lower half of Fig. 2 shows the metabolites of atrazine formed in corn by a combination of benzoxazinone-catalyzed hydrolysis and N-dealkylation (Shimabukuro, 1968). N-dealkylation is not limited to the 2-chloro-s-triazines. The metabolites 2-hydroxy-4-amino-6-ethylamino-striazine (VI) and 2-hydroxy-4-amino-6-isopropylamino-s-triazine (VII) were identified in corn treated with atrazine or hydroxyatrazine
BEHAVIOR OF HERBICIDES IN PLANTS
345
(Shimabukuro, 1968). Metabolite (VI) was also detected in simazinetreated Coix lacryma-jobi L. and corn (Hurter, 1966; Montgomery et al., 1969). In species such as corn either N-dealkylation or hydrolysis may occur first to yield the same end products, (VI) and (VII), as shown in Fig. 2 (Shimabukuro, 1968). Complete dealkylation of 2-hydroxy-s-triazines will give 2-hydroxy-4,6diamino-s-triazine (VIII) (ammeline). Small amounts of this metabolite were detected in Coix lacryma-jobi L. and corn (Hurter, 1966; Montgomery et al., 1969). The formation of ammeline by complete dealkylation may be a slow process. Evidence indicates that N-dealkylation of the remaining side chain of the monodealkylated derivatives does not occur very readily when a hydroxyl group is substituted in place of a chlorine in the 2-position (Shimabukuro et al., 1971b). 3 . Glutathione Conjugation
In highly resistant sorghum, atrazine was not converted to hydroxyatrazine, but to other major water-soluble metabolites (Shimabukuro, 1967). Two of these metabolites were identified as S-(4-ethylamino-6-isopropylamino-s-triazhyl-2) -glutathione ( GS-atrazine) (X) and S-y-L-glutamyl- (4-ethylamino-6-isopropylamino-s-triazinyl-2) -L-cysteine (XI) (Fig. 3) (Lamoureux et al., 1970). The metabolism of atrazine to these compounds occurred very rapidly. Within 7 hours, 62% of the absorbed atrazine was converted to the water-soluble metabolite in sorghum leaf disks (Shimabukuro and Swanson, 1969). Identification of GS-atrazine and (XI) indicated the presence of a third major degradation pathway for 2-chloro-s-triazines in plants (Lamoureux et al., 1970). This is the first example of glutathione being involved in the biotransformation of a pesticide in plants. Glutathione conjugation of halogenated xenobiotic compounds occurs in animals as the initial reaction in the formation of mercapturic acids as excretion products (Boyland and Chasseaud, 1969). The mercapturic acid of atrazine has not been identified in plants, but the formation of GS-atrazine and (XI) indicates the presence of a mercapturic acid-type detoxication pathway for substituted 2-chloros-triazines in plants. A similar detoxication pathway for the metabolism of 2-chloro-4-ethylamino-6- ( 1-methyl- 1 -cyanoethylamino) -s-triazine was reported in the rat (Hutson et al., 1970).
4. Degradation of Methoxy- and Methylmeucapto-s-triazines Very little is known about the metabolism of substituted 2-methoxy-striazines in plants (Kniiesli et al., 1969). Hydrolysis of the methoxy group has been reported, but this reaction does not seem to be a major degradation reaction in plants (Kniiesli et al., 1969).
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1 XNL
CH,CH,N
NCH(CHd, H
H
(0
0 NH, 0 0 0 II I II H I I H I1 HOC- CHCH,CH,C- N- FHC- NC &COH
Insoluble residue
CH,CH,N H
k
NC H(cH,), H
\
\. \ \
\
y4
HOC- CHC&CH,C-
0
H II NFHC- OH
A
N/
N
(XI)
FIG. 3. Metabolism of atrazine by glutathione conjugation in plants. (From Shimabukuro ef al., 1971b.)
More extensive studies of 2-methoxy-s-triazine metabolism have been made in animals (Shimabukuro et al., 1971b). N-dealkylation and hydrolysis of the methoxy group occurred very readily in rats (Larson et al., 1970). It is conceivable that plants could also carry out similar reactions. In animals, w-oxidation of N-alkyl side chains seems to be a major reaction. In the rat, the side chain of a substituted methoxy-s-triazine was oxidized to the primary alcohol (Larson et al., 1970). Further oxidation of the side chains to the carboxylic acid derivatives was reported for substi-
BEHAVIOR OF HERBICIDES IN PLANTS
347
tuted 2-chloro-s-triazines in rats and rabbits (Bohme and Bar, 1967). In plants, w-oxidation has not been demonstrated. However, this does not imply that plants are incapable of carrying out such a reaction. Information is very limited on the metabolism of prometryne and other methylmercapto-s-triazines in plants. The conversion of prometryne to hydroxypropazine was demonstrated in carrot, cotton, and broad bean (Viciu faba L.) (Mueller and Payot, 1966;Montgomery and Freed, 1964;Sikka and Davis, 1968). The removal of the methylmercapto group is postulated to occur through a stepwise oxidation to the sulfoxide, sulfone, and hydrolysis (Gysin, 1962). The sulfone derivative of prometryne and also its N-dealkylated derivative, 2-methylmercapto-4-amino-6-isopropylamino-striazine, were detected in pea (Mueller and Payot, 1966). 5 . Terminal Residues
Higher plants readily absorb and translocate s-triazine herbicides throughout the plant. Root absorption and acropetal translocation ensures uniform distribution of the herbicides. Elimination of s-triazine herbicides and their degradation products by secretion from leaf surfaces or roots has not been demonstrated to be very significant. Therefore, final disposition of s-triazine herbicides may occur by complete oxidation to CO, or incorporation into plant residues. The evolution of 'TO, from W-ring-labeled 2-chloro-s-triazines has been reported in plants (Montgomery and Freed, 1961; Gysin, 1962). Cucumber and corn metabolized between 0.1 and 2.6% of the absorbed ring-labeled simazine and atrazine to 14C0, (Mueller and Payot, 1966; Ragab and McCollum, 1961). Another report indicated that corn, cotton, and soybean metabolized ring-labeled simazine but not atrazine to 14C0, (Davis et al., 1965). No 14C0, was detected from cotton treated with ring-labeled ipazine (Hamilton and Moreland, 1963) and from sorghum and corn treated with ring-labeled atrazine (Shimabukuro, 1967). The significance of ring cleavage and oxidation to CO, is still questionable, as evidenced by the conflicting reports. The amount of 14C0, evolved from such a reaction was relatively low compared to the total herbicide absorbed by plants. The incorporation of the heterocyclic s-triazine ring and/or its cleavage products into insoluble plant residues may be more significant than metabolism to CO,. An increase of radioactivity in insoluble residue occurred with time in sorghum treated initially for 48 hours with 14C-atrazine (Shimabukuro, 1967). More than 50% of the absorbed radioactivity was present as insoluble residue after 14 days. Approximately 21% of the absorbed radioactivity was incorporated as insoluble residue in sorghum under continuous treatment in 100 pM atrazine for 20 days (Bakke et al., 1972). The reports indicate a significantly greater amount of radioac-
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FREAR, R . H. HODGSON, R. H. SHIMABUKURO, G. G. STILL
tivity from l'C-atrazine incorporated into insoluble residue than radioactivity expired as lCO,. The insoluble residue may be the end product of the glutathione conjugation pathway (Fig. 3) (Lamoureux, 1971 ; Shimabukuro et al., 1970). More than 77% of the "C-atrazine absorbed by corn leaves was converted to the peptide conjugates (Fig. 3 ) within 16 hours. An increase in insoluble residue to 38% after 144 hours was accompanied by an almost equivalent reduction in the peptide conjugates (Shimabukuro et al., 1970). The chemical nature of the insoluble residue is unknown at the present time. However, it appears to be highly stable and resists digestion by rats and the ruminant sheep. Between 88 and 100% of the 14C-activitypresent as the insoluble residue in sorghum was excreted in the feces of rats and sheep (Bakke er al., 1972). C.
ENZYME REACTIONS
Many examples of herbicide metabolism in whole plants, animals, and microorganisms are known at present. However, research on metabolism at the moiecuiar level is very limited (Frear et a/., 1972). Isolation and characterization of several plant enzymes involved in herbicide metabolism have been reported (Frear and Shimabukuro, 1971; Frear et al., 1972). One of these enzymes, glutathione S-transferase, seems to be the most significant factor in 2-chloro-s-triazine metabolism and selectivity in higher plants (Shimabukuro et al., 1971a). Several different types of glutathione S-transferase which catalyze the conjugation of a variety of foreign compounds with reduced glutathione have been isolated and partially characterized from animal tissues (Boyland and Chasseaud, 1969). The isolation and partial characterization of a soluble enzyme from corn leaves which catalyzed the glutathione conjugation of atrazine and other 2-chloro-s-triazine herbicides is the first report of a glutathione S-transferase in higher plants (Frear and Swanson, 1970). Glutathione S-transferase from corn leaves has been purified over 73fold and has an estimated molecular weight of approximately 40,000 (Frear et al., 1972). The enzyme is found primarily in leaf tissue of species, such as corn, sorghum, sugarcane, Sudangrass [Sorghum Sudanese (Piper) Stapfl, and Johnsongrass [Sorghum halepense (L.) Pers.], which are resistant to 2-chloro-s-triazine herbicides (Frear and Swanson, 1970). Substrate specificity studies indicate that glutathione S-transferase from corn is specific for reduced glutathione (Frear and Swanson, 1970). Other compounds such as cysteine, mercaptoethanol, reduced lipoic acid, and CoA did not act as substrates (Frear and Swanson, 1970). The enzyme also appears to be quite specific for substituted 2-chloro-s-triazines. Little
BEHAVIOR OF HERBICIDES IN PLANTS
349
or no enzyme activity was observed in compounds where chlorine in the 2-position was substituted with the methoxy, methylmercapto, or hydroxyl groups (Frear and Swanson, 1970). A 12-fold increase in specific activity was observed when an isopropyl group (atrazine) was substituted in place of an ethyl group (simazine) in the 6-position (Frear and Swanson, 1970). No conjugation with glutathione occurred when one of the alkyl side chains was removed by N-dealkylation (Frear and Swanson, 1970). The in vivo metabolism of several 2-chloro-s-triazine herbicides in sugarcane leaves (Lamoureux, 1971) reflected the specific activities obtained in vitro (Frear and Swanson, 1970). The specific activity of glutathione S-transferase from corn and in vivo metabolism in corn or in sugarcane leaves indicated that conjugation occurred most rapidly with 2-chloro4-ethylamino-6-tert-butylamino-s-triazine (GS-13528) and atrazine. The rate of conjugation was least for simazine. The difference in rate of conjugation between atrazine and simazine seems to explain the relatively higher toxicity of simazine over atrazine in some species. The greater toxicity of simazine over atrazine in wild cane (Sorghum bicolor L. Moench) was clearly demonstrated (Thompson, 1972). Glutathione conjugation of atrazine occurred five times as fast as that of simazine in wild cane (Thompson, 1972). The enzyme which catalyzes N-dealkylation of s-triazine herbicides has not been isolated and characterized in plants. Atrazine and other substituted s-triazines have been tested as possible substrates for the cotton N-demethylase system which oxidatively N-demethylates substituted N-methylphenylurea herbicides (Frear et al., 1972). The tests indicated that s-triazine herbicides did not act as substrates for cotton N-demethylase (Frear et al., 1972).
D. SELECTIVITY Herbicides, including the s-triazines, may act on more than one biochemically or physiologically sensitive site to cause death in plants (Frear and Shimabukuro, 1971). The Hill reaction in photosynthesis seems to be the most sensitive site through which atrazine and other s-triazine herbicides act to cause irreversible damage to plants (Moreland et al., 1959). There are many factors in the plant environment and within plants which influence selectivity. Basically, all these factors act to reduce the final concentration of the toxic herbicide reaching the most sensitive site. The primary factor which determines the tolerance of plants to atrazine and other s-triazine herbicides seems to be the plants’ ability to degrade the toxic parent molecule rapidly (Shimabukuro, 1967; Thompson et al., 1971 ). The most significant degradation pathway for substituted 2-chloro-s-tri-
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azines in terms of herbicidal resistance and selectivity is the glutathioneconjugation pathway (Fig. 3 ) (Lamoureux et af., 1970). In both resistant sorghum and corn, the recovery of atrazine-inhibited photosynthesis was accompanied by a rapid conversion of atrazine to its peptide conjugates (Shimabukuro and Swanson, 1969; Shimabukuro et al., 1970, 1971a). A 7 7 4 0 % conversion of atrazine to GS-atrazine occurred in leaves of sorghum and corn over a 6-hour recovery period. N-dealkylation produced greater amounts of partially detoxified derivatives of atrazine (111, IV) (Fig. 2 ) in intermediately susceptible species, such as pea (Shimabukuro et al., 1966) and cotton (Shimabukuro and Swanson, 1970). The detoxication of atrazine by hydrolysis to hydroxyatrazine (Fig. 2 ) seems to be limited to a few selected species (Hamilton, 1964). Even in corn, hydroxyatrazine formation is a significant detoxication mechanism only when atrazine is absorbed through the roots (Shimabukuro et al., 1971a ) . The activity of glutathione-S-transferase in leaf tissue of corn seems to be the major factor in atrazine resistance. The results on atrazine detoxication in a resistant and susceptible line of corn strongly support the conclusion that most corn plants could resist herbicidal injury even if the hydroxylation pathway was absent (Fig. 2 ) (Shimabukuro et al., 1971a).
E. DISCUSSION AND SUMMARY Most of our information on the behavior of s-triazine herbicides in plants is derived from studies with the 2-chloro-s-triazines, atrazine and simazine. Information on metabolism and selectivity of 2-methoxy- and 2-methylmercapto-s-triazines is very limited. The s-triazines are one of the most exhaustively studied class of herbicides in use today. However, information on their ultimate fate in plants and the environment is still inc9mplete. There is no consistent evidence that ring cleavage and oxidation occur at an appreciable rate. There is evidence showing the incorporation of s-triazine derivatives into insoluble plant residue. However, the chemical nature of such residues is completely unknown at present. The metabolism of 2-chloro-s-triazines emphasizes the importance of the plant organ through which the compound enters the plant. Significant quantitative differences appear in metabolism of atrazine between roots and shoots in plants due to the difference in the distribution of the enzyme responsible for its metabolism. Differences in root and shoot metabolism and their influence on selectivity have been demonstrated with other compounds such as propanil and chlorpropham (Frear and Still, 1968; Still and Mansager, 1971). Therefore, it cannot be assumed that the fate
351
BEHAVIOR OF HERBICIDES IN PLANTS
or behavior of atrazine or any other pesticide is similar in the root, shoot, or reproductive organs of a plant.
V.
Heterocyclics
A. PICLORAM 1 . Introduction Picloram was introduced for the nonselective control of woody plants and most perennial broadleaf weeds (Hamaker et al., 1963). This herbicide seems to be only moderately toxic to grasses. Picloram was found to act as an auxin with activities equal to that of 3-indoleacetic acid (IAA) and 2,4-D (Kefford and Caso, 1966). Picloram is absorbed readily by roots and leaves and translocated acropetally and basipetally (Baur and Bovey, 1969; Bovey et al., 1967). Very little is known about the metabolism and disposition of picloram and its derivatives in plants. 2 . Metabolic Pathways Picloram apppears to be very stable in plants. A low rate of decarboxylation was detected in cotton plants treated with carbony1J4C picloram (Fig. 4 ) (Meikle et al., 1966). Soil microorganisms seem to have a greater capacity for decarboxylation of picloram than plants (Meikle
4-Amino-2,3,5trichloropyridine
Picloram
\ COOH
no coon 4-Amino-3,5-dichloro6-hydroxypicolinic acid
FIG.4. Picloram metabolism in plants.
coon Oxalic acid
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et al., 1966). An appreciable loss of radioactivity in bean leaf disks treated with carbonyl-''C picloram was attributed to decarboxylation (Sargent and Blackman, 1970). However, direct evidence for decarboxylation was not presented and loss of radioactivity may be due to other factors. It appears that most of the picloram absorbed by plants remains unchanged and decarboxylation is not an important mechanism for picloram metabolism in higher plants (Meikle et al., 1966; Redemann et al., 1968). Small amounts of acidic and neutral metabolites of picloram have been detected (Redemann et al., 1968; Maroder and Prego, 1971 >.Oxalic acid was identified by derivatization and gas chromatography and 4-amino-3,5dichloro-6-hydroxypicolinic acid was identified by gas chromatography (Redemann et al., 1968). In wheat, at least 10% of the radioactivity absorbed as ring-labeled IT-picloram was found as neutral metabolites. At least 80% of the neutral metabolite fraction was hydrolyzed by pancreatin or HCI to yield picloram, oxalic acid, and 4-amino-3,5-dichloro-6-hydroxypicolinic acid. The proportion of the three acidic hydrolysis products was the same as that in the plant extracts (Redemann et al., 1968). After 84 days, the total radioactivity in wheat plants was distributed among the 4 compounds (free and conjugated) as follows: picloram (83% ) ; oxalic acid ( 8 % ) ; 4-amino3,5-dichloro-6-hydroxypicolinic acid ( 5% ) ; and 4-amino-2,3,5-trichloropyridine ( 4 % ) (Redemann et al., 1968). The acid derivatives were postulated to be lipid conjugates. However, no positive identification of the conjugates was made except that these compounds were hydrolyzed by HCI and pancreatin (Redemann et al., 1968). A major conversion of picloram to an unknown conjugate was reported to occur in slimleaf wallrocket [Diplotaxis tenuifolia (L.) DC] (Maroder and Prego, 1971 ). Hydrolysis of the unknown conjugate yielded only picloram. The unknown conjugate appeared to be more. polar than the parent picloram. Thereforc, it appears that the unknown conjugate (Maroder and Prego, 1971) may not be the same as the postulated lipid conjugates (Redemann et al., 1968), which appeared to be more nonpolar than the parent acidic compounds. There is no indication of substantial insoluble residue accumulation in plants. The detection of oxalic acid indicates a low rate of ring cleavage (Redemann et al., 1968). It seems more likely that picloram persists in higher plants predominantly in its unchanged form.
3. Discussion and Summary Very little is known on the behavior and fate of picloram in plants in contrast to other classes of herbicides. Information on metabolism beyond
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the initial degradation reactions is almost totally absent. No work has been reported on plant enzymes catalyzing picloram metabolism. The mechanism of action and basis for selectivity of picloram may be expected to be very similar to that of 2,4-D. Both of these herbicides are potent growth regulators. However, even the mechanism for causing death in broadleaf plants by 2,4-D is still not completely understood. Since picloram is used quite extensively ir, our environment, it is imperative that studies be made on the ultimate fate of this herbicide. B.
PYRAZON
1. Introduction Pyrazon was introduced in 1964 as a selective herbicide for the control of annual broadleaf weeds in sugar beets and red beets. Pyrazon is readily absorbed by roots of resistant plants and translocated in the xylem to the foliar parts (Frank and Switzer, 1969b; Stephenson and Ries, 1967). Pyrazon and its metabolites are not translocated in the phloem as evidenced by the lack of movement out of treated beet leaves (Stephenson and Ries, 1967). The primary mode of action of pyrazon in plants seems to be the inhibition of the Hill reaction in photosynthesis (Frank and Switzer, 1969a; Eshel, 1969b; Hilton et al., 1969). 2 . Metabolism Information on the metabolism of pyrazon in higher plants is very limited. At least two of the initial metabolites of pyrazon have been identified, but knowledge of subsequent metabolism and the ultimate fate of these metabolites in plants are completely unknown (Ries et al., 1968; Stephenson and Ries, 1969). The major metabolite in resistant plants was identified as the N-glucoside of pyrazon, N - (2-chloro-4-phenyl-3(2H) -pyridazinone)glucosamine (N-glucosylpyrazon) (Fig. 5 ) (Ries et al., 1968). Unchanged pyrazon was not detectable in shoots of sugar beet plants 6 weeks after soil treatment with radioacive pyrazon. At least 97% of the radioactivity in the shoots after 4 weeks appeared to be N-glucosylpyrazon (Stephenson and Ries, 1969). A cleavage product of pyrazon, 5-amino-4-chloro3 (2H)-pyridazinone (ACD) (Fig. 5 ) (Stephenson and Ries, 1969; Fisher, 1964) and an unknown conjugate of this cleavage product were also found in small amounts in the shoots of sugar beets (Stephenson and Ries, 1969). Hydrolysis of the polar metabolites of pyrazon in sugar beets yielded ACP as one of the products (Stephenson and Ries, 1969). The conjugated moiety of the ACP complex has not been identified.
354
D. S. FREAR, R. H. HODGSON, R. H. SHIMABUKURO, G. G . STILL 0.
c1
Pyrazon
0
~ N-~ N - g h c o s e
N-Glucosylpyrazon
ACP
0
J
c1
H4 y : - x ACP-Complex
FIG.5 . Pyrazon metabolism in plants.
A time course study on the formation of pyrazon metabolites in the soil and plants suggests that ACP is formed in the soil and absorbed by plant roots. Subsequent conjugation of ACP was postulated to occur in roots and shoots after uptake from the soil (Stephenson and R i a , 1969). In resistant beets, N-glucosylpyrazon was formed in the shoots, but not in the roots (Stephenson and Ries, 1967). Susceptible plants such as tomato failed to produce N-glucosylpyrazon in either the shoots or roots (Stephenson and Ries, 1967). At least 12% of the total radioactivity absorbed as "C-pyrazon by sugar beet was incorporated into insoluble plant residues after 16 weeks (Stephenson and Ries, 1969). No report has appeared on in vitro studies of pyrazon metabolism. N-glucosylation of pyrazon appears to be similar to the N-glucosylation of chloramben (Swanson et a!., 1966; Frear et al., 1967). The arylamine N-glucosyltransferase, which catalyzes the N-glucosylation of chloramben, was found to have a broad specificity for substituted anilines, and was present in a number of plant species and tissues (Frear et al., 1967; Frear, 1968a). A similar enzyme system may also be responsible for the glucosylation of pyrazon. However, any determination of similarities or differences in the N-glucosylation of pyrazon, chloramben and other substituted anilines must await further in vitro enzyme studies.
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The N-glucosylation of pyrazon in plants appears to be another example of localized metabolic activity in specific plant organs. We have seen that the metabolism of 2-chloro-s-triazines, propanil, chloramben, and substituted urea herbicides may often be localized in the roots or shoots of plants. In roots of resistant beets, only pyrazon was detected after 72 hours, but 50-60% of the translocated pyrazon was converted to its N-glucoside in the shoots (Stephenson and Ries, 1967). 3 . Selectivity
The metabolism of pyrazon to N-glycosylpyrazon appears to be a detoxication mechanism which is present in resistant plants such as beet, but not in susceptible tomato and moderately tolerant millet (Panicurn rniliaceum L.) (Stephenson and Ries, 1967). Resistant sugar beets metabolized pyrazon’ very rapidly in both the roots and shoots (Frank and Switzer, 1969b). The roots of susceptible lambsquarter (Chenopodiurn album L.) metabolized pyrazon slowly, but the shoots did not alter the parent compound (Frank and Switzer, 1969b). Since the primary site of action (Hill reaction in chloroplasts) for pyrazon is located in the shoots of plants, the differential metabolism in shoots of susceptible and resistant plants becomes highly significant in terms of selectivity. The results indicate that the relative rate of pyrazon metabolism to its N-glucoside is the basis for selectivity in higher plants (Frank and Switzer, 1969b). VI.
Diphenylethers
A. INTRODUCTION Substituted diphenylethers are a relatively new class of herbicides. They have been classified into two groups, primarily based on distinct differences in ring substitution and in light requirement for herbicidal activity (Matsunaka, 1969a,b). One group, with substitutions at the 2,4- or the 2,4,6-positions, is active only in the light. The other group, with substitutions at the 3- or the 3,5-positions, is active in dark or light. The two groups also differ in other biological properties (Matsunaka, 1969a,b). Several of the ortho-substituted, light activated, diphenylethers have been developed recently for selective preemergence and postemergence control of annual grasses and broadleaf weeds in transplanted rice, soybeans, Brassica, and other crops (Ebner et al., 1968; Geissbiihler et al., 1969; Matsunaka, 1969a,b; Hawton and Stobbe, 1971a; Rogers, 1971; Eastin, 1971a,b,c). Published reports on the behavior of diphenylether herbicides in plants have been limited. Studies with nitrofen and fluorodifen have shown that the ortho-substituted diphenylethers are readily absorbed by root and leaf
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tissues, but subsequent translocation is limited (Hawton and Stobbe, 1971a,b; Eastin, 1969, 1971a,c). In some studies (Walter et al., 1970), preferential acropetal or basipetal translocation was not established. Other studies demonstrated limited acropetal movement (Eastin, 1969, 1971a,c; Geissbuhler et a]., 1969; Hawton and Stobbe, 1971b; Rogers, 1971). The ortho-substituted diphenylethers have been shown to function as inhibitors of noncyclic electron transport coupled with photophosphorylation in the chloroplast (Moreland r t al., 1970). Mechanisms for the light activation of ortho-substituted diphenylethers have becn proposed recently (Matsunaka, 1969a). Studies with nitrofen and rice seedlings suggest that light activation is a photobiochemical process in which chlorophylls or yellow pigments, especially xanthophylls, act as acceptors of light energy in the photoactivation mechanism.
B.
METABOLISM
Studies on the metabolism of fluorodifen show that the key reaction for the degradation of this herbicide in plants is a rapid cleavage of the ether linkage (Geissbiihler et al., 1969; Rogers, 1971; Eastin, 7971a,b,c). The 4-nitrophenol released by the cleavage of the ether is conjugated as a glucoside (Geissbuhler et al.. 1969). Studies on the fate of the 2-nitro-4-
Fluorodifen
[ ?]
+
H
O
G
N
O
,
-
00
FIG.6. Metabolism of fluorodifen in plants,
0
NO,
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trifluoromethylphenyl moiety have not been published (Geissbiihler, 1971) . A substantial percentage of the radioactivity from fluorodifen-lJT treated plants remains associated with insoluble residues (Rogers, 1971; Eastin, 1969, 1971b,c; Geissbuhler, 1969). A minor pathway is the reduction of one or both of the nitro groups (Rogers, 1971; Eastin, 1971b; Geissbuhler, 1969). A summary of the present information on the metabolism of fluorodifen in plants is shown in Fig. 6. A recent study (Hawton and Stobbe, 1971b) has indicated that nitrofen is also cleaved at the ether linkage and that the formation of several unidentified metabolites is dependent on a light-controlled biochemical process. Diphenylethers are considered as relatively stable compounds in biological systems (Williams, 1959). The rapid cleavage of the ether bond in fluorodifen represents an apparent exception to such a concept. Unfortunately, studies on the enzyme systems responsible for substituted diphenylether metabolism in plants have not been reported, and the mechanism of the ether bond cleavage is unknown. The reduction of the nitro groups in fluorodifen is undoubtedly similar to that observed for many other nitro-substituted pesticide compounds in higher plants (Menzie, 1969). The glucosylation of 4-nitrophenol is presumably catalyzed by a UDPG:glycosyltransferase (Frear, 1968a).
C. SELECTIVITY The selectivity of the ortho-substituted diphenylethers, nitrofen and fluorodifen, has been ascribed to a number of factors (Walter et al., 1970; Eastin, 1971a; Hawton and Stobbe, 1971a; Rogers, 1971). The increased susceptibility of sorghum and tall morning glory [Zpomoea purpurea (L.) Roth] to fluorodifen has been attributed to increased translocation and higher concentrations of fluorodifen in lower and upper stem tissues (Walter et al., 1970). Cucumber was apparently more susceptible to fluorodifen than peanut because of increased uptake and translocation coupled with a slower rate of degradation (Eastin, 1971a). It has been suggested that the rate of fluorodifen degradation in soybeans was sufficient to serve as a protective mechanism, if the degradation products were relatively nontoxic (Rogers, 1971 ) . Studies with nitrofen indicated that the selectivity of this herbicide was apparently associated with internal factors rather than differences in foliar spray retention and penetration (Hawton and Stobbe, 1971a). Internal factors might include increased rates of degradation, but it has been suggested that membrane permeability may be a factor in the selectivity of nitrofen in rape (Brassica campestris L.) and cabbage (Brassica oleracea L.) (Hawton and Stobbe, 1971b; Perida, 1971).
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D. DISCUSSION Two possible mechanisms for the light activation of ortho-substituted diphenylether herbicides havc becn proposed by Matsunaka ( 1 9 6 9 4 . If one assumes that the parent diphenylether molecule is the toxic form of the herbicide, and that it must penetrate the cell and the chloroplast membranes to reach the target site, it is interesting to speculate that light activation of the orrho-subsituted diphenylethers may initiate a reversible (possibly hormonally rcguiated) reaction in foliar tissues that would enable the parent herbicide molecule to be transported across cell membranes. Once inside the cell, the parent molecule could cross the chloroplast membrane and inhibit photosynthesis and/or be degraded to nonphytotoxic products in the cytoplasm. Such a hypothesis would fit the second proposed mechanism of Matsunaka (1969a) which does not involve the conversion of the herbicide into another toxic compound. It would also explain the delayed increase in the number of metabolites in plants exposed to high light intensitites (Hawton and Stobbe, 1971b), the similar metabolic pathways found in resistant and susceptible plants (Eastin, 1971a), as well as the suggested involvement of membrane permeability (Hawton and Stobbe, 1971b; Perida, 1971 ) and increased rates of degradation (Eastin, 1971a; Rogers, 1971) as factors in selectivity. Comparative physiological and biochemical studies with the two groups of diphenylether herbicides should provide a challenging new area of investigation, and should result in the development of additional basic information on the mechanism of light activation, the mechanism of ether cleavage and the metabolic pathways and reactions of substituted diphenylethers in higher plants.
VII.
A.
Substituted Ureas
INTRODUCTION
Substituted ureas constitute a major class of herbicides used in agriculture today. The chemical properties of substituted urea herbicides and their behavior in plants have been the subject of several recent reviews (Geissbuhler, 1969; Casida and Lykken, 1969; Menzie, 1969; Melnikov, 1971 ; Plimmer, 1970). Extensive modification in ring structures, ring substituents, and alkyl moieties have resulted in the development of a broad class of selective urea herbicides for prc- and postemergence control of grass and broadleaf weeds in many crops. The substituted ureas are potent inhibitors of photosynthesis, and their effect on this vital plant process is
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usually considered as the primary mode of action for this class of herbicides (Geissbuhler, 1969; Moreland, 1967). Urea herbicides are readily absorbed by roots and shoots. Foliar absorption is increased with certain surfactants (Bayer and Yamaguchi, 1965; Hill et al., 1965; McWhorter, 1963). Structural differences among the various substituted urea herbicides may result in significant alterations in absorption, mobility (Geissbuhler, 1969; Hogue, 1970), and metabolism (Frear et al., 1969, 1972). Movement is primarily in the apoplast, and transport is acropetal and lateral (Geissbuhler, 1969; Strang and Rogers, 1971a). B.
METABOLISM
Substituted dimethyl- and methoxymethylphenylurea herbicides undergo stepwise N-dealkylation to monodealkylated and dealkylated metabolites in higher plants (Geissbuhler, 1969; Menzie, 1969). Geissbuhler ( 1969) and Kuratle et al. (1969) have shown that the substituted methoxymethylphenylurea herbicides are N-demethylated and N-demethoxylated. Recent studies with linuron (Nashed and Ilnicki, 1970) and chlorbromuron (Nashed et al., 1970) suggest that N-demethylation may be the preferred initial step in the metabolism of this type of substituted phenylurea herbicide. Recent studies (Frear et aZ., 1972; Frear and Swanson, 1972) have demonstrated that N-hydroxymethyl intermediates are formed during the oxidative N-demethylation of monuron and monomethylmonuron and that these intermediates are conjugated as glucosides. Evidence for the oxidation, dehalogenation or cleavage of the substituted phenyl ring has not been reported in plants. Similarly, the cleavage of substituted phenylurea metabolites to the corresponding anilines has not been observed in recent plant studies (Geissbuhler, 1969; Frear and Swanson, 1972). It has been suggested (Frear and Swanson, 1972) that previous reports on the identification of trace amounts of substituted aniline and nitrobenzene metabolites may have been the result of radioisotope-labeled impurities in the phenylurea herbicides studied or possible degradation of the herbicides before absorption by the plant tissues. The assumption that all isolated pesticide residues are the direct result of plant metabolism may not be valid. This may be particularly true in the case of long-term studies under nonsterile conditions and in studies where the significance of photochemical degradation has not been established or the herbicides have not been adequately purified to remove trace quantities of radioisotope-labeled impurities. Extreme care should be exercised in the interpretation of experimental data from such experiments. Significant amounts of unknown polar metabolites and insoluble or
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N-C-N
\
Monuron
I
FIG.7. Monuron metabolism in plants.
"bound" residues have also been observed in recent studies with several substituted phenylurea herbicides (Frear and Swanson, 1972; Nashed et al., 1970; Nashed and Ilnicki, 1970; Geissbiihler, 1969). The significance and nature of these metabolites and residues remains to be established. A summary of the present information on the metabolism of monuron is shown in Fig. 7 (Frear and Swanson, 1972).
