Advances in Agronomy, Volume 20

ADVANCES IN

AGRONOMY VOLUME 20

CONTRIBUTORS TO THIS VOLUME W. H. ALLAWAY GLENNW. BURTON MICHELP. CESCAS ROY G. CREECH LAWRENCE J. GRAY V . A. JOHNSON

W. D. KEMPER I. C. MAHAPATRA S. R. OLSEN WM. H. PATRICK, JR. JERREL B. POWELL J . W. SCHMIDT

ROY W. SIMONSON EDWARDH. TYNER

ADVANCES

IN

AGRONOMY Prepared under the Auspices of the AMERICAN SOCIETY

OF

AGRONOMY

VOLUME 20

Edited by A. G. NORMAN The University of Michigan, Ann Arbor, Michigan

ADVISORY BOARD J. P. MARTIN R. R. DAVIS F. A. HASKINS J . W. PENDLETON W. D. KEMPER W. A. RANEY

ACADEMIC PRESS 0 N e w York and London

COPYRIGHT^^ 1968, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. N O PART O F THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT W RI TTEN PERMISSION FROM T H E PUBLISHERS.

ACADEMIC PRESS, INC. 1 I I Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W . l

LIBRARY OF CONGRESS CATALOG CARDNUMBER50-5598

P RI N TED IN T H E U N I T E D STATES O F AMERICA

CONTRIBUTORS TO VOLUME 20 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

W. H . ALLAWAY ( 2 3 5 ) , Director, Plant, Soil & Nutrition Laboratory, Soil and Water Conservation Research Division, Agricultural Research Service, United States Department of Agriculture, Ithaca, New York GLENNW. BURTON(49),Research Geneticist, Crops Research Division, Agricultural Research Service, United States Department of Agriculture, Georgia Coastal Plain Experiment Station, Tifton, Georgia MICHELP. CESCAS( 1 5 3 ) , Assistant Professor, Department of Soil Science, Lava1 University, Quebec, Canada ROYG . CREECH( 2 7 5 ) , Associate Professor of Plant Breeding, Department of Horticulture, College of Agriculture, The Pennsylvania State University, University Park, Pennsylvania LAWRENCE J. GRAY( 1 5 3 ) , Physicist, Ocean Technology Department, United States, Naval Undersea Warfare Center, Pasadena, California V . A. JOHNSON ( 199), Research Agronomist, Crops Research Division, Agricultural Research Service, United States Department of Agriculture, Agricultural Experiment Station, Lincoln, Nebraska W. D. KEMPER(91), Research Soil Scientist, Soil and Water Conservation Research Division, Agricultural Research Service, United States Department of Agriculture, Professor, Department of Agronomy, Colorado State University, Fort Collins, Colorado I. c. MAHAPATRA ( 3 2 3 ) , Agronomist, Central Rice Research Institute, Cuttack, (Orissa), India S . R. OLSEN(91), Research Soil Scientist, Soil and Water Conservation Research Division, Agricultural Research Service, United States Department of Agriculture, Colorado State University, Fort Collins, Colorado WM. H . PATRICK, JR. ( 3 2 3 ) , Professor of Soil$, Agronomy Department, Louisiana State University, Baton Rouge, Louisiana JERREL B . POWELL(49), Research Geneticist, Crops Research Division, Agricultural Research Service, United States Department of Agriculture, Georgia Coastal Plain Experiment Station, Tifton, Georgia J. W . SCHMIDT ( 199), Professor ofAgronomy, Department ofAgronomy, University of Nebraska, Lincoln, Nebraska V

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CONTRIBUTORS TO VOLUME

20

ROY W. SIMONSON ( I ) , Director, Soil Class$cation and Correlation, Soil Conservation Service, United States Department ofAgriculture, Hyattsville, Maryland EDWARD H. TYNER ( 1 53), Professor of Soils, Department of Agronomy, College of Agriculture, University of Illinois, Urbana, Illinois

PREFACE This is the twentieth volume of this serial publication. Those who had a part in what seemed to be an uncertain venture in 1948 can take some pride in its acceptance and feel satisfaction in the demonstrated willingness of soil and crop scientists and their colleagues in related fields to . prepare authoritative reviews for its pages. If after the twenty-year interval, one set out to rewrite the Preface to Volume 1, there is little that one would need to change or add. The central theme of this publication is crop-soil relationships, their better understanding and beneficial exploitation in the service of man. The power of science-based technology has been as clearly demonstrated in agronomy as in the field of engineering. The remarkable and sustained increase in acre yields that has been achieved in countries practicing scientific agriculture is one of the great achievements of the mid-century period. There is no reason to believe that this phase is over. A11 technology is not directly transferable but, even so, there still remain enormous opportunities for the application of existing technology to areas of the world where productivity is low and the capability of applying new knowledge is not well developed. Barriers to the effective utilization of science are often socioeconomic and political. Social action skills may be more important in fostering application than agronomic education per se. In the next twenty years one may confidently expect the accretion of new knowledge about the characteristics of soils and crop plants, and of their interactions, to proceed at an accelerating rate. These developments will find their way into later volumes and serve the agronomists of the world in their great task of providing sufficient food for all men. A. G. NORMAN Ann Arbor, Michigan August, 1968