C. ENZYMES Striking differences have been observed in the ability of excised leaf tissues to metabolize substituted phenylurea herbicides and their metabolites (Swanson and Swanson, 1968a; Frear et al., 1972). Excised carrot leaf tissues rapidly N-demethylated both linuron and diuron while cotton leaf tissues apparently N-demethylated only the diuron (Frear et al., 1972). Excised soybean leaf tissues apparently carried out only the first N-de-
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36 1
methylation step in the metabolism of monuron (Swanson and Swanson, 1968a). It is interesting to speculate that these striking differences in metabolism are the result of differences in enzyme specificity, but the necessary support for such speculation will only be obtained when we fully understand the basic characteristics and properties of the enzyme systems responsible for urea herbicide metabolism in higher plants. Unfortunately, reports on the enzyme systems responsible for the metabolism of substituted phenylurea herbicides in higher plants have been limited. A number of studies (Frear, 1968b; Frear et al., 1969, 1972; Frear and Shimabukuro, 1971) have shown, however, that several N-methylphenylurea herbicides are oxidatively N-demethylated in cotton, plantain (Plantago major L.), and other tolerant plant species by a microsoma1 mixed function oxidase. Studies with a microsomal N-demethylase from cotton (Frear et al., 1969, 1972) have indicated that this enzyme system is specific for substituted N-methylphenylureas. It is inhibited by reaction products and by structurally related thiourea, semicarbazide, and methoxymethyl analogs. A similar microsomal enzyme system from carrot (Frear et al., 1972) apparently has a broader substrate specificity and will catalyze the N-demethylation of both methoxymethyl- and methyl-substituted phenylureas. The enzyme system responsible for the N-demethoxylation or 0-demethylation of the substituted methoxymethylphenylurea herbicides has not been reported. Similarly, the enzyme system responsible for the glucosylation of N-hydroxymethyl intermediates in the oxidative N-demethylation of dimethyl- and methyl-substituted phenylureas has not been reported.
D. SELECTIVITY The selectivity of substituted ureas is relative and depends on many variables in a complex chain of events. Many studies (Swanson and Swanson, 1968a; Frear and Shimabukuro, 1971; Rogers and Funderburk, 1968; Nashed et al., 1970; Smith and Sheets, 1967; Geissbiihler, 1969) suggest that differential metabolism is a major factor responsible for the selectivity of the substituted phenylurea herbicides once these herbicides are absorbed by tissues of susceptible or tolerant plants. Other important factors which affect the selectivity of this diverse class of herbicides include differential absorption, translocation, and phytotoxicity of metabolites (Feeny, 1968; van Oorschot, 1970; Rubin and Eshel, 1971; Kuratle et d., 1969; Hogue and Warren, 1968; Hogue, 1970; Eshel, 1969a). Susceptible plants, or plants with intermediate tolerance, often have a limited ability to N-demethylate substituted phenylurea herbicides to less
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toxic or nonphytotoxic metabolitcs (Swanson and Swanson, 1968a; Geissbuhler, 1969; J . W. Smith and Sheets, 1967; Rogers and Funderburk, 1968). In certain of these plants, differential metabolism of the generally less phytotoxic monomethylphenylurea metabolites may also be a factor in selectivity. Two new classcs of selective urea herbicides (Kubo et al., 1970; Eshel and Sompolinsky, 1970) are, in fact, substituted monomethyl ureas. The limited capacity of susceptible and moderately tolerant plants to completely detoxify the urea herbicides may reflect reduced enzyme activity and/or differences in enzyme specificity among the various plants and plant tissues. Recent reports (Kuratle el al., 1969; Rubin and Eshel, 1971) indicate that differential phytotoxicity of metabolites may also be a factor in the selectivity of substituted phenylurea herbicides. However, on the basis of these studies differential metabolism of the partially N-dealkylated metabolites could not be eliminated as the reason for the observed differences in phytotoxicity between susceptible and tolerant plants. In highly tolerant plants, a rapid and extensive metabolism of the substituted phenylureas to polar glucoside conjugates and insoluble or “bound” residues (Frear and Swanson, 1972; Nashed and Ilnicki, 1970) not only detoxifies the herbicide, but also immobilizes the herbicide and any partially detoxified metabolites so that they do not reach the primary site of action in the chloroplast.
E.
INTERACTIONS
The recovery of photosynthetic inhibition in monuron-treated cotton leaf disks was shown to be directly related to the rate of monuron metabolism (Swanson and Swanson, 1968a,b). Both the recovery of photosynthesis and monouron metabolism were inhibited by N-methyl carbamate insecticides. Since photosynthesis is generally considered to be the primary site of action for the urea herbicides, the leaf disk technique provides a rapid and relatively simple method for determining possible interactions between urea herbicides and other classes of pesticides. This technique may also permit the investigator to pinpoint interactions at specific metabolic steps. This technique was used in recent reports (Chang et al., 1971a,b) to demonstrate specific metabolic interactions between several different classes of herbicides and insecticides in a number of plant species. These studies established that linuron metabolism was inhibited by certain carbamate and organophosphate insecticides. The metabolism of several organophosphate insecticides was also inhibited by linuron. Experiments with isolated microsomal N-demethylase preparations friom cotton (Frear et al., 1969, 1972) have demonstrated that several substi-
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tuted N-methylcarbamate insecticides and anilide herbicides are effective inhibitors of substituted phenylurea N-demethylation. Thus, it appears that increased persistence of certain insecticides, herbicides, and their respective metabolites may result from biochemical interactions when more than one pesticide is present in the same plant tissue. In the case of herbicides that inhibit photosynthesis, any significant increase in the persistence of either the parent compound or any toxic metabolites in the leaf tissues may result in enhanced phytotoxicity. Similarly, the persistence of insecticidal activity and insecticide residues may also be increased in tissues containing certain herbicides. The significance of the observed molecular interactions between the substituted phenylurea. herbicides and other classes of pesticides and herbicides has not been established. Such studies do, however, illustrate many possible advantages for the use of leaf disks and isolated enzyme systems to screen a variety of compounds as modifiers of in vivo herbicide metabolism and detoxication in higher plants.
F. SUMMARY The substituted ureas represent a broad class of selective herbicides. However, except for a limited number of recent reports on a few of the substituted dimethyl- and methoxymethylphenylurea herbicides, our knowledge of the metabolic fate of these compounds in higher plants is fragmentary. The apparent importance of differential metabolism in the selectivity of the urea herbicides suggests that activity in this area of research, including in vitro enzyme studies, should be increased. The isolation, identification, and significance of unknown polar metabolites and insoluble or “bound” residues also requires additional research effort.
VI 11.
Ca rbama tes
A. CARBANILATES
I . Phenmedipham and Swep Phenmedipham is a carbanilate herbicide which has been recommended for postemergence application in the control of annual weeds in sugar beets. Very little information has been published concerning its metabolism. This compound is particularly interesting because it has two carbanilate functions in one molecule. Bischof et al. (1970) and Kassebeer ( 1971) have reported studies on the metabolism of phenmedipham in vari-
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ous plant species. These investigations showed that phenmedipham was readily absorbed and translocated by susceptible and resistant species. No correlation was observed between herbicide sensitivity and metabolism even though these plant species inactivated phenmedipham in different ways. Kassebeer ( 1971 ) suggested that phenmedipham was inactivated when ( 1) the herbicide was directly bound to plant constituents, (2) the herbicide was hydrolyzed and the decomposition products were bound to plant constituents, or (3) the herbicide was transformed to an unknown compound, possibly a hydroxylated phenmedipham. Kassebeer ( 1971) also suggested that hydroxylated phenmedipham may form complexes with plant constituents. Further studies must be conducted to determine the metabolic fate of phenmedipham in susceptible and resistant species. At this time, the data are incomplete, particularly as to the fate of the metatoluidine portion of the phenmedipham molecule. Swep has been used as a preemergence herbicide on large-seeded legumes and for pre- and postemergence weed control on rice. The metabolism of swep is a striking contrast to the metabolism of phenmedipham. Chin et al. (1964) reported that swep was complexed, without hydrolysis, into a stable lignin complex. From their studies, only limited amounts of free 3,Cdichloroaniline were detected. No work has been published on the metabolism of swep to indicate how the carbanilate is bound to the lignin complex. 2. Propham and Chlorpropham
Propham and chlorpropham have been used as selective herbicides for over 20 years. They are primarily used as preemergence herbicides for the control of annual grasses and broadleaf weeds in a variety of tolerant broadleaf crops. Until recently, little was known about their metabolism in plants. Prendeville et al. (1968) and James and Prendeville (1969) demonstrated the formation of water-soluble metabolites of chlorpropham as a result of plant metabolism. These polar metabolites were shown to be p-glucosides of a modified chlorpropham molecule in which there was no cleavage of the carbamate bond. They concluded that the water-soluble metabolite from foliar treated pale smartweek (Polygonum lapathofolium L.), redroot pigweed (Arnaranfus refroflexus L . ) , tomato, and parsnip (Pastinaca sativa L.) resulted from a modification of the 2-propyl ester portion of the chlorpropham molecule. Still and Mansager (1971, 1972a) showed that root-treated soybean plants absorb, translocate, and metabolize chlorpropham-"C. Polar products and insoluble residues were formed rapidly in root tissues while only polar metabolites were found in shoot tissues. Time-course experiments demonstratcd a precursor-product
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relationship between chlorpropham, polar products, and insoluble residues. Polar metabolites were not translocated once they were formed in either the root or shoot tissues. Studies with ring- and side chain-14C-labeled chlorpropham showed that the carbanilate bond was not cleaved during the formation of either the polar metabolites or the insoluble residual materials. When soybean plants were root-treated with chlorpropham, the predominant polar metabolite was isolated and identified as the 0-glucoside of isopropyl-5-chloro-2-hydroxycarbanilate. Root tissues rapidly converted chlorpropham and the polar glucosyl metabolite to insoluble residues. In shoot tissue, however, isopropyl-5-chloro-2-hydroxycarbanilate was a minor component of the polar metabolites and may be the precursor of other unidentified polar metabolic products. No insoluble residual materials were formed in the shoot tissues of soybean plants root-treated with chlorpropham. Isopropyl-5-chloro-2-hydroxycarbanilatehas been tested in plants and shown to have low phytotoxicity (Zick, 1971). The rapid formation of this metabolite apparently provides an effective detoxication mechanism in soybean plants. Plant enzyme studies (Still, 1971) have not been successful in demonstrating significant in vitro hydroxylation of the chlorpropham aryl moiety. The nature of the hydroxylating system and the mechanism by which soybean plants specifically hydroxylate chlorpropham at the ortho position hopefully will be shown in the future. The chemical character and the biological significance of the insoluble metabolites from chlorporpham metabolism in soybean root tissue should also be studied. The mechanism by which plants bind carbanilates to insoluble materials is a research area that warrants additional attention. This research should deal with both the metabolism of these materials in plants, and the metabolic fate of the insoluble residues in soils.
3. Barban Barban is a very selective postemergence herbicide used primarily for the control of wild oats in wheat. Initial plant metabolism studies on barban were reported by Riden and Hopkins (1961, 1962). These workers demonstrated the conversion of barban to a polar metabolite, compound X. This metabolite was shown to be a nonproteinaceous dialyzable material that yielded 3-chloroaniline upon caustic hydrolysis. The conversion of barban to aniline-containing polar metabolites occurred in 13 different plant species including grasses and broadleaf plants. The appearance and disappearance of the polar metabolite (compound X) was shown to be a function of time. Jacobsohn (1970) also demonstrated the conversion of foliar applied barban to polar metabolites and extended the investigation to root-treated wild oat (Avena futua L.) and barley plants. These plants
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also converted barban to 3-chloroaniline-containing polar metabolites. Riden and Hopkins ( 1962) reported that after foliar application of barban they were able to isolate 2-chloro-4-aminophenol from barban-treated plant tissues. In leaf-treated tissues, Riden and Hopkins (1962) concluded that barban-' *Cconcentration decreased with time and the 3-chloroaniline moiety of barban was complexed as polar metabolites. Still and Mansager ( 1972b) showed that root-treated soybean plants absorb, translocate, and mctabolize barban-"C. Water-soluble products and insoluble residues were readily formed in the roots. Only polar residues were found in shoots. Time-course experimcnts indicated a possible precursor-product relationship between barban, polar products, and insoluble residues. Pulse time-course studies indicate that the water soluble metabolites are not readily translocated from the tissues in which they were formed. The radiocarbon distribution in soybean plants root-treated with phenyl-I *C-, carbonyl-"C-, and butynyl- (C- 1) ,"C-labeled barban were identical in all comparative experiments. These studies indicate that the barban molecule was not cleaved in soybean. Soybean plants were roottreated with phenyl-*'C-labcled barban for 4 days and the polar metabolites isolated. Barban was altered in such a fashion that its polar metabolites were rendered highly reactive and volatile when they were isolated from normal plant constituents. In all cases, the aromatic nucleus of barban was not altered. This is in agreement with Riden and Hopkins (1962), who observed 3-chloroaniline as the hydrolysis product of compound X. This fact was substantiated by caustic hydrolysis of the purified barban-lT polar metabolites. The hydrolytic products were isolated and separated by gas-liquid chromatography and characterized by mass spectral analysis to prove that the radiolabel was in the 3-chloroaniline moiety of the polar metabolites. It was concluded that the 4-chloro-2-butynyl alcohol moiety was the reactive functional group of the barban molecule in soybean. The highly reactive nature of the alcohol moiety has also been indicated by the report of Lamoureux et al. ( 1971 ) on the formation of a barban glutathione conjugate. The polar metabolites found in the root and shoot were products of the active 4-chloro-2-butqnyl alcohol metabolism and perhaps precursors to the insoluble residual materials found in root and stem tissues. The absence of aryl hydroxylation of barban may be the result of the reactive 4-chloro-2-butynyl alcohol moiety and the rapid formation of polar metabolites which were not capable of enzymatic hydroxylation.
4 . Sunzmary The carbanilate herbicides still require a great deal of additional research effort before a complete understanding of their metabolism in plants is achieved. It does appear that a major route of metabolism of the car-
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banilate herbicides is through aryl hydroxylation and conjugation. The direct incorporation of swep into lignin is not understood and more data are required to interpret this observation. In the case of barban, the carbamyl alcohol moiety is sufficiently reactive so that polar metabolites are formed prior to aryl hydroxylation. Plants do not appear to cleave the carbamate bond of the carbanilate herbicides. This is contrary to the belief of a few years ago, and apparently quite different from the metabolism of these compounds in soils.
B. N-ALKYLCARBAMATES The N-methyl carbamates, such as the carbamate alkaloid physostigmine, have been of interest for many years as natural plant components. In the medical field, the carbamate prostigmine has been of interest because of its active anticholinergic actions. Early studies by Gysin (1954) and Kolbezen et al. (1954) showed that several N-methyl carbamates had insecticidal properties. Continued development of this class of compounds has provided a number of important commercial insecticides. Dorough and Casida (1964) and Kuhr and Casida (1967) have reported the plant metabolism of the insecticide carbaryl. Their studies show N-methyl hydroxylation to yield 1-naphthyl hydroxymethylcarbamate and the formation of 4-hydroxy-l -naphthyl methylcarbamate and 5-hydroxy- l -naphthyl methylcarbamate as a result of plant metabolism. The N-alkyl carbamates are generally recognized as insecticides; however, some of the N-methyl carbamates also exhibit herbicidal activity. Dichlormate, terbutol, and tandex are marketed as herbicides. They have low anticholinesterase activity and have been shown to act as plant growth regulators. Tandex is a mixed function herbicide containing both an N-alkyl carbamate and a phenylurea in the same molecule. Tandex is presently used as a defoliant along railroad, highway, and utility right-of-ways and has had no application in crop areas. Terbutol is a selective herbicide used for the control of crabgrass [Digitaria sanquinalis (L.) Scop.] in turf (Haubein and Hansen, 1965). Little or no information is available on the metabolism of either terbutol or tandex. Dichlormate is a unique herbicide that inhibits the synthesis of plant pigments (Herrett and Berthold, 1965), in sensitive species by specifically blocking the oxidation of [-carotene. Corn is moderately sensitive and bean is resistant to dichlormate. However, both species are capable of rapid and equally efficient metabolism of dichlormate following surface application to the epicotyl. Few metabolic differences have been observed in the metabolism of dichlormate-14C between susceptible and resistant plant species. Both species formed water-soluble degradation products with
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equal facility. Herrett ( 1970) reported the identification of metabolites from plants treated with dichlormate. The nonpolar fraction contained dichlormate, which constituted a major portion of the fraction, and four other products. Two of these products were identified as 3,4-dichlorobenzyl alcohol and 3,4-dichlorobenzoic acid. The polar component was treated with p-glucosidase to release aglucones which were isolated and characterized. Preliminary information (Herrett, 1970) indicates that the polar fraction contains glucosides of the N-hydroxymethyl derivative as well as the demethylated dichlormate. In addition to the above aglucones, the presence of 2-hydroxy-3,Cdichlorobenzylalcohol was indicated and thought to exist in the plant as the 2-hydroxydichlormate glucoside. However, this hydroxylated carbamate was not isolated. This observation is particularly interesting in view of the ortho-hydroxylation of chlorpropham which has been reported by Still and Mansager ( 1972a). The plant metabolism of dichlormate and the insecticidal N-alkyl carbamates are similar. In both cases, there was N-alkyl hydroxylation as well as hydroxylation of the aryl alcohol moiety.
IX.
A.
Anilides
INTRODUCTION
The anilides may be divided into two classes based on significant differences in chemical structure and metabolism. The first class is represented by propachlor, alachlor, and Prynachlor. In this class, the chloroacetamide nitrogen is substituted with a phenyl and an alkyl moiety and hydrolysis of the amide bond is hindered. The second class is represented by the 3,4-dichloroalkyl anilides propanil and dicryl. In propanil and dicryl, the aniline nitrogen has a free functional hydrogen and hydrolysis of the amide bond is generally considered to be the initial reaction in plant metabolism. All of these compounds are herbicides with selectivity toward various grasses and broadleaf plants. They are used both as pre- and postemergence treatments.
B.
PROPACHLOR
Most of the recent research on the 2-chloro-N-alkyl acid anilides has been conducted on propachlor. Jaworski and Porter (1965) reported the metabolism of propachlor in corn and soybean plants. Their studies indicated that the N-isopropylacetanilide molecule remained intact after the herbicide was converted to polar metabolites. There was no evidence indi-
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cating that the 2-chloro group was present in the polar metabolites found in the two crop plants. Acid hydrolysis of the polar corn metabolite yielded 2-hydroxy-N-isopropylacetanilide,which was strong evidence for the existence of a conjugated hydroxypropachlor intermediate. Frear and Swanson (1970) reported that propachlor reacted nonenzymatically in vitro with glutathione to yield the propachlor-glutathione conjugate. Lamoureux et al. (1971) studied the metabolism of propachlor in the leaves of corn, sorghum, sugarcane, and barley and identified two of the major polar metabolites as the glutathione conjugate and the 7-glutamylcysteine conjugate of propachlor. Both Lamoureux et al. (1971) and Porter and Jaworski (1965) reported that these propachlor metabolites were of a transitory nature. The character of the final product of propachlor metabolism in plants is not known. However, the ability of plants to detoxify halogenated compounds by conjugation with glutathione does appear analogous to the mechanism used by mammals to detoxify a wide variety of organic halides and related compounds. Although mercapturic acids appear to be the most common end products of metabolism via glutathione conjugation in mammals, the final products of metabolism via glutathione conjugation in higher plants have not been determined. C. PROPANIL The anilide propanil has been studied more extensively than dicryl. Propanil is a potent inhibitor of photosynthesis (Matsunaka, 1969b). When applied to intact rice or barnyardgrass [Echinochloa crus-gulli (L.) Beauv.], both species show rapid and essentially complete inhibition of CO, fixation within 1 hour. Recovery of photosynthesis occurs within 24 hours in the resistant rice treated with a 0.1% solution of propanil and after 48 hours when treated with a 1% solution. Corresponding treatments of barnyardgrass showed no apparent recovery. Several species of plants have been shown to cleave the propanil molecule (McRae et al., 1964; Adachi et al., 1966a,b). The metabolic fate of the propionic acid moiety of propanil was reported by Still (1968b). Propanil labeled in either C-1 or C-3 of the propionic acid moiety was applied to pea or rice plants in nutrient solution. Radioactivity was detected throughout the plant, but the greatest labeling was found in the roots. The fate of the propionic acid moiety of propanil was determined by recovery of 14C0, from plants exposed to pr~panil-'~C. Data indicated that the intact propionic acid was cleaved from the propanil and subsequently catabolized by p-oxidation. Both susceptible pea and tolerant rice converted a high percentage of the administered propanil-14C to 14C0,. Still ( 1968a) also conducted studies to determine the metabolic fate of the aniline moiety of the propanil
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molecule in rice plants. The first stable intermediate of propanil metabolism was N - ( 3,4-dichlorophenyl)glucosylamine. This metabolite was found in root and shoot tissues. Two other compounds were also reported and qualitative tests indicated that each had a reducing sugar component, but characterization was unsuccessful. Yih et al. (1968a) also reported transient propanil metabolites that contained 3,4-dichloroaniline conjugated with glucose, xylose, and fructose. Using propanil-phenyl-l'C as substrate, Yih et a/. (1968a) reported that 34% of the radiolabel was not extracted with methanol and remained in the plant tissues 14 days after foliar treatment. Their studies indicated that a major portion of the 3,4-dichloroaniline moiety was complexed with the polymeric cell constituents, mainly lignin. The aniline was lignin-bound as 3,4-dichloroaniline and not as propanil. In other studies with intact plants, Yih et a/. (1968b) speculated that the biotransformation of propanil to 3,4-dichloroaniline was not a direct hydrolysis but rather an oxidative metabolism to an intermediate molecule 3,4-dichlorolactanilide, which subsequently was hydrolyzed to 3,4-dichloroaniline and lactic acid. This study indicated that the 3,4-dichlorolactanilide was a transient intermediate in rice and not normally observed. However, it accumulated in barnyardgrass, a susceptible species. Adachi et al. (1966a,b) and Still and Kuzieran (1967) reported the presence of hydrolyzing enzymes from homogenates of rice and barnyardgrass leaves. From these studies, it was shown that rice tissue homogenates contained active enzymes capable of hydrolyzing propanil to 3,4-dichloroaniline. Adachi (1966b) showed that rice homogenates were inhibited by the addition of carbaryl. Frear and Still (1968) reported the properties of a partially purified rice aryl acylamidase capable of metabolizing propanil. The enzyme was present in all tissues of rice and barnyardgrass studied. Rice leaves contained 60 times more enzyme units than barnyardgrass leaves. Rice roots and barnyardgrass roots contained the same amount of enzyme activity, but this was only 3% of the enzyme units found in rice leaves. The authors suggested that this difference in enzyme distribution was of significance in the selectivity of propanil between these two species. The enzyme displayed a broad specificity for chlorinated ringsubstituted propionanilide analogs, but was specific for the 3,4-dichloropropionanilide when compared with several alkyl-substituted analogs. The enzyme was strongly inhibited by insecticidal carbamates and to some extent by the organophosphates. The inhibition by insecticidal carbamates appeared to be competitive. The rice enzyme was shown to be inactive in the hydrolysis of several urea and carbamate herbicides. The results from this enzyme study explained the observations of Bowling and Hudgins (1966), and Hisada (1967), who reported that combinations of carbamates and organophosphates increased the phototoxicity of propanil in
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field rice. Thus, inhibition of the aryl acylamidase activity appears to increase the phytotoxicity of propanil to field rice. Summary
In spite of the great amount of research on the metabolism of propanil, both in Japan and in this country, much work still remains to be done. The detoxication of the parent herbicide, propanil, by the aryl acylamidase appears to be a major factor in selectivity. The metabolism of the propionic acid moiety proceeds via catabolic processes in the plant. However, there is little information as to the metabolic fate of the chloroaniline moiety. In plants, present data indicate that the chloroaniline moiety is conjugated with carbohydrates, which are subsequently incorporated into insoluble plant residues. The ability of plants to conjugate and incorporate the chloroaniline molecule into insoluble residues may provide a valuable mechanism for the eventual degradation and effective removal of chloroaniline based herbicides from the environment. However, additional research on the biochemical mechanism and the characterization of the metabolic products of chloroaniline metabolism in plants are needed before such an important degradation process is fully understood. Little information is available on the fundamental biochemistry of lignification. Perhaps basic investigations on the metabolic fate of anilines in plants may also shed light on this problem.
X.
Summary
It is evident from this review that much new information is available on the behavior and fate of herbicides in higher plants. Although the return from this research effort has been impressive, we have only begun to understand this important class of pesticides. Large voids exist in our knowledge of the behavior and fate of other classes of pesticides in plants (Sijpesteijn, 1969; Casida and Lykken, 1969; Menzie, 1969). Little information has been published on the metabolism and ultimate fate of many of the herbicides used today. Much of the information that is available is fragmentary and incomplete. Many of the polar metabolites remain unidentified and none of the insoluble residual materials have been characterized to any significant extent. These materials often represent a major portion of the original herbicide molecule absorbed by the plant. Since plants cannot readily excrete these degradation products, they often remain in the plant tissues as “terminal” residues. The residence times of these residues in nature and their significance in the environment has not been established.
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Increasing evidence suggests that differential metabolism plays an important role in herbicide selectivity. Differences in the rate and extent of metabolism often have a significant effect on the ability of a herbicide or any of its phytotoxic metabolites to reach the primary site(s) of action in the plant. Recent in vitro studies indicate that differences in the activities, specificities, and distributions of key plant enzyme systems responsible for herbicide metabolism also play a direct and important role in selectivity. These enzymes are genetically controlled and their activities may be affected by various reaction products, inhibitors, and other pesticides. Studies of differential herbicide metabolism and the enzymes responsible for herbicide biotransformations in plants represent a new and challenging area of pesticide research. The results of studies in this area of research will provide valuable information for the development of more selective and efficient herbicides. The need for additional research on the behavior and fate of pesticides in plants is evident. The authors hope that this research need has been adequately documented and illustrated in the present review. We also hope that scientists will be encouraged to devote their energies and ingenuity to filling in the many missing pieces in this fascinating and difficult research puzzle. REFERENCES Adachi, M., Tonegawa, K., and Ueshima, T. 1966a. Pestic. Tech. 14, 19-22. Adachi, M., Tonegawa, K., and Ueshima, T. 1966b. Pestic. Tech. 15, 11-14. Adams, R. S. Jr., Baker, D. G., and Nelson, S. E. 1970. Weed Sci. SOC. Amer. Abstr. p. 21. Alder, E. F.. Wright, W. L., and Soper, Q. F. 1960. Proc. N . Cent. Weed Contr. Conf. 17, 23. Anderson, W. P., Richards, A. B., and Whitworth, J. B. 1967. Weeds 15, 24-227. Arle, €3. F. 1968. Weed Sci. 16, 4 3 W 3 2 . Arnold, W. E., and Nalewaja, J. D. 1971. Weed Sci. 19, 301-305. Ashton, F. M., and Tsay, R. C. 1970. Weed Sci. SOC.Amer. Abstr. p. 34. Ashton, F. M., Penner, D., and Hoffman, S. 1968. Weed Sci. 16, 169-171. Bakke, J . E., Shimabukuro, R. H., Davison, K. L., and Lamoureux, G. L. 1972. Chemosphere 1, 21-24. Barrentine, W. L., and Warren, G. F. 1970. Weed Sci. 19, 31-41. Baur, J. R., and Bovey, R. W. 1969. Weed Sci. 17, 524-528. Bayer, D. E., and Yamaguchi, S. 1965. Weeds 13, 232-235. Bayer, D. E., Foy, C. L., Maflory, T. E., and Cutter, E. G . 1967. Amer. J . Bot. 54, 945-952. Bayer, G. H., Hargan, R. P., Cialone, J. C., and Sweet, R. D. 1963. Proc. Northeast. Weed Contr. Conf. 17, 91-97.
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Beynon, K. I., and Wright, A. N. 1968. J. Sci. Food Agr. 19, 727-732. Bischof, Von F., Koch, W., Majumdar, J. C., and Schwerdtle, F. 1970. Z . Pflanzenkr. (Pflunzenpathol.) Pflanzenschutz, Sonderk. 95-102. Biswas, P. K., and Hamilton, W., Jr. 1969. Weed Sci. 17, 206-211. Bohme, C., and Bar, F. 1967. Food Cosmet. Toricol. 5, 23-28. Bovey, R. W., Davis, F. S., and Merkle, M. G. 1967. Weeds 15, 245-249. Bowling, C. C., and Hudgins, H. R. 1966. Weeds 14, 94-95. Boyland, E., and Chasseaud, L. F. 1969. Advan. Enzymol. 32, 173-219. Broadhurst, N. A., Montgomery, M. L., and Freed, V. H. 1966. I . Agr. Food Chem. 14, 585-588. Casida, J. E., and Lykken, L. 1969. Annu. Rev. Plant Physiol. 20, 607-636. Castelfranco, P., Foy, C. L., and Deutsch, D. B. 1961. Weeds 9, 580-591. Chang, F. Y.,and Vanden Born, W. H. 1971. Weed Sci. 19, 107-112. Chang, F. Y.,Smith, L. W., and Stephenson, G. R. 1971a. J. Agr. Food Chem. 19, 1183-1 186. Chang, F. Y.,Stephenson, G. R., and Smith, L. W. 1971b. 1. Agr. Food Chem. 19, 1187-1190. Chin, W. T., Stanovick, R. P., Cullen, T. E., and Hoking, G. C. 1964. Weeds 12, 201-205. Colby, S. R. 1965. Science 150, 619-620. Davies, P. J., Drennan, D. S. H., Fryer, J. D., and Holly, K. 1967. Weed Res. 7, 220-233. Davies, P . J., Drennan, D. S. H., Fryer, J. D., and Holly, K. 1968a. Weed Res. 8, 233-240. Davies, P. J., Drennan, D. S. H., Fryer, J. D., and Holly, K. 1968b. Weed Res. 8, 241-252. Davis, D. E., Gramlich, J. V., and Funderburk, H. H., Jr. 1965. Weeds 13, 252-255. Devlin, R. M., and Cunningham, R. P. 1970. Weed Res. 10, 316-320. Dorough, H. W., and Casida, J. E. 1964. I . Agr. Food Chem. 12,294-304. Eastin, E. F. 1969. Plant Physiol. 44, 1397-1401. Eastin, E. F. 1971a. WeedRes. 11, 63-68. Eastin, E. F. 1971b. Weed Res. 11, 120-123. Eastin, E. F. 1971c. Weed Sci. 19, 261-265. Ebner, L., Green, D. H., and Pande, P. 1968. Proc. 9th Brit. Weed Contr. Conf. pp. 1026-1032. Eshel, Y. 1969a. Weed Sci. 17, 492-496. Eshel, Y. 1969b. Weed Res. 9, 167-172. Eshel, Y.,and Sompolinsky, D. 1970. Weed Res. 10, 196-203. Espinoza, W. G., Adams, R. S., Jr., and Behrens, R. 1968. Agron. J. 60, 182-185. Feeny, R. W. 1966. Proc. Northeast. Weed Contr. Conf. 20, 595-603. Feeny, R. W. 1968. Diss. Abstr. 20, 2261-B. Fisher, A. 1964. I n “Vortriige Anlasslich der Wissenschaftlichen Aussprache uber Chemische Unkrautbekampfung in Zuckerriiben mit Pyramin,” pp. 19-24. Badische Anilin und Soda-Fabrik AG, Ludwigshafen am-Rhein, Germany. Foy, C. L., and Penner, D. 1965. Weeds 13, 226-231. Frank, R., and Switzer, C. M. 1969a. Weed Sci. 17, 344-348. Frank, R., and Switzer, C. M. 1969b. Weed Sci. 17, 365-370. Frear, D. S. 1968a. Phytochemistry 7, 381-390.
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Hilton, J. L., and Christiansen, M. N. 1972. Weed Sci. (in press). Hilton, J. L., Jansen, L. L., and Hull, H. M. 1963. Annu. Rev. Plant Physiol. 14, 353-384. Hilton, J. L., Scharen, A. L., St. John, J. B., Moreland, D. E., and Norr4is, K. H. 1969. Weed Sci. 17, 541-547. Hisada, T. 1967. Proc. 1st Asian Pac. Weed Contr. Interchange p. 107. Hogue, E. J. 1970. Weed Sci. 18, 580-582. Hogue, E.J., and Warren, G. F. 1968. Weed Sci. 16, 51. Horvath, R. S. 1971. J. Agr. Food Chem. 19, 291-293. Hurter, J. 1966. Experientia 22, 741-742. Hutson, D. H., Hoadley, E. C., Griffiths, M. H., and Donninger, C. 1970. J. Agr. Food Chem. 18, 507-512. Jacobsohn, R. 1970. Ph.D. Thesis, University of Minnesota, St. Paul. James, C. S., and Prendeville, G. N. 1969. J . Agr. Food Chem. 17, 1257-1260. Jaworski, E. G., and Porter, C. A. 1965. 149th Meet., Amer. Chem. Soc., Detroit Abstr. No. 20. Jones, D. W., and Foy, C. L. 1971. Weed Sci. 19, 595-597. Kassebeer, Von H. 1971. Z . Pflanzenkr. (Pflanzenpathol.) Pflanzenschutz 78, 158174. Kearney, P. C., and Kaufman, D. D., eds. 1969. “Degradation of Herbicides.” Dekker, New York. Kefford, N. P., and Caso, 0. H. 1966. Bot. Gaz. 127, 159-163. Klun, J. A., and Robinson, J. F. 1969. 1. Econ. Entomol. 62, 214-220. Knuesli, E., Berm, D., Dupuis, G., and Esser, H. 1969. In “Degradation of Herbicides” (P. C. Kearney and D. D. Kaufman, eds.), pp. 51-78. Dekker, New York. Kolbezen, M. J., Metcalf, R. L., and Fukuta, T. R. 1954. 1. Agr. Food Chem. 2, 864-870. Koopman, H., and Daams, J. 1965. Weed Res. 5, 319-326. Kubo, H., Sato, R., Hamura, I., and Ohi, T. 1970. J. Agr. Food Chem. 18,60. Kuhr, R. J., and Casida, J. E. 1967. 1. Agr. Food Chem. 15, 814-824. Kuratle, H., Rahn, E. M., and Woodmansee, C. W. 1969. Weed Sci. 17,216-219. Lamoureux, G.L. 1971. Unpublished data. Lamoureux, G. L., Shimabukuro, R. H., Swanson, H. R., and Frear, D. S. 1970. J . Agr. Food Chem. 18, 81-86. Lamoureux, G. L., Stafford, L. E., and Tanaka, F. S. 1971. J. Agr. Food Chem. 19, 346-350. Larson, J, D., Bakke, J. E., and Feil, V. J. 1970. Joint Conf. Chem. Inst. Can., Amer. Chem. SOC.Pest. Div., Abstr. No. 8. Locke, R. K., and Baron, R. L. 1971. Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, Abstr. 2054. McRae, D. H . , Yih, R. Y., and Wilson, H. F. 1964. Weed Sci. Soc. Amer., Nut. Meet. Abstr. No. 87. McWhorter, C. G . 1963. Weeds 11, 265. Magalhaes, A. C., and Ashton, F. M. 1969. Weed Res. 9, 48-52. Magalhaes, A. C., Ashton, F. M., and Foy, C. L. 1968. Weed Sci. 16, 240-245. Maroder, H. L., and Prego, I. A. 1971. Weed Res. 11, 193-195. Mason, G.W. 1959. Ph.D. Thesis, University of California, Davis. Matsunaka, S. 1969a. J . Agr. Food Chem. 17, 171-175. Matsunaka, S. 1969b. Residue Rev. 25, 45-58.
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Matsunaka, S. 1970. Zusso Kerikyo 10, 40-43. Meikle, R. W., Williams, E. A., and Redemann, C. T. 1966. J. Agr. Food Chem. 14, 384-387. Melnikov, N. N. 1971. Residue Rev. 36, 480. Menzie, C. N . 1969. U.S.. Fish Wildl. Serv., Spec. Sci. Rep.: Wildl. 127, 1-487. Milborrow, B. V . 1963. Biochem. J . 87, 255-258. Milborrow, B. V: 1965. Weed Res. 5, 332-342. Montgomery, M. L., and Freed, V. H. 1961. Weeds9,231-237. Montgomery. M. L., and Freed, V. H. 1964. 1. Agr. Food Chem. 12, 11-14. Montgomery, M. L., Botsford, D. L., and Freed, V. H. 1969. 1. Agr. Food Chem. 17, 1241-1243. Moreland, D. E. 1967. Annu. Rev. Plunt Physiol. 18, 365-386. Moreland, D.E . 1969. Progr. Photosyn. Res. 3, 1693-171 1 . Moreland, D. E., Gentner, W. A., Hilton, J. L., and Hill, K. L. 1959. Plant Physiol. 34, 4 3 2 4 3 5 . Moreland, D. E., Blackmon, W. J., Todd, H. G., and Farmer, F. S. 1970. Weed Sci. 18, 636-641. Mueller, P. W., and Payot, P. H. 1966. Proc. I A E A Symp. Isotopes, Weed Res., 1965 p . 61. Nashed, R. B., and Ilnicki, R. D. 1970. Weed Sci. 18,25-28. Nashed, R. B., Katz, S . E., and Ilnicki, R. D. 1970. Weed Sci. 18, 122-128. Negi, N. S., and Funderburk, H. H., Jr. 1968. Weed Sci. SOC. Amer. Abstr. pp. 37-38. Negi, N . S., Funderburk, H. H., Jr., Schultz, D. P., and Davis, D. E. 1968. Weed Sci. 16, 83-85. Neumeyer, J., Gibbons, D., and Trask, H. 1969. Chem. Week Rept: “Pesticides,” Parts I and 11, 38-68. Oliver, L. R., and Frams, R. E. 1968. Weed Sci. 16, 199-203. Pate, D. A., and Funderburk, H. H., Jr. 1966. Proc. I A E A Isotopes, Weed Res., 1965 p p . 17-25. Perida, J . F. 1971. Diss. Absfr. 32, 652-B. Peterson, R. L., and Smith, L. W. 1971. Weed Res. 11, 84-87. Plimmer, J. R. 1970. Encycl. Chem. Technol. 22, 174-220. Porter, C. A., and Jaworski, E. G. 1965. Proc. Amer. SOC. Plant Physiol. Abstr. pp. XIV-xv. Prendeville, G. N., Eshel, Y., Jones, C. S., Warren, G. F., and Schreiber, M. M. 1968. Weed Sci. 16, 432-435. Probst, G. W., and Tepe, J. B. 1969. In “Degradation o f Herbicides” (P. C. Kearney and D.D. Kaufman, eds.), pp. 255-282. Dekker, New York. Probst, G. W., Golab, T., Herberg, R. J., Holzer, F. J., Parka, S. J., Van der Schans, C., and Tepe, J. B. 1967. 1. Agr. Food Chem. 15, 592-599. Ragab, M. T. H., and McCoilum, J. P. 1961. Weeds 9, 72-84. Rahman, W., and Ashford, R. 1970. Weed Sci. 18, 754-759. Ray, B., and Wilcox, M. 1969. Physiol. Plunt. 22, 503-505. Redemann, C. T., Meikle, R. W., Hamilton, P., Banks, V. S., and Youngson, C. R. 1968. Bull. Environ. Conturn. Toxicol. 3, 80-96. Riden, J. R., and Hopkins, T. R. 1961. J . Agr. Food Chem. 9 , 4 7 4 9 . Riden, J . R., and Hopkins, T. R. 1962. J. Agr. Food Chem. 10, 455-458. Ries, S. K., Zabik, M. J., Stephenson, G. R., and Chen, T. M. 1968. Weed Sci. 16, 40-41. Rogers, R. L. 1971. I. Agr. Food Chem. 19, 32-35.
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CRITICAL CATION ACTIVITY RATIOS Philip Beckett Soil Science Laboratory, Deportment of Agricultural Science, University of Oxford, Oxford, Englond
I. Introduction
....................................................
11. Cation Activity Ratios in Relation to Nutrient Uptake or Plant Growth. .
A. Cation Activity Ratios ........................................ B. Necessary Conditions ........................................ C . Experimental. Problems ...................................... 111. Experimental Evidence ........................................... A. Published Work ............................................. B. Synthesis ................................................... IV. Threshold Ratios ................................................ A. Published Values of Critical or Exhaustion Ratios . . . . . . . . . . . . . . . . B. Field Measurements of Critical Ratios .......................... c. Conclusions ................................................ References .....................................................
I.
379 380 380 381 383 385 385 390 393 393 402 407 408
Introduction
The very successful application of free energy measurements to studies on the availability of soil water has encouraged attempts to do the same for other plant nutrients, such as Na, Mg, and Ca, and reviewed K, elsewhere (Beckett, 1971). It is unfortunate that many experiments on this have attempted to demonstrate a relation between growth, or nutrient uptake, and some function of the free energy of a nutrient, under conditions in which such a relation would seem to be intrinsically improbable. This review (1) briefly recapitulates the problems of measuring the free energies of single ions in the soil, which have led to the use of cation activity ratios; (2) explores the conditions that must be met if the rate of uptake or the total uptake of a nutrient, or if plant growth, is to be regulated by activity ratios or free energy differences in soil or culture solution; ( 3 ) reviews experimental results bearing on these conditions; (4) presents a tentative model which relates uptake or growth to the quantities of labile nutrient held at activity ratios in excess of some.critica1 or exhaustion value; (5) reviews the published data on critical or exhaustion values of cation activity ratios, and presents some unpublished field data. 379
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The discussion relates to all cation nutrients, the data mainly to K, Mg, and Ca. II.
Cation Activity in Relation to Nutrient Uptake or Plant Growth
A. CATIONACTIVITY RATIOS In the use of free energy measurements one complication arises at once. The magnitude of the electrochemical potential px of a single ion X at a given locus in the soil comprises two contributions of which, unlike soil water, only one is determined by a, the chemical activity of X: the other is determined by I), the electrical potential at that locus, so that px = pro
+ RT In a, + vX . f'$ + (any other electrostatic interactions)
(1) where p,n is the electrochemical potential of X in a standard state of unit activity and zero electrical potential, and V , is its valency. In free solution a, may be calculated from c, f x in which c, is the concentration of X, which may be measured, and f x its activity coefficient, which may be calculated. But even in a system at equilibrium RTIn a, will differ between any loci at different q, which is commonly not known or measurable. In particular I) will differ between the soil solution where cxfx may be determined, and the inner parts of the double layers of the exchange phase where most labile cations are retained, and from which their free energies in the soil are controlled (Beckett, 1971). Even if a soil contains a constant quantity of some labile cation in the exchange phase, and at constant activity coefficient, a, as measured in the equilibrium soil solution will fluctuate with any changes in the amount of electrolyte, etc., in it, which alter AI) between the free solution and the inner part of the diffuse layers of the exchange phase. Thus except under special conditions or with particular assumptions, it is not possible to determine a meaningful value for (pX- p x o ) for a single ion. The problem of I) is avoided, however, if we measure the difference between the electrochemical potentials of equivalent amounts of two ions (A and B ) , as in
-
V A
VB
which is constant in all parts of the solution, double layer, or solid phases that are at equilibrium.
CRITICAL CATION ACTIVITY RATIOS
381
This is commonly done. The term R T In (aA1/”a) /( is referred to as AG or AF, and u ~ ~ / ~ A /asu the ~ ~reduced / ~ B activity ratio, or simply as the activity ratio (AR), and here as AR,-,.l Such quantities have the disadvantage that they only allow pA to be measured against the value of pB in the same soil. They have been offered by several workers as measures of the immediate availability (or intensity) of A relative to a “reference ion” B in the same soil. Their use has been reviewed elsewhere (Beckett, 1971 ) . B. NECESSARY CONDITIONS We should examine the conditions that must be met for it to be reasonable to expect nutrient uptake or plant growth to be correlated with values of AR or AG. A priori one can predict that AR,-, or hGA-, will describe the availability of A only:
if (1)
the rate of uptake (or amount of uptake = rate x time) of A depends on pA (in turn regulated by the amount and state of combination of A in the soil) and on no other soil or plant factors (except for pB), nor on nonequilibrium processes;
and (2a) pB is relatively uniform through all parts of the system being compared, and relatively unchanged by uptake or excretion during plant growth, or (2b) ion B is the main or only factor which limits the uptake of A, in a system of given F,. For ARA-, or hGA-, to describe the availability of A, conditions (1) and (2) must both be met. That these are necessary conditions is selfevident and requires no experimental proof. Condition ( 1) is sometimes overlooked; if an experiment or field trial is organized in such a way that the uptake of A is controlled mainly by factors other than the availability of A or B it is unlikely that the uptake of A, or crop yield, will be closely correlated with ARA-, or A G ~ - ~ . Condition ( 1) may be expanded further: ( l a ) Ap, the difference in the equivalent free energies of ions A and B at the moment of offering themselves to the uptake sites, where and whatever these may be, must be equal to the difference between their equivalent free energies in the free soil solution, or
‘D is used for (Ca + Mg); ARcn->rgis UC./UM,
_ _
not ~ U C J ~ U M ~ .
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PHILIP BECKETT
consistently related to it. The term “uptake site” involves no assumptions about the nature of the uptake process; it represents no more than the stage in ion transfer from free solution to stele, at which nutrient ions may cease to be at equilibrium with the free solution. ( 1 b ) The rate of uptake of A must be regulated by: (i) the difference in the equivalent free energies of A and B as offered to the uptake sites, or
(ii) the ratio of the numbers of ions A and B offered to the uptake sites, at the onset of the uptake process. This will depend on the difference in their equivalent free energies in solution, but also on A$ between the uptake sites and the free solution. If, further ( 3 ) the relative amounts of A and B ions translocated to the aboveground parts of the plants over a short time, or the amount of A, are proportional to their average rates of uptake, then the total uptake of A or of B, and the ratio of their total uptakes, over a period, may be regulated by the controlling values o€ AR,,-, (or - I G , - ~ ) . If, also, (4) the growth or yield of the organs harvested are regulated or proportional to the total amount of A translocated, then growth or yield may be regulated by AR,-, (or AGA-R)as measured in soil or solution. All of conditions (1) and (21, and ( 3 ) or (4) must hold if uptake of A, or if plant growth, is to be associated with AR,-,, or AGA-*. Furthermore if the association between uptake or growth, and AR or AG, is to be described by a linear or simple polynomial regression, each of the associations implicit in conditions ( l b ) , ( 3 1 , and (4) will have to be of a simple form. If any one link in these chains of conditions does not hold, then uptake or growth will not be found to be associated with ARA-, or AGA-R. But if uptake or growth are not found to be associated with AR or AG, this does not necessarily demonstrate that more than one condition has not been met. In particular, if such associations are not observed, it does not necessarily prove that ( 1 ), or (la)-( l b ) , are untrue. If the association between uptake or growth, and AR or AG, is found not to be described by a simple regression, this does not necessarily demonstrate that any of conditions (1)-(4) are erroneous; it may show no more than that one
CRITICAL CATION ACTIVITY RATIOS
3 83
of associations (1 ), ( 3 ) , or (4) is more complex in form than the regression which was found not to fit a combination of them all.
C. EXPERIMENTAL PROBLEMS The experimental problems are considerable. For example, it has not always proved possible to sustain the nutrient concentrations in solution culture experiments (Loneragan and Asher, 1967), so the experimental plants have not always experienced the activity ratios intended or reported. It is not always easy to maintain adequate concentrations of all other nutrients-to prevent their deficiency becoming limiting and invalidating condition ( 1 )-without creating conditions in which the activity ratio that actually controls growth is other than the one it was hoped to study; this ambiguity is apparent in many of the data in Table I, p. 396. In several cases the plant material employed in uptake or growth experiments has beeh brought to the experiment in a relatively starved or unbalanced condition, or in a phase of rapid root extension, so that the rates of uptake have been affected by the recharging of cytoplasmic “buffers” or by the demands of rapid cell development in the roots, and the amounts of nutrients translocated to the shoots may have been curtailed. Further difficulties arise in pot or field experiments. When experimental plants are grown in soil, it is less easy to ensure that other factors of nutrients or environment do not affect the uptake of particular nutrients, or the resulting growth. It is not easy, and often impossible, to ensure that the soil, which is to be analyzed after plant growth, has been uniformly depleted, and that the root distribution has been dense enough for the soil to have been exploited uniformly (Nye, 1969). Indeed, since the value of AR or AG must have decreased during the uptake period, it is not clear to what value of AR or AG growth or uptake should be related. For some nutrients, and particularly K, initially nonlabile forms become labile during, and possibly as a result of, depletion; the rate or amount of such release may, but need not, be related to the values of AR or AG observed before, during, or after depletion. These are all practical problems. There are also problems of experimental design. The first of these has already been mentioned; while it may prove possible to relate the rate of uptake of a given nutrient to the AR or AG of a solution in which their values are maintained constant by stirring and replenishment, this will not be possible when plants are growing in soil, in which inevitably AR must fall as a result of the depletion of the nutrient in the numerator. Several workers have attempted to relate the total uptake of a nutrient, or plant growth, over an extended period, to values of AR or AG measured before or after cropping. Unless the initial
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PHILIP BECKETT
or final AR or AG are correlated with the amount of nutrient present, and also uptake is controlled by this amount, there seems to be no more reason why these should be causally related to nutrient uptake than there is for water uptake to be correlated with the free energies of water at field capacity or wilting point at which water uptake started or ended. If rate of uptake were related to AR or AG it is possible that the total uptake of a nutrient (as a time integral of rates of uptake) might be related to a corresponding time integral of the appropriate function of AR or AG during uptake; this has not been attempted. The interpretation of experimental results is yet further confused by the fact that the Q/Z relations [to relate the amount ( QA) of a nutrient present, to its activity ratio ARa-B; see Fig. 31 of different plots in one field or differently treated samples of one soil, are geometrically similar figures. Q/Z relations commonly have a curved lower part over which Q.4 a log AR,_, or AR,-, a log ( l/QA) (Talibudeen and Dey, 1968a,b; Addiscott, 1970) and a linear upper part over which QA a AR,+ Thus for a range of soils with similar Q/Z relations, the amount of labile nutrient A ( Q A ) removed, when the soils are depleted to a fixed low or zero value of ARA-n, may be proportional to their initial ARA-, if the depleted A R s lie on the linear part, and to log (initial AR) if the initial and final AR’s both lie on the curved part, and somewhere between if the initial AR lies on the linear part and the final AR lies on the curved part. There are similar correlations which relate uptake of the nutrient to final AR or log (final AR) . Thus, even if uptake and growth were found to be proportional to AR or AG, it would still not be possible to infer unequivocally whether they were regulated by free energy properties of the nutrient (ARA-B),or by the amount (Q.*)of labile nutrient present (Arnold, 1962). In addition, if the initial AR lies on the linear part of the Q/Z relation and the final AR on the curved part, there could well be no simple regression of uptake or growth on AR or AG, even if the relation were wholly causal. Finally, under various circumstances, the rate or amount of release of fixed K may be proportional to the depleted AR, to the initial AR, or to the difference between the prevailing AR and some equilibrium value for the soil. The rate or amount of K released from nonlabile forms may also be proportional to the clay content of the soil (Arnold and Close, 1961; Smith and Matthews, 1957; Tabatai and Hanway, 1969); this is often proportional to the gradient of the linear part of the Q/Z relation (e.g., Acquaye and Maclean, 1966) which under some conditions is also correlated with the prevailing AR, or with the amount of labile nutrient present. As a result, the uptake of K, or crop growth, can be, and have been, found to be correlated to AR or AG, both properties of labile nu-
CRITICAL CATION ACTIVITY RATIOS
385
trients, even under circumstances when most of the K was not taken up from labile sources at all. Further confusion is introduced by authors who use a “quick” or approximate method of measuring AR (e.g., Beckett, 1971), which does not in fact measure AR but measures a value intermediate between AR and zero, which is some combined function of AR and the gradient of the Q/Z relation, and which gives an approximate measure of the quantity Q of labile nutrient present. In such cases what purports to be a correlation between uptake, etc., and a “quick” AR (as an intensity factor) is in fact a correlation between uptake and Q (a quantity factor). All these make it difficult to conduct experiments, and in many cases to interpret them. They must affect the weight we can give to conclusions for or against an ‘association betwsen growth or uptake, and AR or AG.
Ill.
A.
Experimental Evidence
PUBLISHED WORK
Having, briefly, reviewed the conditions under which nutrient uptake or plant growth might be found to be associated with AR or AG, we may now examine the experimental evidence. It is accepted that there is physicochemical equilibrium between the “free spaces,” between and in the cell walls of cortical and epidermal cells in roots, and the external soil or culture solution. This is hardly surprising, since the so-called “Donnan Free Space” is measured as that part of the root volume in which ions are in physicochemical equilibrium with the solution. There is abundant evidence of electrical charges round the walls of the free space (e.g., Dainty and Hope, 1961), at which “exchangeable ions” are retained in equilibrium with the external solution. There have also been numerous measurements of root “cation exchange capacity,” though mostly by rather brutal methods yielding results that need not necessarily apply to normally functioning roots. Several workers have suggested that these sites are probably not the seat of uptake (Peech and Lagerwerff 1961; Mengel, 1961; Yoshida, 1964) or at least not predominantly so, since the uptakes of particular ions are usually not in proportion to the fractions of such sites they might be thought to occupy. Even so, if the uptake sites are contiguous to or separated from the free solution by charged sites they are likely to be separated from the external solution by a difference in electrical potential. In this case the proportions of ions of different valency (e.g., AJB,) which occupy the
3 86
PHILIP BECKETT
uptake sites at the moment prior to the start of the uptake process will still be regulated by something like Bolt’s (1955) form of the Gapon equation (Eq. 3 ) ,
where I’ is the density of charge on the surfaces which include the uptake sites. There is some evidence, albeit less certain, that the ratio of, e.g., K/Ca uptake from solutions of given ARK-c.,increases as root “cation exchange capacity” decreases (e.g., Drake et al., 1951) in the sense required by Eq. ( 3 ) . This marches with relatively consistent reports of differences between the “cation exchange capacities” of the roots of mono- and dicotyledonous plants, and their capacities for taking up potassium and calcium (e.g., Huffaker and Wallace, 1958). More facts are needed. It is at least clear that, if AJB,, is in any way regulated by l/r as in Eq. ( 3 ) , A,,/B,, is unlikely to equal aJa, if the ions are of different valency; any kinetic analyses of hypothetically antagonistic or synergistic interactions between ions, which assume such equality, are likely to be misleading. Barber and Russell (1961) have shown that a root’s capacity to hold exchangeable ions is in part metabolically controlled; even for a given ARa-, the value of A,,/B,, is likely to vary as the metabolic state of the root modifies I/r. Indeed it is not impossible that some of the synergistic effects (below) claimed for Ca may be due to its blocking effects on otherwise ionized pectate groups (cf. Overstreet et al., 1952; Pierre and Bower, 1943) . Over a considerable range of concentration, the rate of uptake of one nutrient from a culture solution commonly depends on its concentration, up to some ceiling value (Briggs et al., 1961; Overstreet et al., 1952; Lagerwerff and Peech, 1962; Russell and Squire, 1958; Russell et al., 1954). In sand culture experiments, Wild et al. (1969) show no relation between K uptake or dry matter production, and ARK r) or pLRf(-I\Iz,down to ARK., = 0.006 M“’. The rate is affected by transpiration and by other physical factors; it may be increased by small amounts of other ions [e.g., Ca stimulates K and Rb uptake from acid solutions (Moore et al., 1961), K uptake is stimulated by Ca or Mo (Overstreet et al., 1952; Jones et al., 1961; Baroccio, 1962), Rb uptake by Ca (Lagerwed and Peech, 1962), etc.]. On the other hand, there are antagonistic interactions between ions. For example the rate of uptake of Ca is reduced by K (Overstreet et al., 1952), of Sr by Ca (Russell and Squire, 1958), of K by Na (Jacobson et al., 1950), and of Mg by Ca (Moore et al., 1961). Ca seems able to increase K uptake synergistically when in low concentration (e.g., Viets, 1944),
CRITICAL CATION ACTIVITY RATIOS
387
but to reduce it antagonistically at high concentration (Jacobson et al., 1961; Elsam and Hodges, 1967). Also there are specific exclusion mechanisms which operate against particular ions, notably Na, of which the specificity varies among plant species (Marschner and Schafarczyk, 1967) and also depends on transpiration (Ratner, 1935) or respiration. Marked steric effects at uptake sites may serve to select one ion in preference to others (Dainty and Hope, 1959; Schaedle and Jacobson, 1967; Russell, 1963; Shone, 1967). The uptake or DM% of nutrients is more often (Vlamis, 1949; Nearpass and Drosdoff, 1952; Collin and Cline, 1966; Olsen, 1950) though not always correlated with ratios of nutrients than with their absolute amounts. There are numerous reported instances where, e.g., added K has reduced the Mg percent of DM, or induced Mg deficiency symptoms, or reduced yields (cf. Holmes, 1962; Salmon, 1963, 1964; Charlesworth, 1967; Birch et al., 1966; Wolton, 1960, 1963; Whitehead, 1966; Overstreet et al., 1952; Tinker and Ziboh, 1959; Walsh and O’Donohoe, 1945; van der Molen, 1964; Johnson et al., 1957; Barrow et al., 1967). There is usually some threshold concentration or activity for every nutrient ion at which plants cease to be able to take it up. This varies among species (Tyner, 1935). The concentration of an ion at its threshold appears to depend on the presence and concentration of other potentially antagonistic or synergistic ions (see Table I, p. 396). In general, up to some ruling level, the quantity of A taken up during the growth of a crop on different plots of one soil is equal or proportional to the amount of labile A present; above this level, the amounts of A taken up are less than the total amount present, as a diminishing percentage of the amount present (Fig. 1: cf. Blanchet et al., 1962). At this level uptake is greater than the uptake corresponding to the ceiling of yield (Fig.
Q r
D r
0
L
a 3
initially present in tho soil
FIG. 1. The relation between the uptake of A(UA) and the amount of “available” A(QA) (diagrammatic). Almost invariably a small amount of A is not taken up. Some A may become available by release from nonlabile forms, following depletion of the pool of “available” A.
388
PHILIP BECKETT
2); the difference represents luxury uptake (Acquaye et al., 1967; Arnold et al., 1968; Arnold and Close, 1961; Duthion, 1968; Jones, 1961; McConaghy and Smillie, 1965; le Roux and Sumner, 1968; Tinker, 1966; Maclean, 1961a; Piper and de Vries, 1960; Smith and Matthews, 1957; van der Molen, 1964; Bolton, 1967; Rowel1 and Erel, 1971; Wild et al., 1969; Koch, 1968; Matthews and Smith, 1957; Acquaye and Maclean, 1966; Hagin and Bazelet, 1964; Bradfield, 1969; Singh and Talibudeen, 1969). Medvedeva (1968) similarly showed that exchangeable K is as good an index of K suppy as is ARK-,. Almost invariably, some part of A apparently present in labile form is not taken up even during exhaustive cropping. Some part of this apparent residue of a cation nutrient in soil may be an experimental artifact, since it is commonly measured as the amount exchangeable to NH,OAc, and NH,OAc tends to extract more cations than are available to plants. But part of it is apparently held at concentrations or activities below the threshold at which the plant cannot take it up (le Roux and Sumner, 1968; Arnold et al., 1968; and above). This simple model may be confused by the release of a nutrient (e.g., K ) from nonlabile forms during depletion (e.g., Acquaye et al., 1967; Arnold and Close, 1961; Stanton, 1958; Tabatai and Hanway, 1969). Figure 1 indicates diagrammatically how this affects the uptake curve. Herlihy and Moss (1970) indicate how K uptake is better related to the amount (Q) of labile K present than to activity ratios. As depletion continues K uptake tends to be increasingly associated with the capacity of the soil to release nonlabile K, rather than with the amount of labile K present at the start of growth. Pope and Cheney (1957) show how, over 10 harvests, the total uptake of K was better correlated with the release of nonexchangeable K than with the initially labile K. Furthermore the yield (DM) of the harvested parts of a crop plant vary with its uptake of a given nutrient in an approximately sigmoid manner (Fig. 2 ) , in which the yield is most uniquely dependent on the uptake of the particular nutrient over its lower and middle ranges (e.g., Barrow, 1966; Dovrat, 1966). Over this range, DM yield may adjust itself to maintain a near constant percentage of the nutrient in plant tissue (Talibudeen and Dey, 1968a; van der Molen. 1964; Maclean et al.. 1957). The gradient of the graph may vary with other factors (e.g., Dovrat, 1966). Its maximum depends very much upon the balance of other nutrients and very much on the so-called antagonistic nutrients as above (e.g., effect of N on K uptake: McConaghy and Smillie, 1965; Bates, 1971), and on other factors of the environment (Collin and Cline, 1966; Attoe and Truog, 1949). The lowest concave part of the curve is not always present. The falling off in yield at high uptakes of A may indicate the point at which
CRITICAL CATION ACTIVITY RATIOS
3 89
less than optimal
0 j;:
UAI uptake of A
FIG.2. The relation between growth (or yield) of a crop ( Y A )and the uptake of nutrient A (diagrammatic). UAc is the lower critical limit of A, the smallest uptake before the yield is significantly reduced.
A is antagonistically reducing the uptake of some other nutrient B, or possibly the osmotic effect of high concentrations of A (Maclean et al., 1957; Tinker, 1966; Bradfield, 1969; Ward, 1959; Jones, 1961; Freeman, 1967; Sumner, 1965; Hoagland and Martin, 1933; Smith and Matthews, 1957). UAc (Fig. 2 ) is the lower critical limit. If all other factors are optimal, yield will lie within some arbitrarily chosen percentage ( x % ) of the ceiling if the uptake of A is not less than UAc.This and the previous observations may be combined to suggest a critical level to the amount of nutrient available which must be exceeded if yield is not to be less than what would have been achieved with further additions of the nutrient (e.g., Tinker, 1966, 1967). For any one nutrient there is likely to be a lower critical limit (e.g., Freeman, 1967) below which plant yield is reduced by a deficiency of the nutrient itself, and an upper critical limit above which it reduces yield indirectly by suppressing the uptake of some other nutrient. Which of the other nutrients in the soil is most antagonistic to A in any particular situation must depend partly on the specificity of their antagonism, and partly on their relative concentrations. Other things being equal, A1 has more likelihood of being the ion most antagonistic to A in a soil at pH < 5 , and Ca most chance in a calcareous soil (e.g., Scheffer et al., 1962; Tinker, 1964; Hunter et al., 1943). Some nutrients are more easily translocated from the roots than others so that, e.g., the A/B ratio in the organs harvested may not be the same as the ratio A/B of uptake. And some nutrients are better able to move between tissues than others so that meristematic growth may be less drastically affected by a partial deficiency of a mobile nutrient than of an immobile one. Finally, for any cation nutrient there is a range of Q/Z relations with other ions as in Fig. 3. Provided the other ion is not taken up in proportionally larger quantities than A, it is possible to relate the fall in
390
PHILIP BECKETT
t OA
8
=C
-X=D SlOW-
release
FIG. 3 . The relation between ( Q , ) , the quantity of “available” As in the soil, and some activity ratios (AR,.,) in the soil solution (diagrammatic). If [QA]? of A is removed from the soil, then AR,.rr is reduced from ( 1 ) to ( 2 ) . Some A may be released from nonlabile forms, as AR, , is reduced on the depletion of “available” A in the soil.
to the amount of A taken up, by means of the appropriate Q/Z relation. For a given soil, Q.\ (as in Fig. 1 ) and ART\-, are related by Fig. 3. For example, [QJ?, is the quantity of labile A taken up to reduce [m.i-,Iito [AR,\-,l,. Again, this simple model is confused if there is any substantial uptake from initially nonlabile forms, in which case the apparent Q/Z relation (of actual uptake of A against depleted AR,-,) has the form of the modified curves on Fig. 3 (Arnold, 1970). Their intercepts on the Q A axis on Fig. 3 should correspond to their intercepts on the U , axis of Fig. 1. The precise form of the modified Q/Z relations will depend on the relation between rate of release and the prevailing AR,-,.
B. SYNTHESIS We may attempt a synthesis of the propositions and experimental results, presented so far. The forms of Figs. 1-3 vary with the crop, the environment, and the soil. Figure 4 combines them and thereby relates the yields of a given crop on differently treated plots on one soil (via A uptake) to the value of AR+, in the soils before cropping. It assumes that ( 1 ) X is the nutrient most antagonistic to A under the conditions of growth, and (2) there is no uptake of A from nonlabile forms. Under these conditions AR,_, of the soil before cropping must exceed AR.Y-x if yield is not to be more than x % less than the maximum possible. The constituent parts of Fig.
CRITICAL CATION ACTIVITY RATIOS
Exhaust,(o'n if ratio Critical ratio after c+
391
,
I
I
I
\
!
Critical ratio before cropping
FIG. 4. The interaction of Figs. 1-3. Given a critical value for ARA-X,below which growth or yield are impaired by a deficiency of A (see text), and the real forms of the relations which Figs. 1-3 present only diagrammatically, then AREx defines the minimum before-cropping value of A R A - necessary ~ for maximum yield.
4 vary, as above, with circumstances, and particularly with the soil, with the contribution of nonlabile sources of A, and the nature of the main antagonistic ion X. It is consistent with the experiments already reviewed, but adds one new group of assumptions. I t assumes that: (5a) for any nutrient A there is a concentration below which it can no longer be taken up. This ultimate or exhaustion value depends on the concentration of other ions present, such that for any pair of ions A and B there are upper and lower values to the activity ratio ARA-B, outside which uptake of B or A, respectively, is no longer possible. There will be a range of exhaustion.ratios ARA-B, ARA-C, ARA-D, etc., for A, any one of which may become limiting, if the concentrations of B, C, or D,
392
PHILIP BECKETT
etc., arc high enough or that of A is low enough. In any particular situation the uptake of A will come to a stop as soon as the soil or culture solution is depleted to one of these ratios. The most relevant antagonistic ion is the one of which exhaustion ratio with A is likely to be reached first (e.g., Singh and Talibudeen, 1969; Beckett and Nafady, 1969). Tinker (1964) and Salmon (1964) have proposed complex ratios of activities which reduce to different simple ratios under different circumstances, according to which of the potentially antagonistic ions is most significant. This exhaustion value corresponds to Addiscott’s ( 1970) “uptake potential.” (5b) for any ion A there is a critical value of ARA-X,such that if ARA-x is maintained at or below the critical value, or this is reached during the growth of a plant, and all other things are equal, the plateau on Fig. 2 will be significantly below the maximum possible, due to deficiency of A relative to X. There will be a corresponding higher critical value of ARA-s at which uptake of X is reduced by A in excess. Such critical ratios are inevitably empirical, since the relation between AR,\-, and yield is so indirect. This critical ratio corresponds to Addiscott’s ( 1970) “exhaustion potential.” If so, then (6) crop or test plants grown on an untreated soil will be expected to show a yield less than on the same soil fertilized with A if ARA-x (where X is the most critical antagonistic ion) reaches its critical ratio before the crop has taken up the amount of A it needs to make its full growth under prevailing conditions (as in Fig. 4). Assumptions (5a) and (5b) are somewhat equivalent to the ultimate and first wilting point of soil water; ( 6 ) is analogous to the idea of available water, but with the difference that the available amount of any nutrient is not independent of the availability of the second nutrient most effectively antagonistic under the prevailing conditions. If it is anything like a true representation of the facts, Fig. 4 may explain the failure of some previous studies to show a simple linear regression of uptake or yield on AR or AG. Such failures may indeed stem from the failure of AR or AG to regulate uptake or yield, as undoubtedly occurs when the uptake of A or B, or crop yield, are controlled or modified by other factors; they may also be due to confusion between Q and I factors, or between labile and nonlabile nutrients, or between A R and log AR, as indicated above; they may sometimes result from the fact that all three crucial associations are nonlinear, and their points of inflexion may not correspond. Even if yield or uptake were controlled by AR or AG it would be fortuitous if either showed a linear or simple polynomial regression since any viable model of a relation like that summarized in Fig. 4 would predict something considerably more complicated.