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CONTENTS CONTRIBUTORS ro VOLUME20 .

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CONCEPT OF SOIL

ROK W . SIMONSON I . Concept of Soil I1 . Basic Soil Entities I 1 1 . Epilogue . . . References .

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PEARL MILLET BREEDING AND CYTOGENETICS G L E N NW . BURTON A N D JERREL B . POWELL

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Introduction . . . . . Reproduction . . . . . Cytogenetics . . . . . Genetics of Qualitative Characters Genetics of Quantitative Characters Breeding . . . . . . References . . . . . .

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MOVEMENT OF NUTRIENTS TO PLANT ROOTS

S . R . OLSENA N D W . D . KEMPER I. I1. 111. IV . V.

Introduction . . . . . . . . . . lnterdiffusion of Ions of Like Charge . . . . Diffusion of Salts . . . . . . . . . Combined Diffusive and Convective (Viscous) Flow . Role of the Plant . . . . . . . . . . . . . . . . . . . References .

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THE ELECTRON MICROPROBE X-RAY ANALYZER AND ITS USE IN SOIL INVESTIGATIONS MICHELP . CESCAS.EDWARDH . TYNER. A N D LAWRENCE J . GRAY 1. introduction . . . . . . . . . II . Description of the Electron Microprobe Analyzer . 111. Methods for Analysis . . . . . . . 1V. Fundamentals of Quantitative X-Ray Analysis . V . The Quality of X-Ray Microanalysis . . . . . . . VI . Applications to Soil Investigations . VII . Summary and Conclusions . . . . . . References . . . . . . . . . .

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HYBRID WHEAT V . A . JOHNSONA N D J . W . SCHMIDT

I . Introduction . . . I I . Cytoplasmic Sterility . 111. Fertility Restoration . . . . 1 v. Heterosis . . V . Quality of Hybrids . VI . Agronomic Considerations VII . Seed Production . . VIII . Economic Considerations IX . Outlook . . . . References . . . .

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AGRONOMIC CONTROLS OVER THE ENVIRONMENTAL CYCLING OF TRACE ELEMENTS W . H . ALLAWAY 1 . introduction . . . . . . . . . . I 1 . Environmental Cycles of Trace Elements . . . 111 . Agronomic Control Points in Trace Element Problems IV . Current Environmental Problems Involving Specific Trace Elements . . . . . . . . . V . Some Potential Future Trends . . . . . . . . . . . . . . . . References .

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CARBOHYDRATE SYNTHESIS I N MAIZE

ROY G . CREECH

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11. Genetic Mutations

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111. General Carbohydrate Content and Transformations

during Kernel Development . . . IV . Nature of the Carbohydrates in Plants V . Enzymes in Starch Synthesis . . VI . General Statement . . . . . References . . . . . . .

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TRANSFORMATION A N D AVAILABILITY TO RICE OF NITROGEN A N D PHOSPHORUS IN WATERLOGGED SOILS WM.

H . PATRICK. SR.,

1. Introduction . . I 1 . Changes Occurring in 111. Nitrogen . . . IV . Phosphorus . . V . Summary . . . References . . .

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a Soil as a Result of Waterlogging .

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KEYWORD TITLEI N D E X VOLUMES 1-20

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CONCEPT OF SOIL Roy W. Simonson Soil Conservation Service, U S . Department of Agriculture, Washington, D.C.

Page

Concept of Soil .................................................................................. A. Introduction ................................................................................. B. Medium for Plant Growth ............................................................. C. Basic Matter of the Universe ................................ D. Mantle of Loose and Weathered Rock E. Recognition of Soils as Organized Nat ........................... 11. Basic Soil Entities .............................................................................. A. Nature and Dimensions of Basic Soil Entities ................................. B. Relating the Mapping and Classification of Soils ..............................