CRITICAL CATION ACTIVITY RATIOS
IV.
393
Threshold Ratios
The model presented depends on the validity of the critical or exhaustion ratios proposed. The remainder of this review presents data on such ratios: (a) from a survey of published work; and (b) from some exploratory measurements on field trials with K and Mg fertilizers.
A.
PUBLISHED VALUES
OF
CRITICALOR EXHAUSTION RATIOS
Many of the experiments, from which these data are drawn, were planned for other purposes. Some approximations have had to be made in extracting the data. In particular, critical activity ratios should refer to the soil or solution at harvest, but the available data are so limited that I have included some values which relate to the soil before cropping; these are indicated. Activity ratios have been calculated from AG.Concentration ratios have not been converted to activity ratios, partly because the necessary data were not always present and partly because the 10-15% difference is small compared to other errors. Yield assessments relate to the harvested parts of the test plants. In every case, activity ratios are presented with the most deficient nutrient in the numerator. When it was not clear from the experimental data which ratio was limiting, alternative values are given as in “ARB-A or ARc-A”; when the experiment allowed the estimation of critical values for two ratios they are given in the form and ARA-c.” LLARA-B Critical or exhaustion ratios of exchangeable cations are also given; unfortunately many of these had been estimated by NH,OAc extraction.
I , Critical Activity Ratios Here we are concerned with reduction of growth or dry matter production: Is there a particular value for a given activity ratio below which (if all other factors are adequate) growth of some or many plant species is reduced because of a deficiency of the nutrient in the numerator? The question then is: Is there any value of ARA-B which, if achieved before harvest, will have caused the DM yield of the harvested parts of a crop to have been significantly less (by say 10% or so) than it would have been had nutrient A been added to the soil or solution. There are many experimental difficulties. In particular we wish to determine the highest activity ratio at which yield is less than optimum, yet most of the errors of measurement (from, e.g., drying the soil, heterogeneous depletion of the soil, etc.) tend to give rise to a higher activity ratio than that experi-
394
PHILIP BECKETT
enced by the crop. Tinker (1967) has pointed out that the greatest nutrient stress may occur early in the growing period, when the growth of roots is temporarily outstripped by the aboveground parts: also some species return nutrients to the soil during the ripening period that precedes harvest. I have assumed that some kind of adjustment is achieved between perennial crops and the topsoil beneath them (e.g., Tinker, 1964), and have treated samples from soils beneath perennial crops as if they had been collected after harvest. Critical activity ratios are given in Table I. It is unfortunate that some of them refer to uncropped soils. On the model proposed above such critical activity ratios depend on the gradient of the Q / I relation (Fig. 4) : Barrow (1966) gives critical ARK-, values of 0.029 M1/? for soils of low buffer capacity and 0.00042 €or soils of high buffer capacity. On the whole, slight to moderate deficiency symptoms appear at activity ratios greater than those at which significant decreases in yield are observed. Many of the available data relate to ratios such as A/Cec, or exchangeable ions (Table 11). Many of these also refer to soils before cropping. The data in Table I are very heterogeneous and are insufficient to distinguish between species. We may start by ignoring the differences between crops, rejecting values measured before cropping, and rejecting soil values sufficiently greater than the rest to suppose that they are influenced by undepleted soil. Then, as a first approximation, we may extract critical activity ratios from all remaining experiments in which it is clear which is the limiting ratio. This gives ARK-D ARg-yg
0.0005-0.001 for soils arid 0.00002 for solutions 0.000-0.003 for soils.
These are sufficiently different that we may use them to decide which of two alternative ratios was limiting, in the experiments where this was not originally clear. In most cases ARK-,,, was limiting (at values of 0.00060.002 M ’ ? ) though in a few ARK-, was limiting (at 0.00025-0.0007 M1I2). Then we may combine all unrejected data, to obtain empirical median values of A R K - D 0.0005-0 001 Jf”’ h R ~ - y 0.0015 ~ .If1/’
From their nature (as above) these are likely to be overestimates of the critical ratios actually experienced by roots. They may prove to be reasonable indications of a soil in which yield has been less than optimal becaude of (in this case) insufficient K. They lie well below the value of ARK-, =
CRITICAL CATION ACTIVITY RATIOS
395
0.006 at which Wild et al. (1969) found that potassium concentration, not ARK-D,limited growth. Similarly we may extract very tentative values : ARc&-K 13-33 ARcs-~g 0.08 A R M ~ - c ~0 . 0 1 ARK-AI 0 . 0 2 (ARM~-K 4-400)
Similarly we may derive very approximate median critical ratios of exchangeable cations from Table 11, if we combine analyses before and after cropping: K/Cec 0.02; Mg/K 0.5;
K/Mg Mg/Ca
0.2; <0.1;
K/Ca 0.02-0.03; Ca/Cec <0.04;
Mg/Cec 0.03-0.04; Ca/K 1; Ca/Mg
<0.25
It is not possible to compare these with activity ratios since ion-exchange relations are very uncertain at low activity ratios.
2 . Exhaustion Activity Ratios These are the extreme, the lowest activity ratios to which a crop can reduce a soil on exhaustive cropping (all other conditions and nutrients being adequate), or the activity ratios at or below which crops no longer take up the nutrient in the numerator so that growth must cease. Most of the available data relate to K, and particularly to ARK-,, or to K/Cec. The data suffer from some weaknesses. Very close and continuous cropping is required to reduce a soil to exhaustion. There is no obvious yardstick for exhaustion, and it was probably not achieved in all cases. For example 8 cuts of alfalfa did not reduce the activity ratio of a soil as low as continuous percolation with water (Matthews and Smith, 1957). Different workers have used widely different procedures to achieve exhaustion, e.g., 3-4 crops (Thornton, 1937; Breland et al., 1950), 5-6 crops (Arnold and Close, 1961), or 12 crops in 4 years (Stewart and Volk, 1946), and many others. If a soil is not completely exhausted, the results may be confused by a rise in activity ratio during the intermediate stages of depletion (Talibudeen and Dey, 1968a). Furthermore, it is not always clear that an apparent state of exhaustion may not have been imposed by an unrecognized deficiency of some other nutrient, before the ratio being studied reached its exhaustion value. Incomplete exhaustion may result from incomplete root spread; if so there will be marked gradients of activity ratios in the soil, and an average activity ratio determined on a bulked soil sample will be higher than that
TABLE I Critical Values of Reduced Concentration Ratios (M) 1'2 Deficiency conditions Ratio
Experimental conditions
None
Slight
Moderate
Response to nutrient in numerator Severe
Field: perennial
No response
Response 0 004-0 005 (pH
> 5)
and Field
oe ($1
0 0005 0 009-0
(pH 7) 09 (pH 6)
0 004 0 02
0.0016
0.0019
Grass and Hay
Sclieffer rt aE.
w
Moss (1964)
0.0016 -0,005
Grains, beet, potatoes
Scheffer el nl.
~0.0010
Strawberries Irrigated alfalfa
Bradfield (1969) Levin et al.
10,0015
Potatoes: very crude extrapolation from graph Irrigated vetch and clover Corn and Sorghum
Arnold et al.
Wheat
Feigenbaum and Hagin
0 003
0 0099
Tinker (1964)
Cacao Banana Field crops
0015 2 0 005
>o,oona
Oil palm (calc. from a polynomial ratio)
(1902)
>O
Field: sampling time not recorded Field
References
(pH 5 ) (PH 4 )
Field: perennial
Pot trials: 1500 g soil Field
< 4)
0 016-0
Crop and comments
\\'oodruff (1955)
(1962)
(1969)
Field
Field Field: sampled before growth Pot trial 400 g soil
>0,00059
1 0 000523 0.0019
0,0008
10.005 unspec.
0006
0.0016
>0.0006
(1968)
Hagin and Dovrat (1963) Woodruff and Mclntosh (1961)
(1967)
5
c'TI m m
n R
2 4
KdD
Pot trial: 1600 g sampled before growth
KdD
Flowing soh.
0.0004'2
0.000015 0.0000 0.0008
Sand culture
or
WdMg
K/dD
Sand culture
or
WdMg K/dD
0.01 or 0.017 unspec.0 0,0014 OI
0.009
Flowing soh.
or
WdMg K/dD
Sand culture
or
K/dMg K/.\/D or WdMg
Flowing soh.
0 ,00001
or 0.0004
K/dD or W d M g K/dD or WdMg
Flowing soh.
K/dMg
Field: sampled shortly before ripening
Field
Barrow (1966)
Rhodesgrass Legumes Tomatoes
Dovrat (1966)
White clover
Allgren (1941)
Buckwheat
Tyner (1955)
Beet, cabbage, lettuce
Freeman (1967)
Williams (1961)
- 0 ,00009
W d D K/dD
Clover (threshold higher for soils of low K buffer capacity Barley
0.0005 or 0.00044
0.006
or _<0.0014 0.00097 or 0,0018 0,00067 or <0.0018 0.00005-0.00064 or 0.0011-0.0048
or 0,009
0,0005~ or 0.00059 O.OOl1 or 0.0015 0.001s
Wall (1940)
Asher and 14 crop and pasture species (attributes Ozanne higher thresholds by (1967) earlier workers t o poor circulation of solution) Red clover Tyner (1985)
Soybeans
Potatoes 40.0016 Sugar beet 0.0095 0.01-0.003 Barley
Woodruff and McIntosh (1961) Hovland and Caldwell (1960) (continued)
W rQ
TABLE I (confinued)
w ~~
Defic ienry rnnditionr
Ratio
K/.\/Ca
E:nprrimciital crrnclitions
None
Slight
Response to nutrirnt in numerator
Moderate
hc*vcre
Field
Re-ponw
<0.0009
07
W.\/& dMg/K -\/Mg/K
NOr i q m n s r
20 0003
00045 <9.8
Sand culture; I other factors limiting also Field
Relercnres
Maize
Pierre and I h w w (1943)
Ryrgrnas
Grunes e t a / .
IVlieat
Hovlnnd a n d
lJT
07
Crop and r o m m m t s
20
0009 >10.4
(1968)
<400-500
Caldwrll (1980)
Mg/Ca
Mg/Ca
Ca/Mg
Field (Icfiirc cropping
Pot trials
9 5
ur 14
01
0.15-0.95 or
4.5-6.9
Siigar beet
Tinker (1967)
('omnut "Yriiows" indic~itiveof hfg deficienry Lettuce, barley
Netksinghe
Barley
dollansen rt al.
Field crops
IYoodruff (1955)
Currant. tomato
Scheidecker (1968) Sumner (1985)
w -
.oi
Field: perennial
or
dCa/K .\/Ca/K
.
<0.015-0 02
0.95 or 1 6
0 077
(1963)
Vlamis (1949)
01
4 5
>a-P
Flowing s o h .
7
(1968)
.\/Ca/K
Field: sampling time not recorded ( ? before
93
growth )
.\/Cn/K
Solution culture
.\/Ca/K
Field: measured a t tasseling time
2 4 . 4 , unspec. 49, unspec.
Maize (calc. from k'/\h, assuming
Ca = Mg) * Unspec.
= deficiency, b u t degree unspecified.
~
2
c
71
m
m 0
x
m
H H
CRITICAL CATION A,CTIVITY RATIOS
399
near the roots. Something like this may be indicated by the observation (Barrow et al., 1967) that at values of ARK-, above exhaustion, the K percentage of dry matter is higher in plants from solution cultures than from soils at the same ARK-,. In most exhaustion experiments the soil is reported as having been well permeated by roots. More commonly, K exhaustion (at least) is incomplete because K is released from fixed or nonlabile sources in response to depletion. Islam and Bolton (1970) suggest that release commences at ARK-, = 0.0001 M 1 / 2 .In such cases the exhaustion state of a soil represents the balance of uptake and release, of which the latter increases with the extent of depletion. For a similar reason, the extent of depletion of both labile and fixed K tends to increase if the soil is diluted with inert sand (Conyers and MacClean, 1969). Either of these may explain the effect of root/soil (e.g., Boyd and Frater’s (1967) Table 5) ratio on the extent of depletion. Because of K release, soil under continuous cropping often tends to settle down to a steady low K level (Arnold and Close, 1961; Attoe, 1949; Breland et al., 1950; Tabatai and Hanway, 1969), representing the balance of release and uptake. After this, most uptake is from released K (Tabatai and Hanway, 1969; Breland et d.,1950). The level at this steady state is not much affected by prior K fertilization (le Roux and Sumner, 1968; McEwen and Matthews, 1958), nor by other treatments which temporarily affect the amount of labile K before cropping (Attoe, 1946). The steady rate of release may or may not be sufficient to prevent K deficiency symptoms. Commonly the rate is constant over a considerable period of depletion (Tabatai and Hanway, 1969) and then finally falls off when the soils reserve of “intermediate K ’ is exhausted. Few exhaustion trials reach this extreme state, and commonly K,,/Cec at exhaustion is more or less proportional to the rate or amount of K release to all or to the last few crops (Pratt, 1951; Smith and Matthews, 1957), and is 2- to 4-fold higher in soils of high release than low (Hiatt, 1963; Piper and deVries, 1960). There appears to be only negligible release of Mg from fixed sources during exhaustive cropping by ryegrass or clover (Salmon, 1963; Salmon and Arnold, 1963). Experimental data are further confused by the fact that many workers have dried or stored their soil samples before analysis. Both of these tend to restore the initial undepleted state of the soil as long as any part of the soil’s pool of “intermediate K ’ remains (Blakemore, 1966; Tyner, 1935). Barrow et al. (1967) also point out that fine rootlets, inseparable from soil, contain sufficient K to raise the equilibrium activity ratio of a highly depleted but poorly buffered soil, 10- to 30-fold. Also some crops achieve greatest depletion at a stage short of harvest and then excrete nutrients at later stages.
P 0 0
TARLE I1 Critical Ratios of Exchangeable Cations On addition of nutrient in numerator
Deficiency symptoms Ratio of Experiexchangeable mental cations conditions
Sampling time
None
Slight
Moderate
Severe
Response
a
No response
Crop
Reference
2 t: a W
Field
After cropping Pot trials After Field Before Pot trials After Pot trials Before
>o
rn
< .01
09
0.003
0.01~-0.019 0.017
0.09.
Unspec.
Irrigated clover
Hagin and Dovrat (1963)
Alfalfa
All crops
Hear rt a!. (1944) Woodruff (1955)
Wheat Tomato
Feigenbaum and Hagin (1967) Martin et al. (1953)
Tomato Alfalfa
Tliorne (1944) Hunter el al. (1943)
Alfalfa Coffee Citrus seedlings
Prince et al. (1947) Robinson and Chenery (1958) Martin et al. (1953)
0.024
Pot trials Before Pot trials After
0.008 0.097 0.17 <1 0.46
Pot trials After Field Perennial Pot trials Before
0.01-1 0.037
>0.2
Field
Perennial
<9-2.5
Unspec.
Orange trees (quotes other refs.)
McCollocb et al. (1957)
Field
Perennial
<$-9..5
Unspec.
Oil palms
Tinker and Ziboh (1959)
17 7;: rn
el 1
Mg/K and Mg/Cec Mg/K and W/Na Mg/Ca 01 Me/K Mg/Ca Mg/Ca Mg/Ca and Ca/W
Pot trial
0.015-0.04 0.86 0.71
Pot trials Before Pot trials Before
0.5
0.5-0.1
Field After Pot trials Before Pot trials Before
>o.ss <0.95
<10
0.15-0.9 0.03-4
Pot trials Before
0.45
Pot trials Pot trials Pot trials Pot trials =
Before Before Before Before
deficiency, but degree unspecified.
0.03-0.08
lO.05 50.35
Ca/K Ca/K Ca/Na
Unspec.
Prince et al. (1947)
Tomato
Martin et al. (1955)
Sweet lime
Jacoby (1961)
Celery Wheat, sorghum Alfalfa (quotes other refs.)
Johnson et al. (1957) Ssnik et RZ. (1952) Hunter (1949)
Sweet orange
Martin and Page (1969)
SunEower, maize, buckwheat Native serpentine species Corn, soybeans
Walker el al. (1955) Key et RZ. (1964)
Lettuce, barley Sweet orange
Vlamis (1949) Martin and Page (1969)
Alfalfa
Hunter et al. (1943)
Lettuce, barley Tomato Oats, wheat Tomato
Vlamis (1949) Thorne (1944) Ratner (1935) Thorne (1944)
0 . a.5-sa
0.%6
0
Alfalfa
A
Ca/Mg Pot trials Before Ca/Cec and Pot trials Before Ca/Mg Ca/K Pot trials After
Ca/Na
59 0.07
5
Mg/Cec and Pot trials Before MdCa CJMg Pot trials Before Ca/Mg
1.8-15
After
Unspec.
<0.55 0 . % S O .43
1
Unspec.
<0.43
<1.5-9
0.04
402
PHILIP BECKETT
Measurements of exhaustion ratios in solution cultures suffer from the usual problem that unless unusual precautions (Asher and Ozanne, 1967; Asher et al., 1965) are taken the concentrations and ratios of nutrients vary during the experiment, so that DM yields vary with the rate of flow (Allgren, 1941 ) . The available data are given in Tables I11 and IV. As expected, they vary considerably, possibly among crops though the figures are not good enough to confirm this, and certainly with the ratio of roots to soil and with the duration and intensity of depletion. ARK-, values of 0.00001-0.00004 M1/? were attained in both solution and soil. If we ignore differences between crops, and reject what seem to be anomalously high values, the median values of the remaining figures are approximately: .iR~c-” 0 0OOOS for solutions and 0 0005 for soil, h R ~ - u = 0 00006 for solutions, and <0 005 in soil A R y n - ~ <0 002 in soil.
The discrepancies between solution and soil are presumably due to incomplete exhaustion of the latter, the drying of cropped soil samples, and the effect of K from rootlets. Nevertheless the soil values are probably typical of the analyses one can expect from exhausted soils. Table IV records exhaustion ratios of K/Cec%, lowest at 0.3-0.5% and median at 0.7%, with higher values from K releasers. Again, many figures will have been inflated by K release on drying.
B. FIELDMEASUREMENTS OF CRITICAL RATIOS The literature review was supplemented by exploratory field measurements. Soil samples were taken from the control plots of K and Mg feror tilizer trials and analysed for some or all of ARK-=, ARMg-Ca.The aim was to ascertain whether control plots which have lower yields than treated plots, or plots with small applications of fertilizer which gave lower yields than those treated more generously, exhibited values of the appropriate AR at harvest consistently less than some recognizable threshold value. These trials were exploratory and approximate: there were two series of measurements. For the first series, extension officers throughout Britain were circularized for soil samples from the control plots of any current or recent fertilizer trials involving K or Mg. The samples received related to triah on a wide range of crops over several seasons; some had been air-dried or stored. Table V lists the experimenters’ comments on the K or Mg status of their soils or on the K or Mg fertilizer responses shown by the trials.
TABLE I11 Exhaustion Values of Reduced Concentration Ratios (Measured after Depletion unless Stated) M1’* ARK-D
ARK-M~ARN~-D
<0.0018 <0.001-0.006 0.001 0.0005-0.001 0.00084.0013a 0.0009-0.0005 0.00083 0.0008-0.0037 0.0005-0.0007 0.0005-0.0007 (0.001-0.003)b 0.0003 0.0001-0.001 0.0001 0.0006 0.00045 0.0003-0.0005
0.0046
<0.0017
0.0001-0.0005 0.0003-0.0003 0.0001 0.0003-0.0004 (0.001)b 0.00003 0.00001-0.000014 0.0003 0.000015-0.00004 0.000013-0.00003 0.00004 High initial K. High K-release.
Depletion treatment: comments
References
Oats; 60 days, 50 g soil, dried before analysis 3 yr, pots: 60 days, 50 g soil, dried before analysis Japanese millet: 30-60 days, 400 g Cauliflower, bean: 90 days; 700 g, soil dried Oats: 1 crop to heading stage; 3300 g Wheat: 5-6 crops; 400 g, soil dried Calculated from & / I relation, K uptake by crop and specified initial ARK-D before cropping in field Minimum of annual fluctuations in cropped fields Ryegrass, kale: read from graph as ARK-D for no K uptake Japanese millet: 9-63 days, 400 g, soil dried
Acquaye et al. (1967) Acquaye and Maclean (1966) le Roux, 1966 Beckett et al. (1966) Maclean (1961~) Feigenbaum and Hagin (1967) Woodruff (1955)
Strawberries: two crops, 1500 g Ryegrass: 1000 days, 4300 g, followed by 56 days, 500 g Irrigated Rhodesgrass and legumes: 3 yr; field Sugar beet Rye grass: 36 days, 600 g; figures extrapolated to zero uptake of K Minimum value during annual fluctuations in cropped fields (cf. Garbouchev, 1966) Ryegrass and flax Alfalfa: 170 days; German millet: 50 days, 3000 g soil Ryegrass: 400 days, 200 g; soil dried
Garbouchev (1966) Arnold et al. (1968) le Roux and Sumner, 1968 Bradfield (1969) Moss (1970) Dovrat (1966) Rowel1 and Ere1 (1971) Arnold (1962) Blakemore (1966) Wild et a1 (1969) Conyers and Maclean (1969) Talibudeen and Dey (1968a,b)
C]
i?]
2 cl
*r cl 9
8z b
2
8.e e!
0.00006
Subterranean clover: 160-220 days, 1600 g; no K uptake during last 3-6 weeks (threshold is higher for soils of lower K-buffering capacity) Ryegrass: 9 cuts, 526 days (some K still being released Potatoes: 60-70 days, 500 g soil Ryegrass: 180 days, 500 g soil Barley: solution culture Subterranean clover: solution culture
Barrow et al. (1967)
R
Addiscott (1970) Addiscott and Mitchell (1970) Islam and Bolton (1970) Williams (1961) P
E:
P
B TABLE 1I’ Exhaustion Ratio (&,/Ccc) 0: Soils not rclcasing K, o r K-releasing crrpac-ity not specified
Soils reported t o relc~:iseK
K d C c c (%)
I>epletion treatrncnt Alfalfa: 7 crops, 490 days; 3500 g soil Wheat: 5-6 rrops, 400 g soil; sonic K-deficienry symptoms
0.4
0.5-0.7
0.4, 0.6, 0.6, 0.6, 0.6, 0.7, 0.7, 0.7, 0.8, 0.8, 1.7
-
Oats and Sudangrass: 7%) days, 2500 g soil Clover and ryegrass: 360 days; 10,000 g soil Ryegrass: 15 cuts, 1000 days and 4300 6 followed by 1 cut, 56 days and 500 g; & was suhtantially reduced before 16th crop 0.4, 0.44, 0.7, 0.81, 1.4 Sudangrass: 2 crops by short Neubaucr proccdure; 50 g soil 0, 0.53,0.64, 0.72, 0.84, 1.0, 1.04, 0.77, 0.82, 1.04, Alfalfa: 7-8 cuts, 370 days, 9000 g soil (tlircsholti of K 1.14, 1.27, 1.62 release reported as ca. 1%) 1.14, 1.3, 4.4 1.2 1.5-2 70 MiIiued crops; continuous for 4 years; growth ceased for lack of K 0.3, 1.5, 2.6, 2.9, 2.9. 4.2 (higher values associated with K release) Japanese millet 0.46, 0.64, 0.65 0.56, 0.36, 0.36, 0.44, 0.52, 0.53, 0.54, 0.60, 0.64, 0.79, 0.1, 0.19
0.91, 1.95
References Hunter et al. (1943) Feigenbauni and IIagiii (1967)
Marlcan (19611)) I’iper and dc Vries (1960) Moss (1970)
Dowdy arid IIutcheson (1963) Bear et al. (1944)
Weltc et al. (1962) Stewart and Volk (1946)
Stanton (1958)
TABLE V First Exploratory Survey ~~
~
~
~~
~
~
~
~
Soil solution n i a l and response A R K - M ~ ARK-D Crops“
Mg response, or status
K response, or status
(M1’2)
(Ml‘*)
Carrots Hops Nearly all crops Hops Potatoes Potatoes Beet Carrots Potatoes Beet Grass Grass Cereals Cereals Potatoes Early potatoes Grass Grass Grass Grass
Good response Visible deficiency Deficiency Visible deficiency Yield response Some response Very little response No deficiency symptoms Deficiency Leaf evidence of deficiency Response “Mg-treated” “Response not expected” “Response not expected” No response “Response not expected” “High Mg” No response No response No response
Very small response High K K-fertilized High K NDb ND Probably high K No deficiency symptoms Generously K fertilized Possibly some response ND ND No response No response ND No response ND K response K response K response
0.078 0.068
0.01 0.034 0.013 0 . 022 0.01 0.007 0.008 0.011 0.011 0.004 0.009 0.007 0.002 0.002 0.005 0.003 0.001 0.00% 0.0007 0.0006
a
b
18 UK trials; 2 (hops) from West Germany. N D = not detected.
0.055
0.049 0.048 0.032 0.027 0.026 0,023 0.023 0,019 0.012 0,008 0.007 0.006 0.006 0.003 0.003 0.002 0.002
ARM,-CS
0.016 0.26
0.05 0.2 0.04 0.04 0.07 0.14 0.23 0.32 0.25 0.48 0.04 0.07 1.43 0.22 0.06 0.43 0.08 0.09
[I(1 (mM)
[Mgl (mM)
0.76 1.79 0.96 1.70 0.88 0.41 0.75 1.06 0.83 0.34
0.13 0.78 0.53 1.39 0.36 0.18 0.88 1.95 1.5 0.24
0.56
1.02
0.54 0.15 0.15 0.31 0.26 0.05 0.15 0.06 0.05
2.50 0.43 0.48
9.26 2.72 0.24 3.6 0.91 0.78
H
0
F 0
> 2
b
2
5* 5
$
406
PHILIP BECKETT
TABLE V I Second Exploratory Survey"
K response: total Dht increase (all harvests) due t o K application Soil solution of untreated (control) plots after last harvest
Some (5-30%) Nonea
Trial
ARK-D
(.If A
J J
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
B
0
G H J
0 0 0 0 0 0 0
G
K J J D J E J D G D J B G
h
h C
F G
'12)
ARK-M.
(M' 1 2 )
0055 005 005 005 0046
0 016 0 006
0018
0 008.2
0053 0016 0014 0014 0013 001 0009 0007 00065 0006 0006 0006 0006 0005 0004 0003
0009 00024 0002 0002
(
-
* * * * *
0.0028
0.00.23 0,00078 0.0017
-
Case 2"
*
*
* * * * * *
*
0.005 0.0006
-
0,00085 0.0006 0.0005
-
Large (>So%)
*
0.0047 0.0016 0,0035
Case 1*
* *
*
* * *
* *
0 Control plots gave same DM yield as plots with added K or gave DM yield same as plots with higher AR after cropping or could be seen to be at or near rrest of response curve. Result can be seen to be near crest of response curve. No evidence for relation of this result to response curve.
CRITICAL CATION ACTIVITY RATIOS
407
The soil samples were moistened to approximately field capacity and stored for 2 weeks. The concentrations of K, Ca, and Mg in their soil solutions were determined by displacement (Moss, 1969), and activity ratios were calculated (Table V) . The range of soil solution concentrations encountered in this and the following series were:
K Mg Ca
0,015-!2.6
+ Mg
0.1-2.8 0.98-54.7
0.38 0.73 10.1
It is clear that K or Mg response or deficiency symptoms were not well correlated to the K or Mg concentrations of the soil solutions. A value of ARK-,, = 0.015 M1/* seems to separate soils on which the treated plots responded to added Mg, from those which did not. Mg response is not so well associated with K response appears to be associated with soils which reach ARK-Mg 0.003 or ARK-, 0.002 by the last harvest. In the second series, soil samples were specially collected (10-15 cores, 0-6 inches) by field officers of Potash Ltd. in January and February 1965-1966, from control plots of K-fertilizer trials on grassland. Even initially the swards were not all of the same floristic composition, and repeated cutting and K fertilization may have introduced further discrepancies, so the results are only approximate. Subsidiary determinations showed that, except for the sample from trial E, the depleted soils showed little K release on 2 months storage. The soil samples may therefore be taken to represent the control plots at the time of the last cutting in the previous autumn, and compared to the total DM yield of cut grass in that year. Table VI presents the measured activity ratios and the responses (in DM of cut grass) of the treated plots to added K. Repeated removal of herbage has reduced ARK-, as low as 0.0002-0.0007 M I / * in several cases. With the exception of trial E (ARK, = 0.0016) there appear to be critical values of ARK-,, at about 0.005, and of ARK-, at about 0.0015 M1/*.
<
<
C. CONCLUSIONS From this preliminary collection of data, we may extract the tentative values in the accompanying tabulation for soil activity ratios as a possible guide to future work.
408
PHILIP BECKETT
Field trials Literatiire survey Euhaustion ratio ;\1th-L> .11th--\Ip. 1IiU$-h
0 0005 005
-
Critical ratio
0 0005-0 001 0 0015 (4-400)
1st Series 2nd Series critical ciitical ratio ratio < 0 002 < 0 003 (66)
0 0015 0 005
-
The field values of the critical ratio are not as low as some figures from the literature, probably because the small bulk of soil in a pot is more uniformly depleted than soil beneath a field crop. Nevertheless they are of the same magnitude, which is partly reassuring. In the absence of better data, we might tentatively accept values of 0.004, 50, and 0.002 for the critical values of a K / G g , a,,,/aK, and as/=, that, is we may assume that if in any soil the uptake of the necessary quantity of K or Mg by a field crop will require that any of the activity ratios be reduced to below these critical values, then the crop is likely to respond to added K or Mg. At least until better values are available, these figures and the tentative model represented by Fig. 4 might serve as the basis for further greenhouse or field experiments.
ACKNOWLEDGMENTS
I am grateful to Mr. P. Gething and the officers of Potash Limited, to Mr. P. Crooks, and to the officers of the National Agricultural Advisory Service for providing soil samples and details of field trials; to the numerous authors who have supplemented the information in their papers by correspondence; to Dr. M. H. Nafady and the. late Dr. P. Moss for measuring activity ratios on the samples; and to Mr. P. H. Nye, Dr. P. B. Tinker, and Mr. Gething for criticizing drafts of this paper.
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CRITICAL CATION ACTIVITY RATIOS
409
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CRITICAL CATION ACTIVITY RATIOS
41 1
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412
PHILIP BECKETT
Whitehead, D. C. 1966. Commonw. Bur. Pasture Field Crops, Mimeo. Publ. No. 1 . Wild, A,, Rowell, D. L., and Ogunfawona, M. A. 1969. Soil Sci. 108, 432-439. Williams, D. E. 1961. Plum Soil 15, 387-399. Wolton, K. M. 1960. Proc. l i l t . Grassland Congr., & / I , 1960, pp. 544-548. Wolton, K. M. 1963. J . Nar. Agr. A d v . Serv. 14, 122-130. Woodruff, C. M. 1955. Soil Sci. SOC. Amer., Proc. 19, 167-171. Woodruff, C. M., and McIntosh, J. L. 1961. Trans. I n r . Congr. Soil Sci., 7th, 1960, Vol. 3, pp, 80-85. Yoshida, F. 1964. Versl. Larrdbouwk. Onderzoek. 642.
AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed. A
Abdel-Malek, S., 201, 251 Abe, G., 196, 260 Abernathy, C. O., 243, 251 Abernethy, R. F., 269, 271, 285, 311 Abrahams, R. H., 282, 313 Acevedo, A. R., 285, 315 Acharya, C. N., 60, 66, 88 Ackefors, H., 307, 311 Acquaye, D. K., 384, 388,403,408 Adachi, M., 369, 370, 372 Adams, M. W., 125, 126, 132, 142, 165, 185 Adams, R. N., 42, 88 Adams, R. S., Jr., 341, 372, 373 Adams, S. N., 80, 88 Addiscott, T. M., 384, 392, 403, 408 Adkins, B. L., 294, 318 Adkisson, P. L., 226, 251, 261 Adriano, D. C., 161, 162, 181, 182 Affleck, R. J., 305, 311 Agarwal, R. A., 219, 251 Aiyer, P. A. S., 62, 90 Akeson, W. R., 192, 251, 252 Alam, S., 14, 24 Alberda, T., 35, 88 Alder, E. F., 337, 372, 377 Aldrich, D. G., 297, 311, 400, 410 Aleshin, E. P., 38, 88 Alexander, M., 64, 67, 88, 91, 329, 337, 3 74 Ali, M. A. M., 126, 144 Aliev, K. A., 8, 24, 26 Allard, J. W., 123, 142 Allaway, W.H., 283, 285, 287, 289, 290, 291, 292, 296, 299, 311, 313, 314, 317 Allen, H. E., 309, 311 Allen, N., 245, 254 Allen, R. E., 199, 251 Allen, S. E., 177, 186 Allgren, G. H., 397, 402, 408 Allison, F. E., 68, 91
Allison, J. C. S., 121, 125, 136, 142 Alloway, B. J., 283, 311 Alston, A. M., 148, 181 Altschul, A. M., 25 Alvarez, A., 278, 316 Amato, V. A., 339, 340, 374 Ambler, J. E., 161, 165, 181, 182 Ancajas, R. R., 71, 92, 96 Andersen, W . R., 118, 119, 129, 138, 142 Anderson, A. J., 158, 181 Anderson, F. N., 212, 255 Anderson, J. L., 279, 323 Anderson, L. D., 210, 253 Anderson, L. E., 20, 24 Anderson, M. S., 268, 288, 289, 290, 311 Anderson, W. A. D., 284, 310, 314, 315 Anderson, W . B., 155, 175, 176, 181 Anderson, W. L., 295, 311 Anderson, W. P., 339, 372 Angino, E. E.,.302, 311 Annappan, R. S., 227, 251 Anthony, E. H., 35, 90 Aomine, S., 37, 42, 46, 50, 88 Apel, P., 112, 142 Apidianakis, J. C., 294, 312 Aquino, R. C., 108, 127, 142, 144 Ardakani, M. S., 151, 152, 153, 185 Areson, W. R., 192, 251 Argrett, L. C., 286, 310, 317 Arle, H. F., 339, 341, 372 Armstrong, D. E., 37, 78, 94, 95 Armstrong, E. A. J., 35, 88 Armstrong, W., 32, 34, 38, 88 Amett, W. H., 190, 261 Arnold, J., 214, 257 Arnold, P. W., 384, 388, 390, 395, 396, 399, 403, 408, 409, 411 Arnold, W. E., 332, 372 Arnon, D. I., 40, 83, 88 Arnott, H. J., 6, 24 Aronson, A. L., 283, 315 Arridge, H. O., 205, 206, 251
413
414
AUTHOR INDEX
Arthur, D., 307, 311 Asami, T., 71, 74, 88, 95 Asher, C. J.. 383, 387, 397, 399, 402, 403, 409, 410 Ashford, R., 339, 376 Ashri, A,, 16, 24 Ashton, F. M . , 329, 332, 340, 372, 375 Ashwell, M., 9, 24 Ashworth, P. R., 401, 411 Aslam, M . , 227, 260 Atanasoff, D., 6, 18, 19, 24 Athanassiadis, Y. C., 284, 311 Athwal, D. S., 198, 251 Atkins, I. M . , 201, 253 Atkins, R. E.. 217, 251, 254 Attoe, 0. J., 388, 399, 409 Auclair, J. L., 191, 227, 229, 239. 240, 251, 259, ,761 Augustine, M. J., 240, 251 Aurand, L. W., 214, 254 Austenson, H. M., 281, 320 Axelsson, B., 284, 311 Axley, J. H., 286, 287, 325 Azarnotf. D. L., 292, 311
B Babicky, A., 278, 284, 288, 310, 319 Bacalangco, 1:. H., 198, 251 Bach, F., 278. 314 Bache, C. A., 279, 305, 306, 309, 311, 318, 323 Back, W., 43, 45, 84, 88, 89 Backstrom, J., 281, 311 Bar, F., 347, 373 Baetjer, A. M . , 290, 312 Bagley, G. E . , 282, 312, 314, 317, 318 Baiiey, C . F., 226, 251 Bailey, J. C., 222, 251 Bair, F. L., 295, 320 Bajaj, Y. P. S., 165, 185 Baker, A. P., 245, 263 Baker, D. E., 167, 183 Baker, D. G., 341, 372 Baker, J. T., 284, 322 Bakke, J. E.. 345, 346, 347, 348, 372, 375, 377 Balachowsky, A. S., 205, 252 Balashova, G. V., 43, 88
Balassa, J. J., 271, 283, 284, 285, 286, 287, 288, 291, 292, 293, 294, 297, 298, 321. 322 Bandt, H. J., 305, 312 Bandurski, R. S., 342, 374 Banks, V. S., 352, 376 Banse, K., 35, 93 Baratta, E. J., 294, 312 Barber, D. A., 386, 409 Barber, S. A,, 163, 181, I85 Bariola, L. A., 221, 222, 227, 229, 252, 256, 258 Barker, H. A., 60, 63, 65, 74, 89 Barker R. E., 127, 142 Barnes, D. K., 116, 145, 190, 191, 192, 252, 253, 260 Barnes, I., 43, 45, 84, 88, 89 Barnes, 0. L., 189, 206, 252 Barnette, R. M . , 158, 161, 181 Baroccio, A., 386, 409 Baron, R. L., 243, 255, 375 Barrentine, W . L., 340, 372 Barrow, N. J., 387, 388, 394, 396, 399, 403, 409 Barrow, V. L., 268, 312 Barth, E. F., 269, 312 Bartholomew, W. V., 66, 89 Bartlett, R. J., 38, 89 Baskett, R. S., 158, 181 Bass-Becking, L. G. M., 35, 43, 49, 50, 51, 89 Bassi, M., 6, 25 Bastida, R., 215, 252 Bastron, H., 296, 320 Bates, T. E., 388, 409 Batjer, L. P., 50, 89, 175, 181, 287, 312, 323 Bauer, A., 161, 181 Bauer, N., 160, 183 Bauman, L. F., 19, 26, 264 Baumann, C. A., 284, 286, 314 Baunok, I., 355, 356, 374 Baur, J. R., 351, 372 Baxter, R., 20, 24 Bayer, D. E., 339, 340, 359, 372 Bayer, G. H., 339, 372 Bazelet, M., 388, 409 Bear, F. E., 268, 286, 295, 312, 319, 389, 400, 401, 404, 409, 410, 411 Beard, G. W., 227, 252
AUTHOR INDEX Bearse, G. E., 279, 318 Beath, 0. A., 288, 290, 320 Beaty, D. W., 212, 262 Bech, R., 227, 252 Beck, S. D., 188, 209,252 Beckett, P. H. T., 379, 380, 381, 385, 392, 403, 409 Beckham, T. M., 301, 321 Beckwith, R. S., 274, 312 Beeson, K. C., 158, 181, 288, 289, 290, 311 Beevers, H., 6, 24 Behrens, R., 341, 373 Beland, G. L., 192, 252 Belasco, I. J., 359, 374 Bell, H. L., 303, 324 Bender, M. E., 269, 309, 312 Benedict, C. R., 10, 25 Benepal, P. S., 234, 238, 252 Benes, I., 278, 284, 288, 310, 319 Benes, J., 278, 284, 288, 310, 319 Benne, E. J., 387,400,410 Bennett, C. F., 301, 313 Bennett, C. W., 19, 24 Bennett, S. E., 210, 211, 214, 252, 257 Benson, N . R., 175, 181, 268, 287,312 Berdahl, J. D., 109, 142 Berg, M. A., 203, 259 Berg, W., 279, 312 Berger, K. C., 158, 171, 181, 185 Berglund, F., 278, 279, 307, 312 Bergmann, W., 169, 184 Berlin, M., 278, 279, 307, 312 Bernard, R. L., 110, 134, 143 Berner, R. A,, 76, 89 Bernheim, F., 292, 312 Bernheim, M. L. C., 292, 312 Berrer, D., 342, 345, 375 Berthold, R. V., 367, 374 Bertilsson, L., 301, 312 Bertramson, B. R., 395, 399, 409 Bertrand, D., 293, 312 Beynon, K. I., 329, 333, 373 Bhan, K. C., 3, 10, 24 Bhatnaga, V. K., 205, 263 Bhatnagar, M. P., 217, 258 Bianchi, K., 18, 24 Bickley, F., 79, 92 Bigger, J. H., 212, 252
415
Bingham, F. T., 150, 162, 181, 400, 410 Bingham, J., 99, 109, 136, 142, 144, 203, 252 Birch, J. A., 387, 409 Bird, P. M., 293, 312 Birke, G., 307, 312 Birnstiel, M., 7, 27 Bisbjerg, B., 289, 312, 314 Bischof, Von F., 363, 373 Bishop, J. R., 336, 374 Bishop, N., 386, 411 Biswas, P. F., 338, 339, 373 Bjorkman, O., 115, 118, 128, 130, 136, 142, 143 Black, J. H., 222, 252 Blacklock, G. C., 105, 108, 109, 145 Blackman, G. E., 102, 109, 142, 144, 352, 377 Blackmon, W. J., 356, 376 Blakemore, M., 399, 403, 409 Blakeslee, A. F., 19, 24 Blanchard, R. A., 212, 252 Blanchet, R., 387, 409 Blickenstaff, C. C., 190, 191, 252, 260, 264 Bloodgood, D. E., 60, 93 Bloomfield, C., 46, 71, 78, 89, 90, 272, 283, 291, 295, 316 Blum, A., 216, 252 Blume, H. P., 30, 89 Blumenshine, J. A., 293, 323 Boardman, N. K., 4, 8, 24, 138, 142, 143 Boatman, D. J., 32, 34, 88 Boawn, L. C., 158, 160, 161, 162, 170, 171, 173, 174, 175, 177, 179, 180, 181, 183, 184, 186 Bobkoskie, R. L., 278, 314 Bohme, C., 347, 373 Boerngen, J. G., 272, 280, 282, 291, 322 Borst, P., 9, 24 Bohn, H. L., 43, 54,89, 150, 160,186 Boling, J. C., 229, 262 Bolt, G. H., 44, 93, 274, 312, 386, 409 Bolton, I., 388, 399, 403, 409, 410 Bolviken, B., 283, 317 Bongers, W., 236, 252 Booth, C. O., 246, 257 Borchert, H., 51, 89
416
AUTHOR INDEX
Bonner, J., 4,27 Borg, K., 279, 281, 312, 324 Borhnd, J . W., 395, 399, 409 Borneman-Starinkevitch, I. D., 296, 312 Borovicb, S. A., 296, 312 Borovsky, I. B., 296, 312 Bostrom, K.. 53, 89 Botsford, D. L.. 345, 376 Bottrell, D. G., 217, 253 Bouldin, D. R., 35, 36, 45, 89, 91 Bovey, R. W., 351, 372, 373 Bowen, H . J. M.. 271, 283, 285, 286, 291, 296, 297, 308, 312 Bowen, J . E., 167, 181 Bower, C A., 386, 398, 411 Bower, C. H., 76, 92 Bowles, J. bf., 272, 282, 291, 296, 322 Bowling, C . C., 198, 252, 370, 373 Bowman, M. C., 207, 211, 258, 259 Boyd, D. A.. 399, 409 Boyland, E , 345, 348, 373 Boyle, R. W., 280, 316 Boylen, G., 310, 316 Brader, L., 129, 252 Bradfield, E. G., 388, 389, 396, 403, 409 Bradfield, R., 50, 89 Bradford, B. N., 177, 186 Bradford, G R., 297, 31 1 Branson, T. F., 211, 212, 213, 252, 255 Brazzel, J. R., 223, 226, 252, 263 Brech, F., 169, 181 Bredfeldt, J.. 302, 311 Breland, H L., 395, 399, 409 Bremner, J. M.,47, 66, 67, 68, 69, 82, 89, 95 Brett, C. H., 215, 238, 239, 240, 252, 253 Brewer. H . C., 296, 312 Brewer, R. F., 283, 312 Brey, M.E., 35, 90 Brezonik, P. L., 37, 47, 89 Bricken, 0. P., 79, 92 Bricker, 0. P., 80, 89 Briedenbach, R. W., 6, 24 Briggs, G. E., 386, 409 Briggs, J. B., 231, 232, 253, 258 Brim, C . A,, 15, 17, 24 Brimblecrombe, A. R., 207, 253 Brindley, T. A., 208, 209, 252, 257
Brinkman, R., 33, 72, 89 Britten, E. J., 109, 145 Broadbent, F. E., 66, 67, 68, 69, 89, 152, 185 Broadhurst, N. A,, 332,373 Brock, F. E., 292, 31 1 Broda, E., 165, 181 Biody, H., 278, 279, 314 Broeshart, H., 78, 89 Brornfield, S. M., 78, 89, 275, 312 Brooks, R. R., 43, 45, 89, 93 Brown, A. L., 162, 171, 172, 173, 174, 175, 177, 179, 181 Brown, J. C., 161, 162, 165, 181, 182 Brown, R., 269, 271, 312 Brown, V. M., 306, 322 Browne, E. B., 16, 25 Brun, W. A., 112, 142 Bryan, G. W., 306, 312 Bryant, A. E., 38, 89 Bryce-Smith, D., 283, 312 Buckman, J., 271, 284, 294, 297, 298, 321, 322 Budacz, V., 290, 312 Bucher, T., 9, 26 Buford, W. T., 220, 222, 249, 253, 257 Bukovak, M. J., 168, 182 Bullock, H. R., 225, 255 Bullock, J. D., 310, 316 Bunting, A. H., 136, 142 Burdick, G. E., 307, 315, 318, 319 Burgess, E. E., 210, 211, 252, 257 Burgess, T. E., 309, 314 Burk, L. G., 19, 24, 243, 253 Burk, R. F., 287, 313 Burstein, Y., 279, 313 Burton, G. W., 207, 258 Burton, R. L., 207, 258 Busbie, T. H., 190, 191, 252, 253 Busch, A. W., 47, 94 Buss, G. R., 190, 191, 252 Butani, D. K., 227, 262 Butler, G. D., Jr., 227, 230, 253 Butler, H. S.,349, 378 Byard, J. L., 288, 313 Byerly, T. C., 280, 319 Byers, H. G., 288, 289, 313, 314 Byrne, A. R., 280, 313 Byrne, H. D., 191, 253
AUTHOR INDEX C
Cade, J. F. J., 293, 313 Caldwell, A. C., 397, 398, 410 Caldwell, R. M., 199, 253 Caldwell, S. D., 228, 253 Cameron, J. W., 210, 253 Camp, J. P., 158, 161, 181 Campbell, D. R., 293, 319 Campbell, N. E. R., 67, 82, 89 Campbell, W. V., 190, 191, 195, 240, 243, 252, 253, 255 Canerday, T. D., 239, 253, 254 Cannon, H . L., 291, 296, 313 Cannon, W. N., Jr., 208, 253 Carey, B. J., 68, 82, 94 Carlson, C. W., 160, 183, 268, 269, 286, 313, 317, 323 Carison, G. E., 116, 122, 142, 145 Carnahan, H . L., 189, 190, 253, 256, 261 Carpenter, D. M., 288, 310, 318 Carpenter, K., 336, 374 Carpenter, K. E., 304, 313 Carroll, M. D., 163, 164, 167, 168, 182 Carroll, R. E., 284, 313 Carter, D. L., 289, 317 Carter, S. W., 208, 243, 255 Cartier, J. J., 213, 239, 240, 251, 253 Cartwright, W . B., 199, 253, 255, 262 Cary, E. E., 289, 290, 311, 313, 314, 317 Casida, J . E., 328, 329, 337, 358, 367, 371, 373, 375 Caso, 0. H., 351, 375 Caspari, E., 3, 24 Castelfranco, P., 342, 373 Castell, C. H., 306, 313 Castro, R. U., 43, 46, 47, 49, 54, 80, 93 Castro, T. F., 64, 89, 92, 96 Cate, J . R., Jr., 217, 253 Catling, W. S., 203, 261 Cember, H., 278, 313 Chada, H. L., 201, 204, 207, 253, 255 Chalfant, R. B., 193, 253 Challenger, F., 301, 313 Chamberlin, R. I., 293, 313 Chambliss, 0. L., 211, 239, 253 Chandala, R. P., 205, 263
417
Chaney, R. L., 161, I82 Chang, F. Y., 332, 335, 362, 373 Chang, H., 219, 253 Chang, S. C., 78, 89 Chang, T-T., 104, 127, 142, 144 Chaplin, C. E., 232, 233, 253, 261 Chaplin, J . F., 12, 24 Chapman, H. D., 158, 167, 169, 170, 173, 174, 182, 296, 313 Chapman, R. K., 233, 234, 254, 261 Charles-Edwards, D. A., 138, 142 Charles-Edwards, J., 138, 142 Charlesworth, R. R., 387, 409 Charpentier, L. J., 219, 259 Chasseaud, L. F., 345, 348, 373 Chaudry, F. M., 167, 182 Chaugale, 0. S., 217, 263 Chaumont, T. C., 387, 409 Chem, T. M., 353, 376 Chenery, E. M., 400, 411 Cheney, H. B., 388, 411 Cheng, C. H., 198, 260 Chesnin, L., 175, 182 Chesters, G., 64, 90, 172, 184 Chiang, C. T., 46, 89 Chiang, H. C., 249, 253 Chilcote, D. O., 335, 336, 377 Childress, J. D., 295, 320 Chin, W. T., 364, 373 Ching, T. M., 6, 24 Chinn, J. T., 235, 255 Chinu, S. H . F., 281, 320 Chittenden, R. J., 15, 24 Chmelir, I. C., 175, I81 Cho, D. Y., 49, 53, 61, 62, 67, 68, 72, 73, 75, 89 Cholak, J., 281, 293, 313, 316 Chow, T . J., 283, 301, 313 Christ, C. L., 51, 80, 90, 275, 314 Christian, J. E., 331, 377 Christian, R. G., 282, 313 Christiansen, M. N., 340, 375 Christman, R. IF., 64, 89 Chuang, T. T., 60, 95 Cialone, J. C., 339, 372 Clark, D. G., 115, 142 Clark, F. E., 67, 68, 69, 89 Clark, H. E., 168, 169, 185 Clark, M. F., 4, 24 Clark, W. J., 195, 254
418
AUTHOR INDEX
Clark, W. M., 40, 44, 89 Clarke, A. E., 15, 25 Claude, A., 4, 26 Cleij, G., 19, 24 Clemena, G. G., 309, 325 Clifton, C. E., 46, 60, 65, 89 Clifton, C. M., 284, 320 Climy, R. W.. 240, 251 Cline, R. A.. 387. 388, 409 Close. B. hl., 384, 388, 395, 399, 409 Coaker. T. H., 228, 254 Coakley, J. M . , 221, 254 Coen, D.. 18, 22, 24 Coffman. R., 268, 313 Cohen, I.. M., 198, 254 Colby, S. R., 331, 373 Colby. W. G., 386, 409 Coleman, N . T., 44, 91, 274, 320 Coleman, R. G., 288, 313 Collin, G. H., 387, 388, 409 Collins, F. I., 17, 24 Collins. H . I.., 216, 255 Comar, C. L., 290, 294, 313, 324 Compton, L. E., 199, 253 Connell, J., 214, 257 Connell, W. E., 47, 89 Connin, R. V., 192, 254 Conway, V. M.. 37, 89 Conyers, E. S., 399, 403, 409 Cook, M. J., 294, 324 Cook, T. A., 278, 313 Cooke, A. R., 336, 374 Coon, B. F., 194, 214, 254 Cooper, B. P., 243, 263 Cooper, G. S., 68, 69, 89 Cooper, J. P., 102, 116, 117, 119, 128, 145, 146 Cooper, J. S., 269, 316 Copes, R. L., 112, 142 Corbett, M. K., 18, 25 Correns, C., 10, 24 Corwin, A. H., 309, 316 Cory, E. N . , 215, 254 Cottrell, H. J., 336, 374 Coulson, E. J., 307, 313 Cowan, C. B., 229, 258, 259 Cox, E. L., 119, 129, 143 Cox, F. R., 170, 182 Craddock, J. C., 200, 255 Craig, I. L., 16, 26
Craig, J. B., 403, 409 Crawford, C. L., 158, 161, 162, 170, 173. 174, 175, 177, 179, 181, 186 Creighton, C. S., 245, 254 Criddle, R. S., 118, 119, 129, 138, 142 Crocker, W., 280, 325 Crooke, W. M . , 295, 313 Crovetti, A. J., 329, 334, 378 Csonka, E., 279, 318 Cullen, 7'.E., 364, 373 Cunningham, R. P., 329, 373 Cunningham, W. P., 24 Curran, G. L., 292, 311 Curtis, B. C., 201, 254, 265 Curtis, G. I., 18, 24 Curtis, 0. F., 115, 142 Custer, J . L., 278, 324 Cuthbert, F. P., 237, 254 Cutter, E. G., 339, 340, 372 D
Daams, J., 333, 334, 375, 378 Dabrowski, Z. T., 232, 254, 261 Dacosta, C. P., 239, 254 Dahlman, D. L., 236, 254, 256 Dahrns, R. G., 187, 249, 254, 263 Dainty, J., 385, 387, 409 Dale, J. E., 228, 254 Dalton, A. J., 5, 25 Damron, C., 290, 312 Dang, K., 218, 254 Daniel, L. W., 64, 90 Daniels, N. E., 201. 254, 261 Danielson, L. L., 334, 374 Das, H. H., 37, 51, 95 Daugherty, D. M . , 195, 239, 252, 254 Davich, T . B., 220, 221, 254, 257 Davide, J. G., 78, 89 Davids, H. W., 302, 313 Davidson, R. H., 240, 251 Davies, B. E., 283, 311 Davies, E. B., 289, 324 Davies, J., 293, 325 Davies, P. J . , 335, 336, 373 Davis, B. W., 237, 254 Davis, D., 223, 224, 254 Davis, D. E., 340, 343, 347, 373, 374, 376, 377
419
AUTHOR INDEX
Davis, F. S., 351, 373 Davis, J. B., 210, 211, 264 Davis, J. F., 158, 159, 161, 175, 182, 183, 184, 186, 387, 400, 410 Davis, J. R., 282, 313 Davis, R. L., 192, 264 Davis, W. H., 15, 25 Davison, K. L., 347, 348, 372 Day, E. W., 339, 374 Day, P. R., 122, 146 De, P. K., 69, 89 Dean, G., 249, 258 Dean, H. A., 208, 262 Dean, L. A., 171, 185 Deay, H. G., 199, 255 de Freitas, L. M. M., 409 De Gee, J. C., 32, 35, 90 Degens, E. T., 59, 90 DeJager, J. M., 112, 146 DeKock, P. C., 165, I82 Delavaut, R. E., 282, 284, 324 Delevaux, M., 288, 313 Delker, L. L., 292, 319 Delwiche, C. C., 290, 317 Demetrio, J. L., 161, 185 DeMumbrum, L. E., 150, 182 Dennett, J. H., 52, 90 Dennis, E. G., 284, 321 DeRemer, E. D., 151, 160, 182 Derr, R. F., 212, 254 Desser, H., 165, I81 de Treville, R. T. P., 281, 313 Deutch, J., 18, 22, 24 Deutsch, D. B., 342, 373 Devine, J. R., 387, 409 Devlin, R. M., 329, 373 de Vries, M. P. C., 388, 399, 404, 411 Dey, S. K., 384, 388, 395, 403, 411 Dicke, F. F., 12, 26, 208, 210, 211, 215, 217, 243, 251, 253, 254, 255, 261, 262 Dickson, R. C., 189, 254 Dilbeck, J. D., 239, 254 Dilley, R. A., 129, 144 Dimitroff, J. M., 284, 314 Dionne, L. A., 233, 261 Dishner, G. A., 204, 254 Ditman, L. P., 215, 241, 254 D’Itri, F. M., 281, 313 Djamin, A., 197, 254
Doane, J. F., 233, 254 Doelle, H. W., 46, 60, 65, 69, 90 Dogger, J. R., 191, 254 Doggett, H., 218, 254 Dolar, S. G., 172, 182 Doll, E., 175, 186 Don, H., 190, 260 Donald, C. M., 104, 142 Donaldson, H. M., 293, 314 Donega, H. M., 309,314 Donninger, C., 345, 375 Dornhoff, G. M., 112, 142 Dorough, H. W., 367, 373 Doudoroff, P., 303, 304, 305, 314 Douglas, A. G., 222, 254 Douglas, W. A., 210, 211, 254, 258 Dovrat, A., 388, 396, 397, 400, 403, 409, 410 Dowdy, R. H., 404, 409 Downes, R. W., 115, 142 Doyle, J. J., 389, 410 Drake, M., 163, 182, 386, 409 Dreger, R. H., 112, 142 Drennan, D. S. H., 335, 336, 373 Drosdoff, M., 387, 410 Duarte, R., 126, 142 Dubetz, S., 110, 142 Dudley, J. W., 130, 133, 137, 143, 191, 253 Duff, R. B., 152, 186 Duffield, D. P., 278, 314 Dunn, J. A., 237, 254 Dunphy, B., 283, 314 Dunstone, R. L., 112, 120, 121, 143 DuPraw, E. J., 4, 6, 25 Dupuis, G., 342, 345, 375 Dusic, Z., 295, 314 Duthion, C., 388, 409 Duvick, D. N., 2, 13, 15, 25 Dvorak, J., 122, 142 E
Eagles, C. F., 99, 102, 103, 112, 116, 118, 119, 138, 142, 145, 146 Earl, J. L., 301, 313 Eastin, E. F., 355, 356, 357, 358, 373, 378 Ebner, L., 355, 373 Eckenrode, C. J., 241, 254
420
AUTHOR INDEX
Eckhardt, R. C., 110, 211, 254 Eddings, J. L., 162, 171, 172, 181 Eden, W. G.. 215, 259 Edgington, D. N., 306, 318 Edwards, D. G., 398, 410 Edwards, K. J . R., 126, 142 Edwards, R. W., 37, 90 Edwardson, J. R., 2, 3. 1 I , 12, 15, 18, 20, 21, 25, 26 Ehlig, C. F., 288, 289, 290, 311, 314 Eiben, G. J.. 212, 254 Elgabaly, M. M., 150, 182 Elgawhary, S. M., 163, 164, 171, 182 Elkassaby. F. Y., 229, 230, 257 Ellis, B. G., 150, 158, 175, 182, 183 Ellis, M. M., 305, 314 Ellis, R., 78, 94 Ellis, R.. Jr.. 161, 182, I85 Elmore, C. D., 100, 143 Elsam, 0. E., 387, 409 Elsey, K. D.: 245, 254 El-Sharkawy. M., 116, 143 Emara, Y. A., 126, 142 Emecz, T. I.. 105, 143 Emery, D. A., 16, 26 Emmerikh, R. D., 205, 254 Engledow, F. L., 132, 143 English, J. N.,301, 314 Enoch, H., 34, 91 Epstein, E., 164, 165, 167, 182, 185 Erel, K., 388, 403. 411 Erickson, H. T., 239, 253 Erne, K., 279, 281, 312, 324 Erygin, 38, 90 Esau, K., 4, 6. 25 Eshel, Y., 353, 361, 362, 364, 373, 376, 377 Espinoza, W. G., 341, 373 Esser, H.. 342, 345, 375 E,ttinger, M. B., 269, 312 Evans, C. A., Jr., 309, 314 Evans, C . E., 172, 186 Evans, D. D., 34, 90 Evans, D. E.. 227, 255 Evans, L. T., 112, 120, 121, 136, 153 Everett, T. R., 220, 255 Everly, R. T., 204, 217, 254, 255 Everson, E. H., 200, 203, 205, 255, 261 Ewins, A,, 269, 316 Ezygin, P. S., 38, 90
F
Fabrega, E. A., 282, 313 Fabricand, B. F., 35, 90 Farmer, F. S., 356, 376 Faulkner, A., 278, 313 Feeny, R. W., 339, 361,373 Feigenbaum, S., 396, 400, 403, 404, 409 Feil, V. J., 339, 346, 375, 378 Feldherr, C. M., 4, 25 Fernandez, A. T., 225, 255 Ferres, H. M., 160, 182 Ferris, E. C., 294, 312 Fife, L. C., 225, 262 Filby, R. H., 269, 271, 285, 288, 291, 322 Filipovic, Z., 295, 314 Filippovich, I. I., 8, 24, 26 Fimreite, N., 279, 307, 314 Findenegg, G., 165, 181 Fischer, D. D., 288, 319 Fish, P. W., 240, 251 Fishbein, W. I., 282, 313 Fisher, A., 353, 373 Fisher, M. E., 17, 25 Fitzgerald, G. P., 35, 90 Fitzgerald, P. J., 212, 255, 260, 262 Flemming, A. A., 16, 25 Fletcher, K., 272, 324 Flick, D. F., 284, 314 Floyd, E. H., 214, 255 Floyd, R. A., 169, 182 Follett, R. H., 148, 151, 160, 172, 179, 182 Foltz, C. M., 287, 322 Ford, B. T., 217, 255 Fortuno, M. E., 198, 260 Fotiyev, A. V., 64, 90 Fowler, C. W., 126, 143 Foy, C. D., 161, 182 Foy, C. L., 329, 332, 339, 340, 342, 372, 373, 375 Fraenkel, G., 237, 244, 245, 265 Frams, R. E., 339, 376 Francki, R., 4, 8, 24 Frank, J. A., 235, 263 Frank, R., 353, 355, 373 Frankel, R., 18, 20, 25 Frater, A., 399, 409 Frauenthal, H. L., 301, 302, 317, 320
AUTHOR INDEX
Frear, D. S., 329, 331, 337, 342, 343, 345, 346, 348, 349, 350, 354, 357, 359, 360, 361, 362, 369, 370, 373, 374, 375, 377, 378 Freed, V. H., 332, 345, 347, 373, 376 Freeman, G. G., 388, 389, 397, 409 French, S. A. W., 104, 146 Freney, J. R., 74, 90 Frey-Wyssling, A., 4, 25 Friberg, L., 307, 312 Frickie, D. N., 282, 318 Fried, M., 78, 89 Friedel, R. A., 269, 271, 283, 284, 285, 288, 291, 316 Friedman, G. M., 35, 43, 50, 51, 90 Fromm, P. O., 307, 308, 314, 317, 321 Frost, D. V., 287, 288, 294, 298, 314, 322 Fryer, J. D., 335, 336, 373 Fukuta, T. R., 367, 375 Fukuyama, M., 121, 145 Fulkerson, W., 280, 281, 324 Fuller, W. H., 297, 315 Fullum, E., 4, 26 Funderburk, H. H., Jr., 333, 339, 340, 343, 347, 361, 362, 373, 374, 376, 377 Funkhouser, E. A., 168, 169, 185 Funnell, H. S., 279, 320 Furr, R. E., 228, 262 Furusaka, C., 46, 47, 95 Fyfe, R. W., 279, 314 G
Gaastra, P., 122, 143 Gabriel, W. J., 242, 265 Gahan, P. B., 6, 25 Gall, 0. E., 158, 161, 181 Gallagher, P., 278, 313 Galle, 0. K., 302, 311 Gallun, R. L., 199, 200, 203, 204, 205, 253, 255, 256, 260, 262 Ganje, T . I., 282, 283, 319 Ganiron, R. B., 161, 182 Ganther, H. E., 284, 286, 314 Gapon, E. N., 274, 314 Garber, M. J., 162, 181 Garbouchev, I. P., 403, 409 Garcia, C. V., 108, 145
42 1
Garcia, J. A., 225, 262 Gardener, C. J., 108, 145 Gardenshire, J. H., 20 1, 203, 204, 253, 255 Gardner, C. O., 192, 254 Gardner, R., 53, 95 Garin, B. E., 34, 96 Carrels, R. M., 44, 51, 54, 79, 80, 90, 92, 275, 314 Gasser, J. K. R., 78, 90 Gauch, H. G., 165, 181 Gaudy, A. F., Jr., 63, 90 Gaudy, E. T., 63, 90 Gausman, H. W., 211, 255 Gavish, E., 35, 43, 50, 51, 90 Geering, H. R., 151, 153, 182, 183, 289, 314 Geissbiihler, H., 355, 356, 357, 358, 359, 360, 361, 362, 374 Gentile, A. G., 235, 236, 255, 263, 264 Getner W . A., 334, 349, 374, 376 Gentry, C. R., 255 Gerhold, H . D., 242, 255, 263 Gerloff, E. D., 213, 260 Gerola, F. M., 6, 25 Ghassemi, M., 64, 89 Gibbons, D., 329, 337, 376 Gibor, A., 7, 25 Gibson, F. H., 269, 271, 285, 311 Gibson, R. W., 235, 255 Giddens, J., 340, 377 Gilbert, J. C., 235, 255 Gilbert, N., 124, 146 Gilbert, T. W., 309, 315 Gile, P. L., 289, 314 Gilfillan, S. C., 281, 314 Giordano, P. M., 177, 179, 182, 184 Girardeau, J . H., 244, 255 Gissel-Nielsen, G., 289, 290, 312, 314 Gladstones, J. F., 167, 183 Glathe, H., 93 Glomski, C. A., 278, 279, 314 Gloyna, E. F., 72, 92 Godfrey, A. E., 80, 89 Goerlitz, D. F., 64, 92 Gogan, G., 161, 162,186 Golab, T., 337, 338, 339, 340, 374, 376 Goldberg, A. A., 278, 314 Goldberg, E. D., 37, 76, 90, 306, 317 Goldberg, J. B., 124, 145