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I. Concept of Soil

A. INTRODUCTION

“Someone has said that the fabric of human life is woven on earthen looms- it everywhere smells of the clay.” So begins an early chapter in a report on the soils of a part of Saskatchewan, Canada (Mitchell et al., 1950). This is an apt beginning for a description of the soil resources from which farm people gain their livelihood. Throughout human existence, the quality of human life has been dependent in part on the nature of these earthen looms. The fabric of human life has changed in quality as mankind has increased its knowledge and improved its technology. Moreover, the levels of knowledge and technology at any time in the past seem to be reflected in the prevailing conception of soil. The relationship is evident from the study of conceptions held during historic time. The conceptions that do prevail bear not only on approaches and methods followed in the study of soils, but also in their use by any society. The basic concept of soil, since it was first recognized by mankind as an entity worthy of attention, seems to have been that of a thin mantle over the land surface. Most likely, the thin mantle was not distinguished 1

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from the remainder of the earth by our early ancestors. The whole was first taken for granted as a part of a rather frightening world. To early mankind, the ground underfoot provided support for movement and for habitation. The ground may well have been considered fixed and immutable. Little thought would have been given to its nature in the struggle for existence. The conceptions of soil held at various times and places in the past are reviewed in this article. During much, perhaps during all, of historic time more than one conception has existed concurrently. It seems probable that a single individual has commonly used one conception in some circumstances and another conception in other circumstances. Past conceptions of soil are both the foundations and major building stones for the conceptions prevailing now. Examination of some earlier conceptions and how they developed can thus throw light on the nature of present conceptions, which should then be better understood. Possible gains in improved understanding of the taxonomy of animals and plants through review of past approaches and methods are well illustrated by a group of papers published in England within the last decade (Cain, 1958, 1962; Gilmour and Walters, 1963; Walters, 1961). These papers review the roots of taxonomy in the logic of Aristotle, classifications prior to those of Linnaeus, and effects on the present classification of angiosperms on its development in Europe. The careful examination of historical developments in taxonomy makes the present easier to comprehend. Some gain should also follow from better knowledge of conceptions of soil that have been held and changes in such conceptions with time. Several conceptions of soil within the broader concept of the thin surface mantle are known to have been held in the historic past. All versions apply to the upper part or the whole of the regolith, which itself is commonly some feet or tens of feet in thickness. Thus, the regolith forms a negligible part of the mass of the earth. Even the outer rock crust, roughly 25 miles thick, forms a small part of the total mass. The regolith is thus a very minor part of the outer shell of the planet and soils in turn form a small part of that regolith. Yet, soil is the foothold for plants which provide food and fiber for mankind. Furthermore, life as we know it would not be possible without this thin rind of the earth.

B. MEDIUM FOR

PLANT

GROWTH

The conception of soil as a medium for plant growth must have been one of the first held by man. So long as mankind hunted and gathered its food, there was no need for thought about the nature of soil underfoot except as physical support. More than likely, some areas were known to

CONCEPT OF SOIL

3

provide better footing than others and some areas were to be avoided, if possible, but further understanding was unnecessary. When man began to grow his food rather than gather it, the nature of soil assumed a direct bearing on his welfare. It must soon have occurred to the more thoughtful members of the small farming communities that the soil underfoot was a medium for plant growth, something in which seed could be sown and from which plants could be produced to provide food, if all went well. This conception of soil as a medium of plant growth must have come into existence as mankind underwent what Braidwood ( 1 96 1) calls the “agricultural revolution.” The change from food-gathering to food-growing seems to have occurred first along the flanks of the Zagros Mountains about 9000 years ago (Braidwood and Howe, 1962; Young and Smith, 1966). This part of Asia Minor lies along the margins of the Fertile Crescent in portions of lran and Iraq. The transition from a hunting and collecting way of life to village farming seems to have occurred in Central America less than 6500 years ago, beginning with the cultivation of maize and a few other plants in the Tehuacan Valley of southern Mexico (MacNeish, 1964). Primitive ears of corn found in western central New Mexico indicate that cultivation of maize had been started there at least 5600 years ago (Mangelsdorf, 1958). Conceptions held by people in Asia Minor and in Central America 6000 or more years ago, prior to existence of historical records, cannot now be known. Speculation about the state of knowledge among the people is possible, however, on the basis of archaeological evidence. It is known that the people were able to shape stone tools and make pottery vessels of several kinds. They were able to domesticate both plants and animals (Braidwood and Howe, 1962; Coe and Flannery, 1964). The levels of sophistication indicated by the artifacts left by these people make it reasonable to postulate that some individuals in the population recognized soil as a medium for plant growth. More thoughtful individuals in the small farming villages may even have noticed that soils were more productive in some places than in others. That Neolithic people recognized distinctions between soils is suggested by the pattern of prehistoric occupation of soils in western Europe (Clark, 1952: Evans, 1956). The early farmers apparently learned that some soils were too wet or too sandy, and others too hard to till. This education may have come through a process of trial and error with some people starving because they tried to produce their food on poor soils. Even so, the archaeological record indicates that Neolithic people in Europe had learned that soils differed in their usefulness in food production. The available evidence, though fragmentary, indicates that mankind

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ROY W. SIMONSON

long ago thought of soil as a medium for plant growth. Even before that conception was elaborated, people must have recognized the soil underfoot as a substrate on which they might set up their shelters and across which they and the animals they hunted moved. Both of these conceptions require that the human mind go beyond the stage of taking the environment for granted. Both conceptions have persisted in some form down to the present day. Both are still parts of the set used by mankind in thinking about soils and their usefulness. The oldest classification of soils seems to have been an effort to record distinctions in productivity for crops, which implies a recognition of soil as a medium for plant growth. This classification is believed to have been devised in China between 4000 and 5000 years ago (Ping-Hua Lee, I92 1). The soils of the kingdom were divided into nine classes according to productivity, and this became the basis for determining the size of individual holdings and the taxes due to the government. The system of soil classification thus indicates the existence of a rather sophisticated conception of soil several thousand years ago. Approximately 1000 years after the effort to classify the soils in China, civilizations developed in Egypt and Mesopotamia (Braidwood, 196 1) and in the Indus Valley (Raychaudhuri, 1953). Agriculture was the prime support for each of these civilizations. The cultivators of soil seem to have been at the bottom of the social ladder, however, and records are lacking or sketchy about the use of soils. in food production. Perhaps the prevailing views in ancient Egypt and Mesopotamia were comparable to those expressed several millennia later by Columella, who wrote in 60 A.D. that the earth was ". . . the common mother of all things-because she has always brought forth all things and is destined to bring them forth continuously . . ." (Columella, 60 A.D.). No written record was left by the civilization in the Indus Valley, but the people did grow wheat, cotton, and barley and they had a variety of domesticated animals (Raychaudhuri, 1953). Few indications have been found of the conception or conceptions of soil held in classical Greece. Soil was said to serve plants as the stomach does animals in the writings of Hippocrates about four centuries before the birth of Christ (Jarilow, 19 13). A closely similar concept seems to have been held by Theophrastus about 300 B.C. (Ehwald, 1962). The records that have come down from ancient Greece must be fragmentary; most written records were probably destroyed. Only a small part of the accumulated knowledge could have been preserved. Consequently, the available information may not record prevailing ideas with completeness and accuracy. On the other hand, much food was imported by Greek

CONCEPT OF SOIL

5

city-states, such as grain from the Black Sea area and elsewhere (Semple, 1921). How important cultivation of soils on which people lived may have been to the support of the population is therefore an open question. Clear evidence that soil was recognized as a medium for plant growth and that considerable thought was given to its function in producing crops is provided by the writings of Cato, the Elder (Brehaut, 1933) and of Varro in the second and first centuries before Christ (Varro, 35 B.c.). In giving advice on the kind of farm to buy, Cat0 spelled out in decreasing order of merit nine kinds of plots or fields that should be included. The first was a good vineyard and the last a mast grove where animals could forage for acorns. Between the two extremes were listed an irrigated garden, an olive yard, a meadow, and grain land, to name a few more kinds. The grading of soils according to suitability for different plants was carried over by subsequent students of agriculture. Reference is made specifically to the ranking by Cat0 in a book on husbandry written by Varro (35 B.c.) at least one and one-half centuries later. Varro explicitly described soil as a medium €or plant growth in writing about the three meanings of the word terra. The third or “mixed” meaning of terra was “. . . the element in which seeds are sown and germinate. . . .” Later Varro writes that it was most important to determine whether soils are rich, poor, o r medium. Rich soils would support all kinds of plants, could be tilled regularly, and yielded good harvests. Poor and medium soils did not. Almost a century after the death of Varro, Columella outlined a simple classification of soils, also centered on their usefulness for crops and pasture. Listed from best to poorest, soils were rich and mellow; rich and dense; well watered; dry, stiff, and lean (Columella, 60 A.D.). Whether this ranking of soils was meant for general use or only for the locality is not evident. Columella had lived and traveled in several parts of the Roman Empire. The treatment of most topics in his book on husbandry is thorough. It may therefore be that he thought his simple classification would cover all soils. H e may have believed that nothing further would be required for good husbandry. On the other hand, his attention may also have been focused so strongly on use and management that soils were considered only so far as he thought necessary for his instructions. The handbook written by Columella seems to have been a standard reference for at least a thousand years (Ehwald, 1962). I t was a source for other handbooks of husbandry for at least another five or six centuries (Russell, 1961). The similarities in substance between the book by Columella and a pair of books written in the twelfth and thirteenth