422
AUTHOR INDEX
Goldschmidt, V. M., 33, 90, 148, 149, 183, 269, 271, 272, 273, 288, 314 Goldsworthy, A., 121, 143 Goldwater, L. J . , 278, 281, 307, 314, 315. 316 Goodall, D. W., 170, 183 Goodchild, D. J . , 138, 143 Goodman, D., 35, 38, 90 Goodman. J. R., 305, 314 Goodman, N. R., 307, 315 Gordon. M. P., 8, 25 Gorham, E., 32, 90 Gorsline, G. W., 167, 183 Gorz, H. J., 192, 193, 251, 254, 259 Gotoh, S., 60, 62, 90 Could, T. C., 284, 310, 314, 315 Govindjee, 83, 93 Grable, A. R., 31, 90 Graetz, D. A., 64, 90 Grafius, J. E., 124, 125, 132, 142, 143, I45 Graham, H. M., 189, 223, 225, 229, 255, 259, 262 Gramlich, I. V., 338, 339, 347, 373, 374 Grandy, J . W . , IV, 282, 314 Granick, S., 7, 25 Greaves, J. E., 286, 314 Greeley, L., 6, 27 Green, B. R., 8, 25 Green, D. H., 355, 373 Green. V. E., Jr., 71, 90 Greenblatt, I. M., 15, 25 Greenwood, D. J., 34, 35, 38, 46, 47, 66, 68. Y O Gregory, F. G., 170, 183 Greib, B. J . , 37, 78, 80, 91 Grifiths, M. H., 345, 375 Gronka, P. A., 278, 314 Gross, A. T . H., 192, 255 Gross, D., 355, 356, 374 Gruenhagen, R. D., 329, 374 Grun, P., 15, 25 Grunes, D. L., 160, 183, 398, 409 Guaglinni, P., 207, 255 Guan, C . K., 195, 196, 264 Gueldner, R., 220, 256 Guenthner, E., 286, 323 Guevara, C. J . , 241, 255 Guilcher, A., 31, 90 Guinn, G., 165, 183
Guiochon, G., 309, 320 Gulati, S. C., 204, 260 Gullmick, F., 169, 184 Ciunn, S. A., 284, 310, 314, 315 Guss, P. L., 213, 252 Gutenmann, W. H., 279, 305, 306, 307, 309, 311, 315, 318, 323, 324 Guthrie, F. E., 243 Guthrie, W. D., 208, 209, 210, 243, 255, 257, 261, 262 Gysin, H., 342, 347, 367, 374 H
Haag, H. P., 164. 165, 167, 185 Hackerott, H. L., 189, 190, 206, 216, 255, 256, 263 Hacskaylo, I., 221, 254, 339, 340, 374 Hadjimarkos, D. M., 288, 315 Haessler, W. T., 284, 322 Hagan, A. F., 212, 255 Hagan, R. M . , 31, 95 Hageman, R. H., 17, 25, 27, 130, 133, 137, 143 Hagin, J., 388, 396, 400, 403, 404, 409 Hahn, S. R., 125, 132, 145, 205, 255 Haley, T. J . , 283, 315 Hall, C. V., 234, 237, 238, 252, 256, 260, 264 Hall, R. C., 242, 256 Hallauer, A. R., 209, 210, 257, 262 Haller, W . A., 269, 271, 285, 288, 291, 322 Halverson, A. W., 286, 288, 319 Halvorson, A. D., 165, 166, 183 Hamaker, J. W., 351, 374 Hamill, A., 341, 374 Hamilton, C. M., 279, 318 Hamilton, J. C., 272, 282, 291, 296, 322 Hamilton, K. C., 340, 341, 374 Hamilton, M. G., 211, 256 Hamilton, P., 352, 376 Hamilton, R. H., 342, 343, 347, 350, 3 74 Hamilton, W., Jr., 338, 339, 373 Hamlin, J. M . , 303, 316 Hamlyn, F. G., 389, 410 Hamm, J. J . , 211, 264 Hammermeister, K. E., 279, 318 Harnmond, A. L., 281, 315
AUTHOR INDEX
Hammond, P. B., 283, 315 Hammons, R. O., 193, 258 Hampton, R. E., 229, 261 Hamura, I., 362, 375 Hancock, W., 281, 316 Handley, R., 386, 387, 410, 411 Handreck, K . A., 79, 91 Hankin, L., 282, 315 Hanko, E., 279, 281, 312, 324 Hannopel, R., 387, 410 Hansen, J. R., 367, 374 Hansen, L. C., 309, 315 Hansen, N. M., 284, 322 Hansen, R. M., 206, 256 Hanson, C. H., 116, 145, 190, 191, 252, 253, 254, 260, 264 Hanson, J. B., 17, 25 Hanson, K. R., 282, 315 Hanson, W. C., 293, 315 Hanson, W. D., 203, 259 Hansson, K., 279, 323 Hanway, J. J., 384, 388, 399, 411 Harada, T., 70, 90 Hardee, D. D., 223, 259 Harding, R. B., 400, 410 Hargan, R. P., 339, 372 Harper, A. M., 245, 256 Harris, E. J., 307, 315, 318, 319 Harris, R. F., 37, 78, 79, 90, 94, 95 Harrison, W . H., 62, 90 Harrison, W. W., 309, 325 Harriss, R. C., 306, 315 Hart, R. D., 336, 374 Hart, R. H., 116, 122, 142, 145 Harter, R. D., 35, 90 Harvey, J. E., 193, 258 Harvey, T. L., 189, 190, 206, 216, 255, 256, 258, 263 Harward, M. E., 75,'90 Hashimoto, Y., 288, 315 Haskins, F. A., 192, 251 Hasler, A., 273, 280, 285, 315 Hassawy, G. S., 340, 341, 374 Hatch, W. R., 309, 315 Hatchett, J. H., 199, 255, 256 Hattingh, W . H. J., 63, 91, 95 Hattula, T., 278, 318 Haubein, A. H., 367, 374 Haunold, E., 78, 89 Hawkins, B. S., 229, 256
423
Hawley, J. E., 288, 315 Hawton, D., 355, 356, 357, 358, 374 Hay, J., 158, 162, 183 Hayashi, K., 105, 106, 107, 108, 116, 143 Hayes, F. R., 35, 90 Heath, 0. V . S., 115, 143 Hecht, H., 115, 142 Hedin, P. A., 211, 220, 256, 258 Heichel, G. H., 112, 114, 116, 122, 129, 130, 143 Heidbreder, G. A., 281, 323 Heisler, C. R., 190, 261 Helling, C. S., 329, 337, 374 Helminen, M., 279, 315 Hem, J. D., 31, 43, 51, 90 Hemken, R. W., 190, 191, 252 Henderson, C., 301, 314 Henderson, W. R., 239, 252 Hernandez, H. R., 225, 255 Henriksson, K., 279, 315 Herberg, R. J., 337, 338, 339, 374, 376 Herlihy, M., 388, 409 Herman, S., 279, 317 Hernberg, S., 281, 282, 315 Herndon, J., 278, 316 Herrera, R. M., 134, 135, 144 Herrett, R. A., 367, 368, 374 Hesketh, J. D., 100, 116, 143 Heslop-Harrison, J., 20, 25 Hesse, P. R., 52, 90 Heukelekian, H., 47, 91 Hewitt, E. J., 164, 183, 283, 315 Hewitt, G. B., 206, 256 Heydorn, K., 285, 315 Heyne, E. G., 199, 201,251 Heywood, B. J., 336, 374 Hiatt, A. J., 399, 410 Hibbs, E. T., 236, 254, 256 Hicks, D. R., 110, 134, 143 Hiesey, W. M., 128, 143 Higgs, D. J., 293, 319 Highkin, H. R., 138, 143 Hilgeman, R. H., 297, 315 Hill, A. R., 231, 256 Hill, C. R., 294, 315 Hill, G. C., 359, 374 Hill, G. R., 122, 145 Hill, K. L., 349, 376 Hills, W. A., 264
424
AUTHOR INDEX
Hilmoe, R. I., 288, 322 Hilton, J. L., 329, 340, 342, 349, 353, 375, 376 Hirano, C., 196, 197, 256, 260 Hirayama, K., 310, 323 Hirth, C. R., 34, 35, 92 Hisada, T., 370, 375 Hissong, D. E.. 305, 320 Ho, R. K. Y., 301, 321 Hoadley, E. C., 345, 375 Hoagland, D. R., 389, 410 Hodges, T. K., 387, 409 Hodgson, J. F., 148, 150, 151, 153, 155, 156, 157, 164, 175, 182, 183, 184, 285, 286, 291, 315 Hodgson, R. H.. 354, 378 Hoff, D. K., 386, 410 Hoffman, L. H., 293, 325 Hoffman, S., 329, 340, 372 Hoffmeister, G., 177, 185 Hofmeister, F., 288, 315 Hofstra, G., 119, 143 Hogue, E. J., 359, 361, 375 Holdaway, F. G., 192, 261 Holland, R. F., 13, 27 Holland, R. H., 285, 315 Hollowell, E. A., 194, 257 Holly, K., 335, 336, 373 Holmberg, D. M., 174, 175, 184 Holmes, M. R. J., 387, 409, 410 Holmes, N . D., 202, 249, 256 Holmes, R. S., 165, 182 Holmgren, P., 115, 116, 128, 143 Holmstedt, B., 307, 312 Holsing, G. C., 364, 373 Holt, G., 279, 315 Holt, J. M . , 292, 324 Holtzman, R. B., 283, 301, 315, 323 Holzer, F . J . , 337, 338, 339, 340, 374, 376 Honeysett, J . L., 80, 88 Hook, R. H., 293, 323 Hoover, A. W., 278, 315 Hope, A. B., 385, 386, 387, 409 Hopkins, L. L., Jr., 292, 315 Hopkins, T. R., 365, 366, 376 Hormchong. T., 204, 256 Horner, G. M., 286, 324 Horovitz, S., 215, 256 Horrom, B. W., 329, 334, 378
Horton, R. F., 335, 377 Horvath, R. S., 329, 331, 375 Houghtaling, J. E., 223, 224, 225, 229, 249, 258, 259 Houghton, J. M., 349, 378 Hovland, D., 397, 398, 410 Howe, W. L., 189, 190,256 Howeler, R. H., 35, 36, 37, 45, 91 Howitt, A. J . , 213, 256 Hsu, P., 126, 143 Hsu, S. J., 204, 256 Huart, R., 21, 25 Huber, G. A., 23 1, 256 Huddleston, P., 257 Hudgins, H. R., 370, 373 Hudson, T. G. F., 292, 315 Hudspeth, W. N., 221, 256 Huffaker, R. C., 119, 129, 143, 386, 410 Hull, H. M., 329, 342, 375 Humphreys, R. E., 269, 316 Humphries, A. W., 31, 91 Hundt, I., 169, 184 Hunt, 0. J., 189, 190, 256, 261 Hunter, A. S., 389, 400, 401, 404, 410 Hunter, F., 388, 396, 403, 409 Hunter, J. G., 295, 313 Hunter, R. C., 221, 222, 256 Hunter, R. E., 229, 258 Hurd, D. T., 273, 274, 320 Hurter, J., 345, 375 Husted, R. R., 343, 378 Hutcheson, J. B., 404, 409 Hutchinson, G. E., 33, 35, 36, 50, 62, 74, 75, 76, 79, 82, 83, 91 Hutson, D. H., 345, 375 Hvatum, 0. O., 283, 317 Hyatt, E. C., 293, 315 Hyer, A., 228, 229, 258 Hynes, H. B. N., 37, 78, 89, 91 I
Ibbotson, A., 246, 256, 257 Ikehashi, H., 132, 143 Ilcewicz, F. H., 283, 315 Ilnicki, R. D., 359, 360, 361, 362, 376 Imber, D., 138, 143 Imbimlo, E. S., 35, 90 Imura, N., 301, 315 Incoll, L. D., 119, 144
AUTHOR INDEX Ingram, J. W., 214, 255 Ingram, W. M., 300, 316 Irby, H. D., 282, 317 Irvine, J. E., 111, 143 Isaac, R. A., 169, 183 Isaacs, A., 19, 25 Isaak, A., 189, 256 Ishii, S., 197, 256 Ishikawa, K., 71, 91 Islam, A,, 399, 403, 410 Israel, P., 197, 256 Ito, R., 132, 143 Ives, D. J. G., 42, 91 Iwasa, Y., 31, 91 Iwata, T., 197, 256 Iwata, Y., 197, 256 Izhar, S., 111, 112, 114, 116, 122, 127, 143 J
Jackim, E., 303, 316 Jacks, T. J., 25 Jackson, M. L., 76, 78, 89, 91, 150, 182 Jackson, T. L., 159, 162, 183 Jackson, W. A,, 114, 121, 143 Jacob, F., 20, 25 Jacobsohn, R., 365, 375 Jacobson, L., 386, 387, 410, 411 Jacoby, B., 161, 184, 400, 410 Jahontov, V., 227, 257 Jain, K. B. L., 204, 260 Jakob, H., 18, 26 James, C. S., 364, 375 Jana, S., 132, 143 Janitzky, P., 76, 91 Jansen, L. L., 329, 342, 375 Janz, G. J., 42, 91 Jasmin, J. J., 237, 261 Jaworski, E. G., 368, 369, 375, 376 Jeffery, J. W . O., 42, 43, 45, 52, 90, 91 Jenkins, D., 75, 91 Jenkins, J. N., 220, 221, 222, 223, 224, 225, 229, 249, 251, 253, 254, 256, 257, 259, 260, 262 Jenkins, M. T., 211, 254 Jenkins, R. Y., 237, 245, 265 Jenkins, S. H., 269, 316
425
Jenne, E. A., 31, 37, 80, 83, 91, 149, 183 Jennings, P. R., 108, 127, 134, 135, 142, 144, 197, 198, 257 Jenny, H., 44, 91, 150, 182, 275, 296, 316 Jensen, H. L., 70, 83, 91, 148, 183 Jensen, S., 300, 306, 316 Jernelov, A., 273, 300, 301, 306, 316 Jinks, J. L., 3, 10, 25 Joachirn, A. W . R., 66, 91 Joensuu, 0. J., 280, 316 Joffe, J. S., 31, 91 Joharn, H. E., 165, 183 Johannsen, A., 268, 316 Johansen, C., 398, 410 Johnek, A. G., 279, 305, 306, 312, 316 Johnson, C. M., 290, 317 Johnson, H., 351, 374 Johnson, H. W., 194, 257 Johnson, J., 19, 25 Johnson, J. A., 199, 259 Johnson, K. E. E., 387, 400, 410 Johnson, L. A., 310, 322 Johnston, C. O., 199, 251, 260 Johnston, I. M., 291, 295, 318 Johnston, T. J., 110, 134, 143, 145 Johnstone, M . S., 283, 301, 313 Jonasson, I. R., 280, 316 Jones, C. M., 239, 253, 254 Jones, C. S., 364, 376 Jones, D. W., 329, 375 Jones, E. T., 199, 251, 259, 260, 261 Jones, G. B., 167, 185 Jones, G. K., 245, 257 Jones, H. A., 15, 25 Jones, J. B., 386, 410 Jones, J. B., Jr., 169, 170, 183 Jones, J. C., 293, 323 Jones, J. E., 222, 224, 249, 257 Jones, J. R. E., 305, 316 Jones, L. H., 103, 144, 388, 389, 410 Jones, L. H . P., 79, 91, 289, 314 Jones, M. M., 277, 317 Jonsson, E., 307, 312 Jordan, J. H., 69, 91 Jordan, R. A., 269, 309, 312 Joselow, M. M., 278, 316 Josephson, L. M., 211, 252, 257 Joshi, M . S., 283, 319
426
AUTHOR INDEX
Judy, W. H., 158, 175, 183 Jugenheirner, R. W., 210, 257 Jurinak, J. J., 148, 160, 183, 185 Jyung, W . H., 165, 185 K
Kadunce, R. E., 331, 343, 350, 354, 374, 377, 378 Kahn, A., 6, 24 Kahn, H. L., 309, 316 Kaloushova, J., 284, 288, 310, 319 Kamalanathan, S., 227, 251 Karnel, S. A., 229, 230, 257 Karnoshita, Y., 31, 91 Kamprath, E. J., 170, 182 Karnura, T., 34, 45, 46, 61, 71, 91, 95 Kandaswarny, P. A., 219, 251 Kandewity, F., 9 , 26 Kanisawa, M.. 294, 316, 322 Kanno, I., 33, 74, 91 Kaplan, I. R., 33, 34, 35, 37, 43, 49, 50, 51, 71, 79, 89, 91 Kappel, H. M., 45, 93 Karayannis, N. M., 309, 316 Kark, R. A. P., 310, 316 Karppanen, E.. 179, 315 Kassebeer, Von H., 363, 364, 375 Kato, A., 44, 92 Katsunuma, H., 278, 323 Katz, E. L., 305, 320 Katz, M., 303, 304, 305, 314 Katz, S. A,. 309, 320, 321 Katz, S. E., 359, 360, 361, 376 Kaufman, D. D., 64, 91 Kaufrnan, T.,206, 257 Kawaguchi, K., 32, 33, 44, 66, 61, 84, 91, 92 Kawano, K., 108, 145 Kawashirna, R., 110, 111, 127, 128, 140, 141, 144 Ke, B., 129, 144 Kearney, P. C., 64, 91, 286, 287, 307, 316, 325, 329. 337, 374 Keaton, C. M., 286, 324 Keck, R. W . , 129, 144 Kee, N . S., 272, 283, 291, 295, 316 Keeney, D. R., 172, 182 Keep, E., 231, 232, 257, 258
Kefauver, M., 68, 91 Kefford, N. P., 351, 375 Kehoe, R. A,, 281, 316 Kehr, W. R., 190, 256, 257 Keight, D. G., 269, 316 Keith, J. A., 279, 314 Keller, J. C., 220, 257, 259 Kelly, W . H., 268, 324 Kernper, W. D., 163, 164, 171, 182, 183, 185 Kernpthorne, O., 124, 144 Kennedy, F. S., 301, 325 Kennedy, J. S., 246, 256, 257 Kenworthy, A. L., 169, 170, 183 Kerber, J. D., 169, 183 Kessler, T., 269, 271, 283, 284, 285, 288, 291, 316 Keup, L. E., 34, 35, 92 Key, J. L., 401, 410 Khadr, A,, 167, 183 Khalifa, A., 226, 257 Khan, M. A., 99, 102, 108, 109, 112, 116, 117, 121, 122, 144, 227, 260 Kheiralla, A. I . , 125, 136, 144 Kieckhefer, R. W., 212, 254 Kim, J., 301, 315 Kimura, Y., 279, 280, 317, 318 Kinard, W . S., 245, 254 Kindler, S. D., 189, 257 King, H. M., 386, 410 Kircher, H. W., 190, 257 Kirk, J. T . O., 8, 10, 18, 20, 24, 25 Kirk, V . M . , 215, 257 Kisaki, T., 27 Kishaba, A. N . , 190, 257 Kita, D., 44, 91 Kitagishi, K., 197, 265 Kitarnura, H., 60, 62, 95 Kitby, F., 230, 260 Kiwimae, A., 279, 317 Kjensmo, J., 43, 44, 50, 91 Klein, D. H., 306, 317 Klein, R., 279, 317 Kleinkopf, G. E., 119, 129, 143 Klosterrneyer, E. C., 211, 257 Klun, J. A., 209, 257, 342, 375 Knapp, C. E., 280, 281, 317 Knapp, J. L., 211, 257, 258 Kneip, T . J., 281, 323
427
AUTHOR INDEX
Knezek, B. D., 159, 161, 175, 184, 186 Knight, A. H., 295, 313 Knight, R. L., 231, 232, 257, 258 Knipling, E. F., 249, 258 Knoll, J., 307, 317 Kniiesli, E., 342, 345, 374, 375 Kobel, R. J., 10, 25 Koch, J . T., 388, 410 Koch, W., 363, 373 Koehler, F. E., 158, 160, I85 Koenigs, F. F. R., 32, 44, 74, 91, 94 Koeppe, D. E., 12, 26 Kogan, B. A,, 281, 323 Kojyo, S., 109, 145 Kolbezen, M. J., 367, 375 Kolbye, A. C., 280, 319 Kooistra, E., 239, 258 Koons, C., 2, 12, 26 Koopman, H., 334, 375 Kopp, J. F., 302, 317 Kornfeld, J. M., 282, 315 Kosta, L., 280, 313 Kosuge, T., 138, 145 KotzC, J. P., 63, 91 Koyoma, T., 34, 45, 46, 61, 71, 95 Kozelnicky, G.. M., 16, 25 Krantz, B. A., 162, 171, 173, 174, 175, 177, 179, 181 Krauskopf, K. B., 148, 149, 183 Kraybill, H. F., 284, 314 Kring, J. B., 245, 258 Krista, L. M., 286, 317 Kristensen, J., 34, 92 Kristensen, K. J., 34, 91 Kroll, S. S., 284, 322 Kroner, R. C., 302, 31 7 Kroon, A. M., 9, 24 Kruger, F., 305, 317 Kruitwagen, E. C., 227, 228, 263 Kubo, H., 362, 375 Kubota, J., 284, 287, 289, 292, 311, 314, 317, 398, 409 Kuenen, P. H., 33, 34, 91 Kuffler, S. W., 65, 67, 72 Kuhn, U. S. G., 111, 290, 324 Kuhr, R. J., 367, 375 Kumar, K., 217, 258 Kung, S. D., 7, 25 Kunin, R., 273, 317 Kuratle, H., 359, 361, 362, 375
Kurland, L. T., 280, 319 Kurtz, L. T., 401, 410 Kuzerian, O., 370, 377 Kwan, T., 301, 315 Kyuma, K., 32, 33, 44, 67, 84, 91, 92 1
Labanaukas, C. K., 169, 184 Lacadena, L. R., 19, 25 Lafever, H. N., 220, 221, 222, 223, 224, 225, 249, 257, 259 LaFrance, J., 237, 261 Lag, J., 283, 317 Lagerwerff, J. V., 280, 283, 284, 285, 317, 385, 386, 410, 411 Laing, H., 38, 92 Laird, E. F., Jr., 189, 254 Lakin, H. W., 288, 289, 290, 311, 314, 317 Lamar, W. L., 64, 92 Lamm, C. G., 148, 183 Lammerink, J., 234, 258 Lamoureux, G. L., 345, 346, 347, 348, 349, 350, 366, 369, 372, 375, 377 Landes, D. A., 227, 258 Landner, L., 301, 317 Langille, W. M., 306, 325 Laming, F. C., 199, 218, 258 Lantican, R. M., 2, 12, 27 Larkin, D. V., 279, 318 Larson, J. D., 346, 375 Larson, R. I., 256 Latimer, W . H., 40, 92 Latter, B. D. H., 128, 144 Lauer, D. A., 172, 178, 184 Lauer, F. I., 236, 261 Laughnan, J. R., 13, 26 Lawrence, A. W., 269, 317 Laws, W. D., 159, 186 Lawson, R. I., 203, 258 Laysides, J. B., 240, 251 Lazar, V. A., 284, 289, 317, 398, 409 Lazrus, A. L., 301, 317 Learned, R. E., 280, 318 Lebsock, K. L., 203, 259 Leckie, J., 79, 92 Ledouk, L., 21, 25 Lee, D. R., 122, 142
42 8
AUTHOR INDEX
Lee, G. B., 64, 90 Lee, G. F., 37, 47, 89 Lee, H. S., 221, 222, 263 Lee, J., 158, 159, 185 Lee, J. A., 224, 258 Lee, Y. W., 281, 320 Leeper, G. W., 160, 184 Lees, H., 47, 66, 67, 82, 89, 90 Leggett, G. E., 162, 181 Leggett, J. E., 165, 182 LeGrand, H. E., 299, 317 Lehman, W. F., 190, 192, 258, 260 Lehrnann. O., 112, 142 Lehninger, A. L., 25 Lehr, J. R., 177, 178, 184 Leigh, T. F., 221, 222, 227, 228, 229, 252, 256, 258 Lemon. E., 34, 92 Lener, J., 278, 284, 288, 310, 319 Leng, E. R., 130, 133, 137, 143 Leonard, C. D., 155, 175, 185 Le Pelley, R . H., 19, 26 le Roux, J . , 388, 399, 403, 410 Lessrnan, G., 158, 175, 183 Letey, J., 31, 95 Leuck, D. B., 193, 207, 258 Levander, 0. A.. 286, 310, 317 Levin, D. A., 20, 24 Levin, I., 396, 410 Levine, R. P., 138, 144 Lewin, G., 271, 317 Lewis. B. G., 290, 317 Lewis, C. E., 292, 317 Lewis, D., 21, 25 Lewis, R. N , 273, 274,320 Li, C. C., 104, 142 Li, L. F., 292, 325 Lieber, M., 301, 302, 313, 317, 320 Lieberrnan, F . V., 189, 190, 256, 257, 26 I Lieserling, R., 228, 258 Lieth, H., 106, 144 Lilius, H., 281, 282, 315 Lirn, J., 278, 324 Lincoln, C., 221, 222, 249, 256, 258 Lindberg, Z. Y., 269, 317 Lindernann, J., 19, 25 Lindgren, W., 268. 272, 317 Lindquist, D. A,, 221, 254 Lindquist, R. K., 193, 258
Lindsay, W. L., 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 166, 170, 171, 172, 175, 179, 181, 182, 183, 184, 185, 186 Lindsten, J., 279, 323 Lindstrom, O., 281, 317 Lingle, J. C., 174, 175, 184 Linko, Y. Y., 218, 258 Lisanti, L. E., 389, 396, 411 Lisk, D. J., 279, 305, 306, 307, 308, 309, 311, 315, 318, 319, 323, 324 Little, E. I., 277, 31 7 Little, J . B., 283, 317 Little, J. W., 284, 322 Liu, P. S., 119, 120, 144 Livers, R. W., 206, 258 Livingston, P. O., 284, 322 Ljunggren, L., 279, 31 7 Lloyd, B., 68, 92 Lloyd, R., 304, 317 Lo, S. Y., 170, 184 Loach, K., 103, 121, 144 Locke, L. N., 282, 312, 314, 317, 318 Locke, R. K., 375 Lodge, J . P., Jr., 301, 317 Loeffel, F. A., 168, 184 Lofroth, G., 281, 318 Loehr, R. C., 63, 92 Lofquist, G. A., 281, 316 Loneragan, J. F., 163, 164, 167, 168, 182, 183, 383, 398, 402, 409, 410 Lorange, E., 301, 317 Losee, F. L., 284, 287, 292, 294, 311, 317, 318 Lovett, R. J., 307, 318 Lowe, H. J . B., 245, 258 Loy, T. A., 43, 44, 45, 51, 52, 53, 54, 58, 61, 72, 74, 80, 83, 93 Lucas, H. F., Jr., 301, 306, 318, 323 Lucas, R. E., 159, 161, 184 Lucken, K. A., 14, 25 Luckman, W. H., 210, 258 Luginbill, P., Jr., 199, 203, 249, 258, 259 Lukefahr, M. J., 223, 224, 225, 226, 229, 249, 255, 258, 259, 262 Lunt, H. A., 268, 269, 318 Lunt, 0. R., 268, 322
AUTHOR INDEX Lupton, F. G. H., 99, 109, 120, 126, 136, 144, 203, 252 Lyall, L. H., 234, 259 Lykken, L., 328, 329, 337, 358, 371, 3 73 Lynch, K. M., 307, 313 Lyon, W. S., 280, 281, 324 Lyttleton, J. W., 4, 25 M
Maan, S. S., 14, 25 McBride, B. C., 301, 318 McCain, F. S., 214, 215, 259, 262 McCall, R. C., 292, 320 McCarthy, J. H., Jr., 280, 318 McCarty, P. L., 269, 317 McClung, A. C., 409 McClusky, R. K., 237, 245, 265 McCollister, S. B., 293, 323 McColloch, R. C., 400, 410 McCollum, J. P., 347, 376 McCombs, C. L., 239, 252 McConaghy, S., 148, 181, 388, 410 McConnell, K. P., 288, 310, 318 McCree, K. J., 122, 144 McDaniel, R. G., 17, 18, 26 McDermott, G. N., 269, 301, 312, 314 McDermott, R. E., 242, 255 McDonald, D. J., 127, 128, 144 McDonald, M. D., 203, 256, 258 McEwen, H . B., 399, 410 McFadden, M. W., 245, 259 Macfarlane, R. B., 306, 315 McGandy, R. B., 283,317 McGarity, J. W., 68, 92 Macias, W., 246, 259 McIlhinney, J. G., 281, 316 McIntosh, J. L., 396, 397, 412 McKee, G. D., 34, 35, 92 McKee, J. E., 291, 292, 296, 309, 318 Mackenthun, K. M., 34, 35, 92, 300, 316 Mackenzie, F. T., 79, 92 McKenzie, H., 203, 257 Mackereth, F. J. H., 44, 92 McKinney, H. H., 6, 27 McKnight, M. E., 190, 256 McKone, C. E., 306, 318
429
Maclean A. J., 384, 389, 403, 404, 408, 410 Maclean, E. O., 399, 403, 409 McLean, G. W., 295,320 McLean, J. C., 161, 184 McMillian, W. W., 210, 211, 215, 218, 259, 263, 264 McMurtry, J. A., 189, 190, 259, 263 McNeal, F. H., 201, 203, 259, 264 McNulty, P. J., 334, 378 McPherson, D. C., 38, 92 McRae, D. H., 334, 369, 370,375,378 MacRae, I. C., 47, 64, 67, 68, 82, 92, 94 McWhorter, C. G., 359, 375 McWhorter, 0. T., 174, 184 McWilliam, J. R., 128, 144 Magalhaes, A. C., 332, 375 Magnuson, L. M., 302, 311 Mah, R. A., 60, 94 Mahapatra, 1. C., 37, 68, 78, 79, 92, 93 Mahr, I., 60, 92 Majer, A. J., 278, 324 Major, A. J., 224, 257 Majumdar, J. C., 363, 373 Malborn, S. O., 124, I44 Malcolm, J. L., 400, 404, 409 Malenfant, A. L., 293, 318 Mallory, T. E., 339, 340, 372 Maltais, J. B., 239, 251, 259 Mancy, K. H., 51, 93, 309, 311 Manglitz, G. R., 190, 192, 193, 251, 252, 256, 257, 259 Manheim, E. T., 45, 92 Mansager, E. R., 350, 364, 366, 368, 377 Manwiller, A., 215, 257 Marble, V. L., 190, 260 Margolin, M., 289, 320 Margoshes, M., 306, 318 Maroder, H. L., 352, 375 Marschner, H., 387, 410 Marshall, H. O., 245, 261 Martell, A. E., 154, 155, 185, 277, 323 Martens, D. C., 172, 184 Martin, D. F., 34, 37, 54, 68, 69, 79, 92, 223, 224, 226, 252, 258 Martin, E. G. K., 271, 318
430
AUTHOR INDEX
Martin, F. A,, 114, 122, 127, 128, 129, 130, 137, 140, 144 Martin, J. C., 389, 410 Martin, J. P., 400, 401, 410 Martin P. E., 171, 173, 179, 181 Martin, R. P., 386, 411 Martin, R. T., 351. 374 Martin, W. E., 161, 184 Martinez, E. M., 43, 45, 51, 52, 53, 80, 93 Mason, B., 273, 274, 318 Mason, G. W., 329, 375 Mason. K. E., 284. 318 Massey, H. F., 168, 172, 184 Mastromatteo, E., 293, 318 Mathes, R., 219, 259 Matheson, M. J., 128, 144 Mathur, R. K., 226, 262 Mathur, V. S., 104, 260 Matile, P., 6. 26 Matson, W. R., 269, 309, 311, 312 Matsunaka, S., 355, 356, 358, 369, 375, 3 76 Matsuo, H., 44, 92 Matteson, J. W., 211, 259 Matthewman, W. G., 234, 259 Matthews, B. C., 384, 388, 389. 395, 399, 410, 411 Matthews, R. E. F., 4, 24 Matushima, S., 109, 145 Maxwell, F. G., 188, 193, 211, 213, 220, 221, 222, 223, 224, 225, 229. 249, 251, 252, 253, 254, 256, 257, 258, 259, 260. 262 Mechsner, K., 68, 92 Mederski, H. J., 170, 183, 386, 410 Medsher, L.. 75. 91 Medvedeva, 0. P., 388, 410 Meikle, R. W., 351, 352, 376 Mellin, G., 281, 282, 315 Melnikov, N. N., 329, 337, 358, 376 Melsted, J. W., 150, 184 Melton, B. A., 190, 259 Melton, J., 158, 175, 183, 186 Mengel, K., 385, 410 Menhard. E. M., 284, 321 Menzie, C. N., 328, 329, 331, 337, 357, 358, 359, 371, 376 h?enzies, J. D., 268, 269, 313 Merkt, M. E., 222, 259
Merkle, M. G., 351, 356, 357, 373, 378 Mertz, W., 290, 318, 320 Metcalf, R. L., 367, 375 Meuschke, J. L., 280. 318 Meyer, J. R., 222, 223, 258, 259 Meyer, V. G., 15, 26 Michaelis, P., 19, 26 Miesch, A. T., 294, 322 Miettinen, J. K., 278, 318 Mikkelsen, D. S., 66, 95 Milborrow, B. V., 333, 376 Miles, J. R. W., 286, 318 Miller, B. S., 199, 259, 261 Miller, C. F., 291, 296, 320 Miller, F. C., 278, 324 Miller, L. A,, 238, 259 Miller, R. C., 214, 254 Miller, R. J., 12, 26 Miller, V. L., 279, 280, 317, 318 Miller, W. J., 284, 320 Millikan, C. R., 160, 162, 184 Milmore, B. K., 281, 323 Milne, D. B., 292, 322 Mink, G. I., 246, 259 Misiorow, R. L., 190, 257 Miskin, K. E., 114, 144 Misra, R. D., 32, 52, 75, 92 Mitchell, E. B., 257 Mitchell, E. R., 193, 253 Mitchell, H. L., 199, 259 Mitchell, J. D. D., 403, 408 Mitchell, R. L., 80, 92, 148, 165, 182, 184, 186, 274, 282, 286, 291, 295, 296, 318, 323 Mitchell, R. N., 293, 315 Mitchener, M., 282, 284, 290, 294, 321, 322 Mitsui, S., 32, 37, 66, 70, 78, 92 Miyagawa, H., 197, 256 Miyama, T., 278, 323 Mohr, H. E., 292, 315 Mollenhauer, H. H., 24, 26 Montgomery, M. L., 332, 345, 347, 373, 376 Moon, R. N. B., 272, 324 Moore, D., 35, 43, 49, 50, 51, 89, 387, 410 Moore, D. P., 158, 162, 165, 183, 184, 386, 410 Moorman, F. R., 32, 92
43 1
AUTHOR INDEX
Moreland, D. E., 329, 342, 347, 349, 353, 356, 359, 374, 375, 376 Morgan, J. J., 40, 41, 42, 44, 46, 54, 76, 77, 78, 79, 80, 92, 95 Morgan, J. M., 284, 319 Morgan, L. W., 193, 258 Morishima, H., 127, 144 MorrB, D. J., 24 Morris, J. C., 42, 92 Morrison, G. H., 309, 314 Mortensen, J. L., 152, 184 Mortenson, L. E., 70, 92 Mortimer, C. H., 32, 34, 35, 36, 43, 44, 47, 49, 50, 75, 77, 92, 93 Morton, J. D., 284, 320 Mortvedt, J. J., 177, 179, 182, 184 Moss, D. N., 109, 114, 122,142, 144 Moss, P., 388, 396, 403, 404, 407, 409, 410 Motomura, S., 48, 51, 62, 71, 92 Mound, L. A., 230, 259 Mounolou, J. C., 18, 26 Mount, D. I., 304, 319 Mountain, J. T., 292, 319 Moussa, M. A., 230, 260 Moxon, A. L., 288, 322 Mozes, G., 396, 410 Miieller, P. W., 343, 347, 376 Miihlethaler, K., 4, 25 Mueller, K. E., 66, 95 Mukerjimk, M. K., 233, 260 Mulawka, S. T., 305, 320 Munger, H. M., 99, 100, 101, 102, 103, 104, 105, 145 Muniz, C. E., 293, 319 Munkres, K. D., 9, 27 Muramoto, H., 100, 116, 143, 230, 253 Murata, K. J., 296, 320 Murata, Y., 107, 116, 117, 144 Murfet, I. C., 104, 144 Murphy, L. S., 161, 162, 173, 181, 182, 184 Murphy, W. S., 400,410 Murray, B. E., 16, 26 Murthy, G. K., 285, 292, 294, 319 Murty, B. N., 204, 260 Musgrave, R. B., 112, 122, 128, 143 Muttuthamby, S., 227, 260 Myers, M. A. G., 284, 322
N
Nafady, M. H. M., 392, 403, 409 Nagao, K., 301, 315 Nakano, K., 196, 260 Nakasawa, M., 197, 256 Nakashima, T., 66, 89 Nakayama, F. S., 86, 92 Nalewaja, J. D., 332, 372 Nashed, R. B., 358, 360, 361, 362, 376 Nason, A. P., 282, 284, 285, 290, 292, 294, 298, 321, 322 Nass, M. M. K., 9, 26 Nass, S., 21, 26 Nath, P., 238, 260 Natr, L., 122, 142 Navasero, S. A., 62, 95, 108, 145 Navrot, J., 159, 161, 184 Neal, W., 306, 313 Neales, T. F., 119, 144 Nearpass, C. D., 387, 410 Negi, N. S., 339, 340, 376, 377 Nelson, C . D., 119, 143 Nelson, C. E., 158, 161, 170, 186 Nelson, J. L., 150, 162, 171, 173, 177, 179, 181, 184 Nelson, N., 280, 319 Nelson, 0. E., 136, 144 Nelson, R. R., 2, 12, 26 Nelson, S. E., 341, 372 Nestorescu, B., 310, 325 Nethsinghe, D. A., 398, 410 Netter, P., 18, 22, 24 Neubert, P., 169, 184 Neujahr, H. Y., 301, 312 Neumeyer, J., 329, 337, 376 Neunzig, H. H., 245, 261 Newsom, L. D., 222, 257 Newton, J. F., 109, 144 Newton, R. C., 192, 260 Nhung, M. T., 32, 47, 5 5 , 56, 74, 75, 92, 93 Nichol, I., 272, 315, 324 Nicholas, D. J. D., 67, 92, 295, 319 Niederbrudde, E. A., 404, 411 Nicol, W. E., 53, 92 Nicolau, A., 278, 314 Nielsen, F. H., 293, 319 Nielsen, 3. M., 292, 320
432
AUTHOR INDEX
Nielsen, T. R., 44, 91 Nielson, M. W., 190, 192, 260 Nightingale, G. T., 282, 312 Nikkanen, J., 281, 282, 315 Niles, G. A., 226, 230, 251, 264 Nimmo, W . B., 333, 378 Nitelea. I.. 310, 325 Nitla, H., 109, 145 Noble, L. W., 225, 249, 258, 260, 262 Noble, W . B., 199, 263 Nobs, M. A., 115, 128, 130, 136, 142, 143 Noll, H., 7, 26 Norcio, N . V., 113, 144 Norris, K. H.,353, 375 Norvell, W. A., 149, 151, 153, 154, 155, 156, 157, 164, 166, 172, 175, 176, 179, 183, 184, 185 Norwood, B. L., 191, 252, 260, 264 Nosbers, R., 269, 319 Novero, E., 238, 260 Nye, P. H., 383, 410 Nyland, G., 6, 26
Olsen, S. R., 86, 93, 162, 163, 171, 185, 186 Olson, 0. E., 286, 288, 317, 319, 323 Olson, R. A,, 161, 162, 186 Onikura, Y., 60, 62, 90 Onisha, Y., 197, 265 Ortega, C. A., 208, 253 Ortman, E. E., 190, 201, 211, 212, 213, 252, 255, 260, 262 Ortman, E. E., 189, 256 Osada, A., 117, 118, 144 Oshima, S., 304, 305,319 Oskamp, J., 50, 89 Ostadalova, I., 288, 310, 319 Ott, W. L., 309, 315 Otto, J. C . G., 93 Overby, L. R., 287, 314 Overstreet, R., 275, 316, 319, 386, 387, 410, 411 Ozanne, P. G., 162, 185, 387, 397, 399, 402, 403, 409 Ozbun, J. L., 122, 137, 144
P 0
ODonohoe, T. F., 387, 41 I Oertel, A. C., 268, 272, 319 Oeser, A., 27 Ogata, G., 76, 92 Ogburn, B. R., 293, 319 Ogden, R. L., 190, 257 Ogunfawona, M. A., 388, 395, 403, 412 Ohi, T., 362, 375 Ohlrogge, A. J., 331, 377 Ohta, N., 300, 319 Ohta, Y.,19, 26 Ojima, M., 111, 127, 128, 140, 141, 144 Oka, H-I., 127, 144 Olbrick, B., 9, 26 Oldham, W. K., 72, 92 Olembo, J. R., 204, 260 Oliver, B. F., 224, 225, 249, 260 Oliver, L. R.,339, 376 Oliver, S., 185 Olsen, C., 387, 411 Olsen, R. A,, 44, 93 Olsen, R. J., 161, 162, 186
Page, A. L., 150, 181, 274, 282, 283, 312, 319, 320, 401, 410 Painter, H. A., 62, 68, 93 Painter, L. I., 295, 319 Painter, R. H., 187, 188, 189, 190, 192, 193, 199, 201, 205, 208, 210, 212, 213, 214, 215, 216, 226, 237, 238, 249, 251, 253, 255, 256, 258, 259, 260, 261, 262, 263, 264 Pakkala, I. S., 307, 315, 318, 319 Palmer, I . S., 288, 319 Palpant, E. H., 242, 263 Pamatat, M. M . , 35, 93 Pan, S., 301, 315 Pande, P., 355, 373 Pant, N. C., 218, 254 Parao, F. T., 108, 145 Parizek, J., 278, 284, 288, 310, 319 Parka, S. J., 337, 338, 339, 340, 374, 376 Parker, J. H., 199, 260 Parker, R. C., 16, 26 Parks, G. A., 37, 93 Parr, J. C., 244, 260 Parrish, L. P., 34, 35, 92
AUTHOR INDEX Parrott, W. L., 220, 221, 222, 223, 224, 226, 229, 249, 257, 258, 259, 260, 2 62
Patanakamjorn, S., 196, 260 Pate, D. A., 333, 376 Pathak, M. D., 195, 196, 197, 198, 205, 213, 249, 251, 254, 260, 263 Patrick, W. H., Jr., 34, 35, 37, 44, 46, 47, 48, 68, 69, 73, 7 4 , 78, 79, 81, 83, 89,91, 92, 93, 95
Patterson, C. C., 281, 282, 283, 301, 319, 320, 323
Patterson, F. L., 199, 204, 253, 260 Paul, E. A., 70, 93 Paul, R. M., 305, 320 Paulsen, G. M., 161, 162, 181, 182, 185 Pavlik, L., 288, 310, 319 Payne, E. O., 207, 261 Payot, P. H., 343, 347, 376 Peacock, H. A., 229, 256 Peaden, N. R., 190, 261 Peaden, R. N., 189, 190, 256, 263 Pearce, R. B., 116, 122, 142, 145 Pearcy, R.W., 115, 128, 130, 136, 142 Pearsall, W. H., 32, 35, 44, 47, 50, 51, 52, 69, 83, 93 Pearson, G. A., 167, 185, 295, 296, 324 Pearson, W. N., 287, 313 Peat, W. E., 125, 145 Peebles, T. F., 158, 159, 185 Peech, M., 44, 93, 385, 386, 410, 411 Peel, R. D., 236,262 Peeler, J. T., 284, 292, 294, 319 Pendleton, J. W., 109, 110, 134, 143, 145
Penegrin, W. T., 203, 261 Penner, D., 329, 340, 341,372,373, 374 Penny, L. H., 208, 210, 261, 262 Perdue, H. S., 287, 314 Perida, J. F., 357, 358, 376 Perkins, A. T., 400, 411 Perkins, R. W., 292, 320 Perlmutter, N. M., 301, 302, 317, 320 Perner, E. S., 26 Perron, J. P., 237, 261 Perry, H. M., Jr., 284, 294, 321, 324 Pesho, G. F., 217, 251 Pesho, G. R., 12, 26, 189, 208, 210, 217, 243, 254, 255, 256, 261, 262 Peters, D. B., 110, 145
433
Peters, D. C., 189, 212, 254, 256, 260, 261
Peterson, L. A., 172, 184 Peterson, L. K., 202, 249, 256 Peterson, M. J., 269, 271, 285, 311 Peterson, R. L., 335, 376, 377 Petrochilo, E., 18, 22, 24 Pfadt, R. E., 206, 261 Pfeffer, J. T., 63, 93 Phillips, J. R., 249, 258 Pickford, R., 206, 261 Pierce, R. S., 32, 93 Pierre, W. H., 386, 398, 411 Pimental Gomes, F., 409 Pineda, A., 197, 198, 257 Piper, C. S., 388, 399, 404, 411 Piscatov, M., 284, 311, 320 Pitha, J., 310, 319 Pitre, H. N., 216, 254 Plantonow, N. S., 279, 320 Plimmer, J. R., 328, 329, 337, 358, 376 Plocke, D. J., 169, 185 Ploeg, H. O., 359, 374 Pohland, F. G., 51, 60, 93 Pommier, C., 309, 320 Pond, D. D., 233, 261 Ponnamperuma, F. N., 31, 32, 35, 38, 40, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 58, 61, 62, 67, 68, 69, 72, 73, 74, 75, 76, 78, 79, 80, 83, 84, 85, 86, 87, 89, 92, 93, 96
Ponnaya, B. W. X., 199, 259 Pons, L. J., 31, 32, 93 Poos, F. W., 194, 261 Pope, A., 388, 411 Porter, C. A., 369, 376 Porter, C. C., 279, 320 Porter, D. M., 193, 263 Porter, J. R., 274, 320 Porter, K. B., 201, 254, 261 Porter, K. R., 4, 26 Portman, 0. W., 288, 318 Poskanzer, D. C., 310, 316 Postgate, J. R., 75, 93 Potsma, T., 31, 93 Powell, G. W., 284, 320 Powell, J. D., 214, 255 Prask, J. A., 169, 185 Pratt, P. F., 292, 295, 320, 399, 411
434
AUTHOR INDEX
Prego, I. A,, 352, 375 Preiss, I., 138, 145 Prendeville, G. N., 364, 375, 376 Prescott, J. 4 . , 268, 272, 319 Presley, B. J.. 43, 45, 89, 93 Price, C . A.. 168, 169, 185 Prince, A. L., 268.312, 400,404,409,411 Pringle, B. H., 305, 320 Prior, R. E., 284, 322 Probst. G . W . , 337, 338, 339, 340, 341, 374, 376 Prochazkova, V., 278, 284, 288, 319 Purnphrey, F . V., 158, 160, 185 Pura, C . D., 198, 251 0
Quick, J., 161, 172, 181, I84 Quintana, V.. 218, 261 R
Rabb, R. L., 245, 254, 261 Rabinowitch, E., 83, 93 Radcliffe, E. B., 192, 234, 236, 261 Radcliffe, J . E., 207, 261 Radford, P. J., 99, 145 Ragab, M . T . H., 347, 376 Raghu, K., 92 Ragland, J. L., 169, 182 Rahrnan, W., 339, 376 Rahn, E. M . , 359, 361, 362, 375 Rahola, T., 278, 318 Rainwater, C . F., 221, 259 Raj, A. Y., 14, 26 Rakow, A. B., 278, 314 Ralph, R. K., 4, 24 Rarnanis, Z., 18, 22, 26 Rarnel, C., 307, 312 Rameriz, E., 108, 145 Randall, D. D., 212, 254 Randall, W . K., 242, 265 Randhawa, N. S., 152, 185 Randolph, N. M., 218, 263 Rankarna, K., 33, 73, 93, 273, 280, 282, 284, 285, 291, 320 Rao, P. S. P., 197, 256 Rao, T . S., 274, 320 Rao, Y . S., 197, 261
Rasrnusson, D. C., 109, 114, 126, 142, 143, 144 Ratcliffe, D. A., 279, 320 Ratcliffe, R. H., 190, 191, 252, 253 Rathore, V . S., 165, 185 Ratner, E . I., 387, 401, 411 Raulston, J . R., 227, 251, 261 Raun, A. P., 338, 339, 374 Raun, E. S., 208, 243, 255 Raupach, M . , 52, 55, 93 Raven, P. H., 21, 26 Ravikovitch, S., 159, 161, 184, 289, 320 Rawlings, J. O., 191, 253 Rawson, H . M . , 121, 136, 143 Ray, B., 3 3 2 , 376 Razuchs, T., 158, 183 Re, R. R., 206, 251 Reddy, V . R., 197, 261 Redemann, C . T., 351, 352, 374, 376 Reed, D. K., 226, 261 Reed, J. B., 284, 322 Reed, J . F., 287, 320 Reeves, A. L., 293, 320 Refai, E. Y., 199, 261 Reinberg, E., 108, 145 Reisenauer, H . M., 75, 90, 170, 184 Reith, J. W. S., 282, 291, 295, 318 Reitz, L. P., 108, 145, 199, 255 Rernington, R. E., 307, 313 Rentos, P. G., 278, 320 Reusch, W . H., 342, 374 Reuss, J . O., 158, 185 Reyes, 0. C., 66, 67, 89 Rhea, U., 285, 292, 294, 319 Rhoades, M . M . , 2 , 10, 19, 26 Rhodes, A. M . , 210, 258 Rhodes, I., 100, 145 Rhyne, C., 223, 226, 258 Rice, H . M., 388, 403, 408 Rice, R. L., 236, 256 Rice, W . A,, 70, 93 Ricernan, D. S., 167, 185 Richards, A. B., 339, 372 Richards, F. A., 50, 75, 93 Richmond, C . A., 229, 262 Riden, J. R., 365, 366, 376 Ries, S. K., 353, 354, 355, 376, 377 Riga, A. J., 168, 182 Ringlund, K., 200, 261 Ringwood, A. E., 273, 320
435
AUTHOR INDEX
Rink, M. M., 218, 261 Risebrough, R. W., 4, 26 Rismondo, R., 64, 94 Rissanen, K., 278, 318 Rittenberg, S. C., 33, 34, 37, 50, 77, 79, 91 Rittersh, E. L., 191, 253 Riviere, J., 152, 185 Roberts, D. W. A., 202, 261 Roberts, J. L., 60, 94 Roberts, L., 249, 258 Robertson, J. D., 4, 26 Robertson, L., 158, 183 Robertson, R. N., 386, 409 Robinson, A. G., 204, 256, 261 Robinson, G. W., 30, 31, 94 Robinson, J. B. D., 400, 411 Robinson, J. F., 342, 375 Robinson, R. J., 199, 259 Robinson, W. O., 31, 94, 291, 296, 320 Rochow, E. G., 273, 274, 320 Rode, A. A., 31, 73, 94 Rodriguez, J. G., 229, 232, 233, 253, 254, 261 Roesch, W. C., 292, 320 Rogers, J. S., 2, 12, 26 Rogers, R. L., 340, 355, 356, 357, 358, 359, 361, 362, 376, 377 Roginski, E. E., 290, 320 Rolley, H. L. J., 37, 90 Romenskaya, N. N., 43, 94 Romney, E. M., 295, 320 Rosario, E. L., 113, 145 Rose, A. H., 60, 95 Rosen, C. G., 301, 325 Rosenfeld, I., 288, 290, 320 Rosenquist, T., 73, 79, 94 Rosenthal, M. W., 310, 322 Rosoff, B., 310, 323 Ross, R. G., 281, 320 Ross, W. M., 13, 14, 26, 27, 216, 255 Roth, E. R., 242, 261 Roth, M., 287, 292, 311 Roth, W., 342, 377 Rowell, D. L., 388, 395, 403, 411, 412 Roy, J . K., 197, 256, 261 Rubin, B., 361, 362, 377 Ruckman, J. E., 66, 95 Rudder, J. R., 239, 252 Rudgal, H. T., 269, 320
Rudgers, L. A., 161, 185 Riihling, A., 283, 320 Ruppel, R., 200, 203, 205, 255 Russell, E. W., 31, 68, 94 Russell, G. E., 245, 246, 256, 261 Russell, M . P., 198, 218, 254, 261 Russell, R. S., 386, 387, 409, 411 Russell, W. A., 12, 26, 209, 210, 257, 261 Ruttner, F., 31, 34, 36, 38, 51, 62, 75, 94 Ryan, P., 158, 159, I85 Rydalv, M., 307, 324 Ryle, G. J. A., 136, 145 Ryser, G. K., 15, 27 5
Sadek, S. E., 293, 323 Sager, R., 18, 22, 26 Saha, J. G., 281, 320 Sahama, T. G., 33, 73, 93, 273, 280, 282, 284, 285, 291, 320 Sahni, V. M., 227, 262 Sakamoto, S-I., 111, 144 Salaman, R. N., 19, 26 Salandanan, D., 92 Salmon, S. C., 108, 145 Salisbury, R. M., 282, 320 Salmon, R. C., 387, 392, 399, 411 Salotto, B. V., 269, 312 Salvia, J. D., 283, 320 Samitz, M. H., 309, 320, 321 Sampath, S., 197, 261 Samuelson, O., 273, 321 Sanborn, W. T., 47, 94 Sand, S. A., 26 Sandal, P. C., 14, 24 Sandes, J. E., 35, 90 Sandmeye, E. E., 190, 261 Sane, P. V., 227, 263 Sanford, L. L., 236, 262 Sanik, J., 400, 411 Sargent, J. A,, 352, 377 Sarkar, S. N., 69, 89 Sarkissian, I. V., 17, 26 Sasahara, T., 116, 145 Sasamoto, K., 196, 262 Satawa, D., 204, 260
436
AUTHOR INDEX
Sato, K., 42, 47, 48, 49, 60, 62, 68, 96 Sato, R., 362, 375 Sauer, H. I., 294, 322 Sauvard, S., 310, 325 Savant, W. K., 78, 94 Saver, T. N., 724, 258 Saxena, H . P., 197, 256 Scales, A. L., 228, 262 Schaedle, M.. 387, 410, 411 Schafarczyk, W.. 387, 410 Schafer, D. E., 335, 336, 377 Schafer, J . F., 199, 253 Schafer. L., 293, 313 Scharen, A. L., 353, 375 Scheffer, F., 389, 396, 41 I Scheidecker, D., 398, 411 Scheifele, G . L., 2, 12, 26 Scheiner, D. M., 309, 321 Schiffman, R. H., 308, 314, 321 Schilier, I., 225, 262 Schillinger. J. A., 190, 191, 200, 203. 252, 262 Schillinger, J. A., Jr., 200, 255 Schlehuber, A. M., 201, 254 Schlenk. F., 60, 95 Schmid, G. H., 138, 145 Schmid, W. E., 164, 165, 167, 185 Schmidt, G. P., 45, 94 Schmidt, W. R., 338, 340, 377 Scholander, P. F., 34, 38, 94 Scholander, S. I., 34, 38, 94 Schonhorst, kl. H., 190, 192, 260 Schranzer, G. N., 301, 321 Schreiber, M. M., 364, 376 Schreiner, E. J., 242, 255 Schrenk, W. G., 400, 411 Schroeder, E. D., 47, 94 Schroeder, H. A., 269, 271, 281, 282, 283, 284, 285, 286, 287, 288, 290, 291, 292, 293, 294, 297, 298, 310, 316, 321, 322, 324 Schroo, H., 159, 185 Schubert, J., 310, 322 Schuffelen, A. C., 44, 94 Schulte, H. F., 293, 315 Schultz, D. P., 340, 376, 377 Schultz, E. S., 19, 26 Schultz-Schaeffer, J., 15, 26
Schuster, M. F., 207, 208, 211, 228, 229, 262, 264 Schutz, W. M., 17, 24 Schwartz, C. D. 231, 256 Schwartz, K., 287, 292, 322 Schweizer, E. E., 339, 377 Schwerdtle, F., 363, 373 Sciaroni, R. H., 268, 322 Scott, A. D., 34, 90 Scott, C. E., 158, 181 Scott, G. E., 208, 209, 210, 261, 262 Scott Russell, R., 60, 94 Scribner, B. F., 296, 320 Scribner, W. G., 309, 315 Searight, W. V., 288, 322 Seatz, L. F., 148, 185 Sebald, W., 9, 26 Sedova, G . P., 292, 322 Seligman, E. J., 278, 320 Selye, H., 310, 322 Sengupta, K., 296, 322 Sethunathan, N., 64, 94 Seven, M. J., 310, 322 Shacklette, H. T., 272, 280, 282, 291, 294, 296, 322 Shade, R. E., 200, 264 Shafer, J. I., Jr., 1 IS, 142 Shah, K. R., 269, 271, 285, 288, 291, 322 Shahied, S. I., 340, 377 Shamberger, R. J., 288, 322 Shands, R. G., 199, 262 Shank, D. B., 212, 262 Shapiro, M., 278, 314 Shapiro, R. E., 78, 94, 280, 319 Sharkey, A. G., Jr., 269, 271, 283, 284, 285, 288, 291, 316 Sharma, R. C.,205, 263 Sharma, U. K., 217, 263 Sharples, G. C., 297, 315 Shaver, T. N., 224, 225, 226, 262 Shaw, E., 171, 185 Shaw, K., 47, 68, 69, 82, 89 Shaw, T. L., 306, 322 Sheets, L. W., 225, 228, 264 Sheets, T. J., 361, 362, 377 Shibko, S. I., 280, 319 Shibles, R. M., 112, 142 Shih, C. Y., 219, 253
AUTHOR INDEX Shimabukuro, R. H., 329, 337, 342, 343, 344, 345, 346, 347, 348, 349, 350, 361, 372, 373, 375, 377 Shiori, M., 76, 94 Shone, M. G. T., 387, 411 Shukla, S. S., 37, 78, 94 Shulte, B. M., 286, 319 Shults, W. D., 280, 281, 324 Shumway, L. K., 19, 26 Sievers, R. E., 309, 315 Sifton, H. B., 37, 94 Sifuentes, J. A., 212, 262 Sijpesteijn, A. K., 328, 371, 377 Sikka, H. C., 347, 377 Sikka, S. M., 227, 262 Sillen, L. G., 39, 40, 54, 72, 80, 94, 154, 155, 185, 277, 323 Silverberg, J., 177, 185 Simmonds, N . W., 115, 145 Simpendorfen, K. J., 31, 93 Sims, J. R., 150, 181 Sinclair, T . R., 109, 134, 145 Singer, P. C., 87, 94 Singh, A., 13, 26 Singh, A. G., 226, 262 Singh, D. N., 214, 215, 259, 262 Singh, I . D., 104, 105, 145 Singh, M., 26, 110,145 Singh, M . M., 388, 392, 411 Singh, R. N., 70, 71, 94, 214, 262 Singh, S. R., 201, 214, 217, 262 Sinha, R. N., 205, 262, 264 Sisakayan, N. M., 8, 24, 26 Size, J. G., 284, 322 Sjostrand, B., 279, 312 Skerfving, S., 279, 307, 312, 323 Skerman, V. B. D., 47, 68, 82, 94 Skinner, J. L., 193, 258 Skopintsev, B. A., 43, 94 Slatyer, R. O., 120, 145 Sleesman, J. P., 194, 240, 241, 264 Slife, F. W., 349, 378 Sloane, L. W., 224, 257 Slonimski, P. P., 18, 22, 24, 26 Smart, N . A., 281, 323 SmiIIie, G., 388, 410 Smirnov, E. V., 43, 94 Smith, B., 306, 313 Smith, D. H., 200, 255 Smith, E. D., 31, 95
437
Smith, F. F., 194, 261, 288, 289, 290, 311 Smith, G. I., 109, 145 Smith, H., 60, 94 Smith, H. H., 10, 11, 26 Smith, J. A., 384, 388, 389, 395, 399, 410, 411 Smith, J. C., 193, 194, 263 Smith, J. W., 361, 362, 377 Smith, K. A., 60, 94 Smith, K. M., 6, 24 Smith, L. W., 335, 341, 362, 373,' 376, 377 Smith, M. S., 334, 377 Smith, 0. F., 189, 190, 256, 263 Smith, P. F., 297, 315 Smith, R. F., 189, 263 Smith, R. G., 283, 323 Smith, R. H., 242, 263 Smith, R. L., 68, 69, 89, 151, 160, 182 Smith, W. O., 232, 261 Smith, W. T., 243, 263 Smolyak, L. P., 34, 96 Snelling, R. O., 212, 252 Snow, J. W., 223, 263 Snyder, J. R., 210, 263 Sogawa, K., 198, 263 Soles, R. L., 242, 263 Sompolinsky, D., 362, 373 Sonis, S., 303, 316 Soper, Q. F., 337, 372, 377 Sopper, W . E., 269, 323 Sorensen, E. L., 189, 190, 192, 193, 255, 256, 258, 263, 264 Spence, J . A., 281, 316 Spencer, D., 7, 8, 20, 27 Spencer, H., 310, 323 Spencer, H. C., 293, 323 Sperber, J. I., 75, 95 Sperling, R., 279, 313 Spilhaus, A., 302, 323 Spitznagle, L. A., 331, 377 Sprague, G. F., 6, 27, 187, 263 Squire, F. A., 226, 263 Squire, H. M., 386, 411 Srivatsava, H. K., 17, 26 Stadtman, T., 63, 95 Stafford, L. E., 366, 369, 375 Stahl, Q. R., 280, 323 Standifer, L. C., Jr., 339, 340, 377
438
AUTHOR INDEX
Stanescu, C., 310, 325 Stanford, E. H., 119, 129, 143, 189, 190, 259, 263 Stankovic, B., 295, 314 Stanley, J. M . , 255 Stanovick, R. P., 364, 373 Stanton, D. A., 388, 404, 411 Staples, E. L. J., 282, 320 Staples, R., 189, 257 Starkey, R. L., 44, 45, 50, 52, 75, 95 Starks, K. J , 210, 211, 215, 218, 259, 263 Stavrakis, G., 203, 263 Steele, T. E., 229, 256 Steinberg, R. A , , 295, 323 Steinkoenig. L. A., 291, 296, 320 Stephen, C. E., 304, 319 Stephens, J. C., 13, 27 Stephens, S. G., 221, 222, 263 Stephenson, G. R., 335, 353, 354, 355, 362, 373. 376, 377 Sterling, T. D., 281, 313, 316 Stevenson, F. G., 151, 152, 153, 183 Stevenson, G. A., 192, 255 Stewart, D. K. R., 281, 320 Stewart, E. H . , 395, 404, 411 Stewart, I., 155, 175, 185 Stewart, 3. A., 165, 185 Stewart, P. A., 243, 245, 253, 263 Stewart, P . L., 295, 311 Stewart, R. N., 1 1 , 27 Stickel, W. H., 280, 319 Stiles, W., 148, 158, 186 Still, C. C., 370, 377 Still, G. G., 350, 364, 365, 366, 368, 369, 370, 373, 377 St. John, J . B., 353, 375 Stobbe, E. H., 355, 356, 357, 358, 374 Stockell, F. R., 292, 319 Stoewsand, G. S., 279, 323 Stokes, R. M., 308, 314 Stokinger, H. E., 292, 319 Stoller, E. W., 331, 377 Stoltz, L. P., 232, 233, 253, 261 Stolzy, L. H., 31, 95 Stone, R. W.. 309, 325 Stoner, A, K., 220, 235, 236, 255, 263, 264 Storvick, C. A., 292, 323 Stoskopf, N. C., 104, 105, 108, 145
Strang, R. H., 340, 359, 377 Strehlow, C. D., 281, 323 Stringfellow, T., 235, 263 Stringfield, G. H., 2, 27 Strong, F. E., 227, 228, 258, 263 Stroup, D., 11, 27 Stuckenholtz, D. D., 161, 162, 186 Studer, R., 387, 409 Stumm, W., 40, 41, 42, 46, 54, 76, 77, 78, 79, 80, 87, 92, 94, 95, 275, 323 Sturgis, M. B., 35, 45, 93, 95, 287, 320 Subramaniam, S., 126, 144 Sukegawa, E., 301, 315 Sullivan, M. J . , 238, 252 Sullivan, R. J., 285, 291, 323 Sumner, M. E., 388, 389, 398, 399, 403, 410, 411 Sunderman, F. W., 293, 323 Sunderman, F. W., Jr., 293, 323 Suneson, C. A., 199, 263 Sutton, M., 282, 320 Suzuki, T., 196, 263, 278, 323 Svetalio, E. N . , 8, 26 Swailes, G. E., 233, 263 Swaine, D. J., 148, 186, 282, 295, 296, 323 Swanson, C. R., 329, 331, 332, 333, 335, 354, 360, 361, 362, 374, 377, 378 Swanson, H . R., 343, 345, 348, 349, 350, 359, 360, 361, 362, 369, 374, 375, 377, 378 Swarup, V., 217, 263 Sweet, R. D., 339, 372 Swensson, A., 279, 307, 312, 317 Swetly, P., 9, 27 Swift, H., 9, 27 Swithenbank, C., 334, 335, 378 Switzer, C. M., 353, 355, 373 Syers, J. K., 37, 78, 94, 95 Syme, J . R., 108, 109, 145 T
Tabatai, M. A., 384, 388, 399, 411 Tabor, E. C., 269, 271, 284, 323 Taft, H. M., 221, 259 Takahashi, H., 310, 323 Takahashi, R., 104, 145 Takai, S., 60, 62, 95
AUTHOR INDEX Takai, T., 71, 91 Takai, Y., 34, 45, 46, 61, 71, 74, 88, 95
Takano, Y.,108, 116, 145 Takeda, K., 45, 47, 96 Takeda, T., 121, 145 Taker, N., 196, 260 Takijima, Y.,60, 62, 95 Taksdal, G., 241, 263 Tal, M., 115, 138, 143, I45 Talbert, R. A., 339, 340, 378 Taliaferno, C. M., 207, 258 Talibudeen, O., 384, 388, 392, 395, 403, 411 Tamura, I., 196, 263 Tanada, T., 76, 94 Tanaka, A., 62, 95, 108, 145 Tanaka, F. S., 348, 349, 359, 360, 361, 362, 366, 369, 373, 375 Tanaka, J. S., 235, 255 Tanaka, M., 74, 95 Tanaka, T. S., 108, 109, 145 Tangrnan, E. P., 293, 315 Tank, G., 292, 323 Tanner, J. W., 108, 145 Tatsumoto, M., 301, 323 Tatum, L. A., 3, 27 Taylor, C. G., 278, 323 Taylor, D. L., 21, 27 Taylor, H. F., 336, 378 Taylor, S. A., 34, 95 Teetes, G. L., 218, 263 Tejning, S., 279, 307, 312, 323 Teotia, T. P. S., 217, 263 Tepe, J. B., 337, 338, 339, 340, 341, 374, 376 Terai, M., 300, 319 Ter Haar, G. L., 301, 323 Terman, G. L., 177, 186 Tewari, K. K., 7, 8, 9, 20, 27 Tezuka, Y., 60, 62, 95 Thacker, E., 288, 289, 290, 311 Thapar, N. T., 286, 323 Theurer, J. C., 15, 27 Thiel, P. G., 63, 91 Thomas, G. H., 340, 377 Thomas, H. V.,281, 323 Thomas, J. F., 75, 91 Thomas, J. G., 189, 263 Thomas, M. D., 122, 145
439
Thomas, R. L., 125, 132, 145 Thomas, W. D. E., 295, 319 Thomas, W. I., 167, 183 Thomas, W. W., 255 Thompson, A. C., 220, 256 Thompson, A. H., 287, 323 Thompson, J. E., 280, 319 Thompson, J. F., 398, 409 Thompson, L., Jr., 349, 378 Thompson, R. K., 15, 27 Thorne, D. W., 147, 148, 159, 161, 162, 167, 168, 173, 186, 400, 401, 411 Thorne, G. N., 105, 108, 109, 145 Thorne, S. W., 138, 142 Thornton, I., 272, 324 Thornton, S. F., 395, 411 Thorp, J., 31, 95 Thurlow, D. L., 161, 182 Thurston, R., 243, 244, 245, 251, 257, 260, 263 Tianco, E. M., 43, 44, 53, 54, 58, 61, 72, 74, 80, 83, 93 Tidke, P. M., 227, 263 Tiffin, L. O., 165, 168, 182, 186 Tillander, M., 278, 318 Tiller, K. G., 150, 186 Tilney-Bassett, R. A. E., 8, 10, 18, 25 Timar, E., 75, 76, 95 Tinker, P. B., 388, 389, 392, 394, 396, 398, 400, 411 Tinline, R. D., 281, 320 Tipton, C. L., 209, 257, 262, 343, 378 Tipton, I. H., 271, 281, 282, 283, 284, 285, 290, 291, 292, 293, 294, 297, 298, 321, 322, 324 Tipton, K . W., 222, 257 Tissieres, A., 4, 26 Todd, H. G., 356, 376 Todd, J. W., 194, 263 Toerian, D. F., 63, 95 Tolbert, N. E., 27 Tornchick, G. J., 278, 314 Tomisawa, J., 197, 265 Tonegawa, K., 369, 370, 372 Tong, S. S. C., 308, 309, 324 Toth, S. J., 268, 295, 319, 324, 389, 400, 401, 404, 410 Trager, R., 292, 325 Trask, H., 329, 337, 376 Trehan, K. B., 205, 263
440
AUTHOR INDEX
Treharne, K. J., 112, 116, 118, 119, 138, 142, 145, 146 Trierweiler, J. F., 151, 153, 171, 183, 186 Trites, A. R., Jr., 288, 317 Troughton, J. H., 122, 144 True, L. F., 297, 315 fruelson, D., 178, 179, 186 Truog, E., 388, 409 Tryphonas, L., 313 Tsao, F. H . C., 343, 378 Tsay, R. C . , 340, 372 Ts’o, P. 0. P., 4, 27 Tsubota, G., 76, 95 Tsunoda, S., 99, 102, 107, 108, 109, 112, 116, 117, 121, 122, 144, 145 Tucker, B. B., 401, 410 Tucker, T. C . , 150, 160, 186 Tunney, H., 388, 396, 403, 409 TUPPY,H., 9, 27 Turner, F. T., 34, 46, 47, 48, 68, 69, 73, 81, 93, 95 Turner, R. C., 53, 92 Turner, R. L., 280, 322 Tuve, T., 288, 324 Tyler, G., 283, 320 Tyner, E. H., 387, 397, 399, 411 Tyrell, C., 202, 261 U
Udo, E. J., 150, 160, 186 Ueckert, D. N., 206, 207, 256, 264 Ueshima, T., 369, 370, 372 Ukita, T., 301, 315 Ulfuarson, U., 279, 317 Ullmann, W. W., 282, 315 Ulmer, D. D., 286, 307, 324 Ulrich, B., 389, 396, 411 Underwood, E. J., 158, I81 V
Vaccaro, R., 37, 95 Valberg, L. S., 292, 324 Valencia, C. M., 54, 80, 93 Vallee, B. L., 168, 186, 286, 306, 307, 318, 324 Vamos, R.,75, 95
Van, T. K., 195, 196, 264 van Bennekom, J. L., 19, 27 Vancura, V., 152, 186 Van Daalen, J. J., 333, 334, 378 Van Dam, L., 34, 38, 94 Vandecaveye, S. C., 286, 324 Van Den Berg, L. A., 280, 319 Vanden, Born, W. H., 332, 373 Van den Burgh, R. S., 191, 260, 264 Van der Kevie, W., 31, 32, 93 van der Meer, Q. P., 19, 27 van der Molen, H., 387, 388, 411 van der Schuiling, R. D., 37, 51, 95 Van der Schans, C., 337, 338, 339, 340, 376 Vanderzant, E. S., 264 van Deursen, F. W., 333, 334, 378 Van Dobben, W. H., 105, 145 Van Emden, H. F., 234, 244, 264 van Oorschot, J. L. P., 361, 378 van Raalte, M. H., 38, 95 Vanselow, A. P., 274, 297, 311, 324 Van? Woudt, B. D., 31, 95 Varis, A. L., 233, 264 Vasey, E. H., 163, 181 Vaughn, W. W., 280, 318 Venkataraman, N., 227, 251 Ventris, J., 386, 409 Vergara, B. S., 104, 142 Verguano, O., 295, 313 Verhagen, A. M. W., 109, 145 Verloop, A., 333, 378 Vielemeyer, H. P., 169, 184 Viets, F. G., Jr., 147, 148, 158, 159, 160, 161, 162, 170, 171, 173, 174, 175, 177, 179, 181, 183, 184, 186, 386, 411 Vigliani, E. C., 284, 324 Viles, F. J., Jr., 293, 324 Villareal, R. W., 2, 12, 27 Villiers, T. A., 6, 27 Vinande, R., 175, 186 Vinograd, J., 4, 27 Vinton, W. H., 284, 321 Vinyard, E., 292, 322 Virtanen, A. I., 209, 264, 342, 378 Visek, W. J., 290, 324 Viste, K. L., 329, 334, 378 Vit6, J. P., 242, 264 Viteri, F., 287, 313
AUTHOR INDEX
Vlamis, J., 295, 296, 324, 387, 398, 401, 411 Volk, N. J., 395, 404, 411 Volk, R. J., 115, 121, 143 von der Pahlen, A., 124, 145 von Euler, U., 307, 312 Vossen, P. G. T., 269, 271, 312 Vrejoiu, G., 310, 325 W
Wacker, W. E. C., 168, 186, 286, 307, 324 Waddle, B. A., 221, 222, 249, 256, 258 Wadham, S. M., 132, 143 Waggoner, P. E., 115, 145 Wahlroos, O., 209, 264, 342, 378 Wain, R. L., 336, 378 Walker, D., 44, 95 Walker, D. W., 218, 261 Walker, H. M., 401, 411 Walker, J. K., Jr., 230, 264 Walker, J. M., 163, 181 Walker, R. B., 401, 411 Wall, M. E., 397, 411 Wallace, A., 159, 167, 183, 186, 386, 410 Wallace, D. H., 99, 100, 101, 102, 103, 104, 105, 111, 112, 114, 116, 122, 127, 137, 143, 144, 145,146 Wallace, H. A. H., 205, 262, 264 Wallace, L. E., 201, 203, 259, 264 Wallace, R. A., 280, 281, 324 Walsh, L. M., 173, 184 Walsh, T., 387, 411 Walsh, W. C., 348, 350, 377 Walter, E. V., 211, 264 Walter, J. P., 356, 357, 378 Walton, P. D., 126, 143 Wang, T. S. C., 60, 95 Wann, E. V., 210, 211, 253, 258, 264 Wann, F. B., 161, 186 Wannamaker, W. K., 221, 222, 264 Wanntorp, H., 279, 281, 312, 324 Ward, G. M., 389, 411 Waring, S. A., 66, 67, 95 Warmke, H. E., 15, 25 Warner, J. D., 158, 161, 181 Warnich, S. L., 303, 324
44 1
Warren, G. F., 340, 361, 364, 372, 375, 376 Warren, H. V., 282, 284, 324 Warren, W. V., 269, 271, 284, 323 Wasson, C. E., 215, 216, 264 Watanabe, F. S., 86, 93, 162, 186 Watkinson, J. H., 289, 324 Watson, D. J., 99, 104, 109, 120, 136, 142, 146 Watson, J. D., 4, 26 Watson, J. P., 403, 409 Waugh, T. C., 302, 311 Wax, L. M., 331, 377 Wear, I. I., 172, 186 Weast, R. C., 277, 324 Webb, J. S., 272, 324 Webb, R. E., 235, 236, 255, 264 Weber, J. A., 243, 263 Weber, J. H., 301, 321 Webley, D. M., 152, 186 Webster, J. A., 192, 264 Weibel, D. E., 201, 253 Weijden, C. H., 37, 51, 95 Weinberg, S. B., 278, 316 Weissler, A., 280, 319 Welbank, P. J., 104, 105, 108, 145, I46 Wells, N., 288, 324 Wells, R., 7, 27 Welte, E., 404, 411 Wene, G. P., 211, 225, 228,255, 264 Werkman, C. H., 60, 95 Werner, W., 404, 411 Wessling, W. H., 221, 222, 264 West, E. S., 161, 186 West, I., 278, 324 Wester, P. O., 294, 324 Westermark, T., 279, 305, 306, 312, 316 Westoo, G., 279, 306, 317, 324 Wetter, L. R., 70, 93 Whaley, W. G., 26, 125, 146 Wheeler, H. W., 281, 313 Whetstone, R., 296, 320 Whitcomb, W. H., 214, 265 White, D. B., 306, 315 White, M. N., 307, 319 White, R. G., 233, 261 Whitehead, D. C., 387, 412 Whitehead, E. I., 286, 288, 319, 322 Whitfield, M., 44, 50, 95 Whitfield, P. R., 7, 8, 20, 27
442
AUTHOR INDEX
Whitney, I. B., 290, 324 Whitney, R. S., 53, 95 Whittig, L. D., 76, 91 Whittington, W. J., 125, 136, 144, 145 Whitworth, J . B., 339, 372 Widstrom, N. W., 210, 211, 264 Wiebe, G. A., 199, 253 Wieczorck, G. A,, 189, 313 Wien, H. C., 11 I , 133, 146 Wight, K. M . , 44, 45, 50, 5 2 , 95 Wijma, J., 333, 334, 378 Wilcox, M., 332, 376 Wild, A.. 388. 395, 403, 412 Wildman, S . G., 4, 7, 8, 20, 24, 27 Wildner, G. F.. 118, 119, 129, 138, 142 Wilkinson, H. F., 163, 186 Wilkinson, J. F., 60, 95 Williams, A. S., 20, 27 Williams, D. E., 44, 91, 397, 403, 412 Williams, E. A., 351, 352, 376 Williams, G. R., 20, 27 Williams, H. H., 288, 324 Williams, H. L., 278, 324 Williams, J. D. H., 37, 78, 94, 95 Williams, J . P., 7, 25 Williams, K . T., 286, 325 Williams, P. P., 339, 378 Williams, R. F., 121, 143 Williams, R. N . , 207, 264 Williams, R. T., 357, 378 Williams, W., 124, 146 Williams, W. A., 66, 95 Willis, W. H., 69. 91 Wilson, D., 102, 112, 116, 117, 128, 146 Wilson, F. D.. 225, 264 Wilson, H. F., 369, 370, 375, 378 Wilson, J . A., 14, 27 Wilson, J. H., 109, 145, 386, 410 Wilson, L. F., 242, 265 Wilson, M. C., 192, 200, 264 Winchester, J. W., 288, 315 Winieski, J. A., 242, 255 Winter, D., 310, 325 Winter, J. D., 264 Winter, S. R., 109, 145 Wise, R. A., 284, 322 Wiseman, B. R., 215, 216, 238, 264 Wiser, W. J., 264
Witts, K. J., 104, 109, 146 Wittwer, S. H., 165, 185 Woldendorp, J. W., 65, 67, 95, 96 Wolf, H . W., 291, 292, 296, 309, 318 Wolfe, R. S., 301, 318 Wolfenbarger, D., 194, 211, 240, 241, 262, 264 Wollman, E. L., 20, 25 Wolstenholme, D. R., 9, 27 Wolton, K. M., 387, 412 Wood, D. L., 242, 264 Wood. E. A., Jr., 201, 204, 207, 216, 253, 254, 256, 262, 265 Wood, J. M., 301, 325 Wood, R. F., 287, 313 Wood, W. A,, 60, 96 Woodmansee, C. W., 359, 361, 362. &7J Woodriff, R.,309, 325 Woodruff, C. M., 396, 397, 398, 400, 403, 412 Woodward, D. O., 9, 27 Woolcott, G. N., 265 Woolson, E. A., 286, 287, 307, 316, 325 Work, T . S., 9, 24, 27 Wrazidlo, W., 169, 184 Wressell, H. B., 192, 265 Wright, A. N.,'329, 333, 373 Wright, J. W., 242, 265 Wright, L. D., 292, 325 Wright, T. L., 293, 325 Wright, W. L., 337, 372, 377 WU, B-F., 122, 128, 130, 146 Wurhmann, K., 68, 92 Wyatt, R., 70, 93 Wynne, J. C., 16, 26 Y
Yaalon, D. H., 53, 96 Yadava, H. N . , 226, 262 Yamaguchi, J., 108, 145 Yamaguchi, S., 359, 372 Yarnamoto, R. T., 237, 244, 245, 265 Yarnane, I., 34, 42, 47, 48, 49, 53, 60, 62, 68, 96 Yarnazaki, R. K., 27 Yang, C. C., 46, 89 Yates, P. O., 278, 313 Yatsu, L. Y., 25, 27
443
AUTHOR INDEX
Yatsuda, S., 104, 145 Yeager, D., 293, 313 Yearian, W., W. C., 249, 258 Yih, R. Y., 334, 335, 369, 370, 375, 378 York, J. O., 214, 265 Yoshida, F., 385, 412 Yoshida, S., 197, 265 Yoshida, T., 64, 71, 89, 94, 96 Yoshizawa, T., 46, 96 Young, E. G., 306,325 Young, J . O., 284, 318 Young, J. R., 211, 263 Young, L. T., 282, 318 Young, M. F., 15, 24 Young, R. D., 177, 185 Young, R. G., 306,318 Youngson, C. R., 352,376
Yuan, W. L., 47, 74, 93, 96 Yunkervich, I. D., 34, 96 Yurachek, J. P., 309, 325 Yushima, T., 197, 265 L
Zabik, M. J., 353, 376 Zaher, M. A., 230, 260 Zaki, M. A., 336, 378 Zelitch, I., 122, 146 Zevin, A. C., 19, 27 Ziboh, C. O., 387, 400, 411 Zick, W. H., 365, 378 Zilio-Grandi, F., 64, 94 Zimmerman, M., 400, 411 Zimmerman, P. W., 280, 325 Zobell, C. E., 35, 42, 45, 96
SUBJECT INDEX
A
Acalyinma trivitiata, 238 Acyrihosiphon pisrim, 190, 239 Agropyron reperis, 334 Agropyron smithii, 206 Alfalfa, 116, 188-193, 334 Alfalfa seed chalcid, 192 Alfalfa weevil, 191 Alliurn, 158, 237 Aluminum, 55. 76 Ammonia, 65-67 Amophorophora rirbi, 23 1 Ananas cornosur, 158 Anasa iristis, 237 Aniherigona varia var. soccata, 216 Anrhonomiis grandis, 219 Antimony, 270, 275, 297, 308 Antonina grantinis, 207 Aphid, 204 Aphis fabae, 245, 246 Aphis gossypii, 329 Apion godman, 241 Apple, 158 Arachis hypogeae, 16 Arsenic, 270, 274, 275, 285-287, 297, 298, 302, 307, 308 Arirndinnria, 200 Atrazine, 342-351 A vena byzantina, 203 Avena futira, 365 Avena sativa, 203 B
Banded cucumber beetle, 237 .Batban, 365-366 Barium, 270, 273, 274, 275, 293, 295, 297, 308 Bark beetle, 242 Barley, 109, 112, 200, 204-205, 332, 335, 343, 365 Barley stripe mosaic virus, 6 Barnyard grass, 369, 370 444
Bean, 98, 99-102, 103-105, 111, 126, 127, 158, 333, 367 Bean leaf beetle, 195 Beet, 353 Bemisia tabaci, 227, 230 Benefin, 337, 338-339 Benzamides, 334-335 Benzoic acid, 328-337 Bermudagrass, 207, 208 Beryllium, 270, 274, 275, 293, 295, 297, 303, 308 Beta isirlgaris, 99, 160 Bismuth, 270, 275, 297, 308 Blackgrain stem sawfly, 201 Blissiis Ieircopterrcs, 218 Boll weevil, 219, 220-223, 230 Bollworm, 223-225, 230, 249 Bouielona gracilis, 206 Brassica campcstris, 357 Brassica oleracea, 357 Brevicoryne brassicae, 234 Bromoxynil, 335-336 Brown planthopper, I98 Brrichophagus roddi, 192 Buffel sandbar. 208 C
Cabbage, 357 Cabbage aphid, 234 Cabbage Iopper, 234 Cabbage maggot, 233 Cabbage worm, 234 Cacodylic acid, 286 Cadmium, 270, 274, 275, 283-285, 297, 298, 302, 303, 304, 307, 308 Calartdea oryzae, 2 18 Calcium, 295, 386, 398, 401 Camnrilu pellucida, 206 Carbamate herbicides, 363-368 Carbonate redox systems, 85-87 Carbon dioxide exchange, 111-1 19, 127-129 Carbon dioxide, soil, 61
445
SUBJECT INDEX
Carex eleocharis, 206 Carya illinoensis, 158 Cation activity ratios, 379-412 Cation exchange capacity, 385 CCC, 244 Cenchrus citiare, 208 Cephus cinctus, 201 Cephus pygmaeus, 201 Cephus tabidus, 201 Cereal leaf beetle, 200,203,205 Cerium, 270,297,308 Cerotoma trifurcata, 195 Cesium, 270,274,275,294,297,308 Chaetocnema confinis, 237 Chelating agents, 154-157, 164,165-166 Chilo suppressalis, 195-196 Chilo zonellus Swin, 217 Chinch bug, 218 Chloramben, 328,331-332 Chloris gayana knuth, 208 Chlorobromuron, 359, 360
Cotton fleahopper, 229 Cotton leafworm, 230 Crabgrass, 367 Crop yield, cation activity ratios, 388-392 physiological genetics, 97-146 Cucurbita, 237-239 Cylindrocopturus jurnissi, 242 Cynodon dactylon, 207 Cytoplasm, organelles, 3-6 pathological inclusions, 6-7 D
Dactylis glomerata, 102 Dendroctonus monticolae, 242 Detergents, soil, 64 Diabrotica balteata, 237 2-Chloro-4-ethyamino-6-isopropylamino- Diabrotica longicornis, 211 Diabrotica undecimpunciaia howardi, s-triazine, 64 Chlorophyll, deficiencies, 10-1 1 193, 211, 237 Diabrotica virgifera, 211 Chloroplast, genes, 7-8 Diaphana nitidalis, 238 Chloropropham, 364-365 Diatraea saccharalis, 21 8 Chlorops oryzae, 196 Diatrea grandiosella, 214 Chlorthiamid, 333 Chromium, 270,273,275,290-292,294, Dicarnba, 329, 332-333 Dichlobenil, 329, 333-334 297, 301,304,307-308 Dichlormate, 367-368 Coin lacryma-jobi, 343,345 0,O-Diethyl-0-p nitrophenyl phosphoroColorado potato beetle, 236 thioate, 64 Conoderus falli, 237 Digiiaria sanqiiinalis, 367 Copper, 158,303,304 Dimethylarsinic acid, 286 Corn, 109, 112, 277 Dinitroanilines, 337-342 herbicides, 342,343,344,348,349, Diphenylethers, 355-358 350,367,368 Dolichos lablab, 241 insect resistance, 208-216 male-sterile, 2-3, 12-14, 15 E zinc, 158,160,168,174,175 Corn earworm, 193, 210-211 Corn leaf aphid, 204-205, 213-214,217 Echinochloa crus-galli, 369 Corn rootworm, 211-213 Echinocloa, 200 Costelgtra zealandica, 207 Elasmopalpus legnosellus, 193 Cotton, 10 Elongate flia beetle, 237 herbicides, 337,341,343,347,349, Empoasca devastans, 226, 227 350, 360,361,362 Empoasca fabae, 192,194,236,240 insect resistance, 219-230 Empoasca fascialis, 226 Cotton aphid, 229 Empoasca lybica, 227,230
446
SUBJECT INDEX
Empoasra terra-rc.gitiae, 226 Epicarrta vittatu, 195 Epilachna varirestis, 194,238,240 Epitrix hirtipennis, 235 European corn borer, 12,208-210,217 European pine sawfly, 242 F
Fall armyworm, 193,207,215-216, 218 Fertilizer, 174-180 Filly panicum, 208 Fish, 305-308 Fluorodifen, 35.5-358 Franhlinelli spp., 193 Frit fly, 203, 705 G
Gallium, 270,274,275,297,308 Gustrimarcirr n~arrnoratirs, 219 Genetics, extrachromosomal inheritance, 1-27 crop yield, 97-146 resistance to insects, 246-250 Germanium, 270,275,294,297,308 Gley, 30-31 Glycine niax, 109 Gossypirrni anomalnn~,230 Cossypirrni arborerim, 220,222,226,221 Gossvpiitni barbadensr, 220,222,225,
Heliothis zea Boddie, 210 Heliorliis virescens, 223,224,225,249 Helrniritliosporiirni maydis, 2,3, 12, 15 Herbicides, behavior in plants, 327-378 Hessian fly, 198-200,204
-y-1,2,3,4,5,6-Hexachlorocyclohexane, 64 Hordeirrii vrrlgare, 112,332,335 Hydrellia griseolu, 203 Hylemya antiqua, 237 H>ilernya brassicae, 233 Hylernya cilcrura, 233 Hylemya floralis, 233 Hypera postica, 191 I
Insects, plant resistance, 187-265 Ion absorption, 164-167, 302-303 loxynil, 3 35-33 6 Iponiea batatas, 109 Iron, 274,275 submerged soil, 30,31,32,33,36,
41,49,53,5 5 , 61,71-73, 84-85 J
Japanese beetle, 194 Jassid, 226-227 Johnsongrass, 348
227, 229 Gowypirrrn lierbacermi, 220 Gossypiitni /iirsirtrrtn, 10, 226,227,229,
337 Gosypiirni spp., 220 Gos'Fypiirmtlirrrberi, 222,226,228 Gossjpiirtn tomentostrm, 226 Grass grub, 207 Grasshopper, 206-207, 219 Greenbug, 193,201,203,205,216-217 Green manure, 66 Green leafhopper, 198 Green peach aphid, 234,236,243-244 Green rice leafhopper, 196 Growth analysis, 99-105, 11 1, 125-126 H
Heliothrs zeu, 193, 195,223,224,22.5,
249
L Lactrrca spp., 237 Lead, 270,274,275,281-283, 290,297,
298,301,303,304,307,308 Leaf area, 126 Leaf miner, 236, 238 Leptinotarsa decemlineata, 236 Lesser cornstalk borer, 193 Lettuce root aphid, 237 Linuron, 359-362 Liriotnyza brassicae, 236, 238 Lissorlioptrirs oryzophilus, 198 Lithium, 270,273,274,275,294,297,
308 Loliirm miiltiflorrrm, 102 Lolirrrn pererine, 102 Lycoperisicon escrrleritrrm, 235,236 Lycopersicon Itirscrtum, 235, 236
447
SUBJECT INDEX
Lygus hesperus, 227, 228 Lygus lineolaris, 193, 227, 228, 241, 249 M
Macrosiphum euphorbiae, 235 Magnesium, 387 cation activity ratios, 393-408 Malathion, 341 Male sterility, 11-15 Malus spp., 158 Manduca sexta, 236, 244-245 Manganese, 274 submerged soil, 30, 32, 33, 36, 47, 49, 61, 73-74, 83-84 Marsh soil, 31-32 Mayetiola destructor, 198, 204 Meadow spittlebug, 192 Medicago, 189, 191 Medicago falcata, 191 Medicago sativa, 116, 334 Medicago sativa var. gaetula, 191 Medicago transchanica var. agropyretorum, 192 Melanoplus differentialis, 206 Melanoplus mexicanus, 206 Melanoplus sanguinipes, 206 Melilotus, 189, 191 Meliodogyne graminicola, 197 Melittia cucurbitae, 23 8 Mercury, 270, 274, 275, 278-281, 297, 300-301, 303, 304, 306-307, 308 Methane bacteria, 62-63 Methansarcina methanica, 62 Mexican bean beetle, 194, 238, 240 Mite, 193 Mitochondria, 9-10 genes, 9-10, 12-13, 17-18 Molybdenum, 158, 295, 386 Monuron, 362 Myzus persicae, 234, 236, 243-244, 245, 246 N
Nematode, 197 Neodiprion sertifer, 242 Nephotettix bipunctatus cinticeps: 196 Nephotettix impicticeps, 198
Nickel, 270, 274, 275, 293, 295, 297, 308 Nicotiana, 243 Nilaparvata Iugans, 198 Niobium, 270, 275, 294, 297, 298, 308 Nitrate, 37, 47, 49, 64, 67-70 Nitrofen, 355-358 Nitrogen, 162-163, 196, 208, 209 ammonia accumulation, 65-67 denitrification, 67-70 fixation, 70-71 redox systems, 81-83 soil, 65-71 Nutrient uptake, cation activity ratios, 380-385 0
Oats, insect resistance, 200, 203 Onion, 158 Onion maggot, 237 Orchardgrass, 102 Organic matter, soil, 151-153, 160, 161 Oryza ridleyi, 195 Oryza sativa, 105, 195, 333 Oscinella frit, 203, 205 Ostrinia nubilalis, 12, 208, 217 Oulema melanopus, 200, 203, 205 Oxidation-reduction potential, 38-39 Oxygen, 34-35 P
Paddy soil, 32-33, 37, 67 Parathion, 64 Parnara guttata, 196 Pea, 191, 335, 343, 347, 369 Pea aphid, 190, 239-240 Peach, 158 Peanuts, 16, 193-194 Pearl millet, 207 Pecan, 158 Pectinophora gossypiella, 225 Pemphigus betae, 245 Pemphigus bursarius, 237 Pesticides, soil, 64 Phaseolus, 240
448
SUBJECT INDEX
Pliaseolus vulgaris, 98, 158, 333 Pheasant, 295 pH, effect, 153-154 values, change interpretation, 53-55 soils and sediments, 51-53 Phenrnedipharn, 363-364 Phenylurea herbicides, 359-363 Pki/aneiis spuniariirs. 192 Phosphate, 32, 35. 36 Phosphorus, 76-79, 161-162, 209, 274, 295 Phyllosticta zene, 2, 3, 12 Pickleworm, 238, 239 Picloram, 351-353 Pieris rapae, 234 Pineapple, 158 Pine reproduction weevil, 242 Pink bollworm, 225-226 Pinirs, 242 Pisrrm sariviim, 239 Plant breeding, crop yield, 97-146 extrachromosomal inheritance, 1-27 Plant growth. cation activity ratios, 380-385 Plant, herbicide behavior, 327-378 Planthopper, 197 Plant nutrition, zinc, 147-186 Phtella macrr/iperinis, 234 Podagrica spp., 229 Popillia japonica, 194 Potassium, cation activity ratios, 384, 386, 387, 388, 393-408 Potato, 15, 99, 158 Potato aphid, 235 Potato leafhopper, 192, 194, 236, 240 Prodenia litura, 230 Prometryne, 347 Pronamide, 334-335 Propachlor, 368-3 69 Propanil, 369-37 1 Propham, 364-365 Prirnits persica, 158 Pseirdatomoscelis seriatrrs, 229 P yrazon, 35 3-3 5 5 Q
Quackgrass, 334
R
Rape, 357 Redox equilibria (pE), 39-45 Rhodesgrass, 208 Rhodesgrass scale, 207-208 Rliopalosiphrrni maidis, 204, 21 3, 217 Rhopalosiphi~mpadi, 204 Ribulose- 1 ,5-diphosphate carboxylase, 118 Rice, 287 crop yield, 105-108, 113, 117, 127, 128, 134, 140 herbicides, 333, 355, 364, 369, 370 insect resistance, 195-198 soil, 32, 38, 51, 55-56, 57, 61, 63, 66, 70, 71 Rice-plant skipper, 196 Rice stem borer, 195-197 Rice stem maggot, 196, 197 Rice water weevil, 198 Rice weevil, 198, 214-215, 218 Root exudate, 152 Rubidium, 270, 274, 275, 297, 308 Rubis aphid, 231 Ritbis spp., 231-232 Rye, 205-206, 343 Ryegrass, 102, 112, 128 5
Saccharicni oficinnrrrm, 111 Scandium, 270, 274, 275, 297, 308 Schizaphis graminrim, 193, 205 Schizaphis graminum Rondani, 216 Scripophaga nivella, 219 Selenium, 270, 274, 276, 286, 287-290, 297, 298, 307, 308 Seraria, 200 Setaria scheelei, 208 Sewage, 268, 269, 280, 292, 306 Silicon, 79-80, 196-197 Silver, 270, 274, 276, 297, 303, 308 Simazine, 342, 347, 349 Sitona cylindricollis Fahraeus, 191 Sitophilirs oryzae, 198, 214, 218 Sitophilirs zeamais, 2 18 Sogatodes oryzicala, 197 Soil, submerged, chemistry of, 29-96
SUBJECT INDEX
trace metals, 268-277 zinc, 147-157 Solanium penellii, 235 Solanum, 235, 236 Solanum tuberosum, 15,99,158 Sorghum, 345,348,350 insect resistance, 213,216-218 male-sterile, 13-14 Sorghum halepense, 348 Sorghum-shoot-fly, 216 Sorghum stem borer, 217 Sorghum subglabrascens, 218 Sorghum Sudanese, 216,348 Sorghum virgatum, 216 Southern corn rootworm, 193 Southern leaf blight, 12,15, 23 Southw~esternbristlegrass, 208 Southwestern corn borer, 214 Soybean, 109-110,112,127,194-195 herbicides, 331, 337,341,343,355,
360,364,366,368
Spider mite, 228-229,235 Spodoptera frugiperda, 193,207,215,
218
Spotted alfalfa aphid, 188-190, 191,193 Spotted cucumber beetle, 237 Squash bug, 237 Squash vine borer, 238 Stem borer, 197 Stipa comata, 206 Stomates, photosynthesis, 114-115, 129 Strawberry mite, 228,232 Striped blister beetle, 195 Striped cucumber beetle, 238 Strontium, 270,273,274,276,294,297,
308
Subterranean clover, 168 Sudangrass, 14,216,348 Sulfur, 74-76, 85 Sugar beet, 99,103, 109,121, 160,
245-246, 353
Sugar beet root aphid, 245 Sugarcane, 11 1, 218-219,348,349 Sugarcane borer, 218-219 Sweetclover aphid, 192 Sweetclover weevil, 191-192 Sweet potato, 109 Sweet potato flea beetle, 235 Swep, 364 Systena elongata, 237
449 T
Tandex, 367 Tarnished plant bug, 193,227,241,249 Tellurium, 270,274,276,294,297,298 Terbutol, 367 Tetranychus sinhai, 205 Tetranychus tumidus, 193 Tetranychus turkestani, 228,232 Tetranychus urticae, 228,230,235 Thallium, 270, 276,297,308 Therioaphis maculata, 188 Therioaphis riehmi, 192 Therioaphis trifolii, 189 Thrip, 193 Tin, 271,276,292,297,298,308 Titanium, 271,274,276,294,297,298,
308
Tobacco, 10,243-245 Tobacco budworm, 223 Tobacco flea beetle, 235-236 Tobacco hookworm, 236,244-245 Tomato, 118,161, 364 Tonoptera grarninum, 201,203 Trace metals, 267-325 Translocation, 119-121 Trialeurodes abutilonea, 229 s-Triazines, 342-351 2,3,6-Trichlorobenzoic acid, 329-33 1 Trichoplusia ni, 234 Trifluralin, 64,337-338,339,340,341
(~,a,a-Triflur0-2,6-dinitro-N,N-dipropylp-toluidine, 64 Trifolium, 189 Trifolium subterraneum, 168 Trigomella, 189, 191 Triticum aestivum, 14,332,336 Triticum vulgare, 99,333 Tryporyza incertalas, 197 Tryporyza inotata, 195 Turnip maggot, 233 U
Urea herbicides, 358-363 V
Vanadium, 271,274,276,292-293, 295,
297, 298, 308
Vicia faba, 347
450
SUBJECT INDEX W
Water, trace metals, 299-302 WaterIogging, 30-3 I , 38 Wheat, 108-109, 120 herbicides, 332, 333, 336, 343, 352, 365 insect resistance, 198-203 male-sterile, 14 Wheat stem sawfly, 201-203 Winter wheat, 99 Wireworm, 237
Y
Yellow clover aphid, 189 Yellow leaf blight, 2, 12 2
Zeu maps, 2, 109, 121, 122, 158
Zinc, soils and plant nutrition, 147-186 trace metals, 274, 284, 304, 305, 306 Zirconium, 271, 274, 276, 294, 297, 298, 308