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ROY W. SIMONSON

centuries demonstrate the degree to which the knowledge gained by the Romans was still accepted and used. Ibn-al Awan wrote the “Book on Agriculture” in the latter half of the twelfth century (Olson and Eddy, 1943). The book, written in Moorish Spain, refers frequently to what others have said about specific practices but does not give direct sources. The twelfth-century book seems as thorough for its day as was the book by Columella some eleven centuries earlier. Both men were keen observers and careful students. Moreover, it seems virtually certain that Ibn-a1 Awan was acquainted with the writings of Columella, but it would have been indiscreet for a Moor to refer openly to the writings of an infidel as a prime source of information. In the thirteenth century, Crescentius of Bologna collected and summarized Roman literature on agricultural husbandry (Russell, 196 1; Ehwald, 1964). The book was widely used and reprinted, whereas that by Ibn-a1 Awan was forgotten and dropped out of sight for several centuries. The emphasis in both these books was on agricultural husbandry. Both books also carried as part of their backgrounds the idea of soil as a medium for plant growth. The three books written across a span of thirteen centuries express about the same conception of soil. Moreover, the conception seems to have been general rather than explicit. To a large degree, the three authors must have shared the view expressed by Columella that earth or soil was “. . . the common mother of all things . . . .” They all recognized differences in quality or productiveness of soils, and they all realized that soils responded to good management or husbandry. Some agricultural practices were known to be better than others because their application resulted in higher yields. More specialized conceptions of soil as a medium for plant growth arose in the eighteenth century and were developed further in the nineteenth century. Jethro Tull ( 1 733) introduced a horse-drawn cultivator, the use of which increased crop yields. His explanation of the increase was that the cultivation or “hoing,” as he called it, increased the total surface of soil particles exposed to roots and thus expanded the pasture of the plants. Very small soil particles were thought to be taken in as food by plant roots. Tull did recognize that cultivation controlled weeds, but improved yields were ascribed primarily to improved nutrition of crop plants. With the rise of chemistry during the eighteenth century, it was applied to the study of agriculture. Efforts were being made to increase food production. The need for increased food supplies was widely recognized, even the cause of some alarm (Marbut, 1925). Agriculture was a prime

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7

concern of many able chemists for the better part of a century, as witness the books by Wallerius ( 176 I ) , Davy (1 8 13), and Liebig (1 843). General concern about food supplies persisted until the Mollisols (Chernozems, Brunizems, and other dark-colored soils of temperate grasslands) came into full production in several parts of the world about the middle of the nineteenth century. The proposition that plants feed directly on the humus in soils was put forward by Wallerius in “Chemical Foundations of Agriculture,” published in Sweden near the middle of the eighteenth century (Wallerius, 1761). Other components of the soil were thought to be accessory instruments rather than direct nutrients. The idea that humus wae absorbed directly by plant roots was developed further during the first half of the nineteenth century by von Wullfen and Thaer (Usher, 1923). These men thought that levels of organic matter in soils must be kept at o r near original levels to maintain fertility. Otherwise, the fertility of soils and expectable yields would decline. The men noted that amounts of humus of soils in central Europe could be lowered rapidly under cultivation. Crop yields were then reduced. From these observations, von Wullfen and Thaer concluded that soils could be exhausted in a short time. They consequently made comparisons of changes in levels of soil organic matter under different cultural methods. For each method, the additions of organic matter and the probable losses of humus were calculated. Balances were struck between gains and losses of humus. These were believed to correspond to gains and losses in soil fertility. Books of the day are full of such computations (Usher, 1923). T h e persistence of an idea after it has gained a degree of acceptance is impressive. An idea may persist in whole, in part, or in modified form. The history of the proposition that plants feed on humus illustrates this state of affairs well. The spreading of the proposition put forward by Wallerius is evident from a statement made some seventy years later in an agricultural journal published in Chicago (Hudson, 1844): “Every farmer can understand that plants, like animals, are organized beings: that they live, grow, and require food for their sustenance; that this food is principally animal and vegetable matter, duly prepared by fermentation and decomposition.” The persistence of some belief in the proposition that humus is the key to soil fertility is indicated by experiments conducted during the first half of the twentieth century to raise levels of organic matter in soils of the southeastern United States. It was widely believed that the Ultisols (RedYellow Podzolic soils and related kinds) had been exhausted by long use for growing crops. It was also believed that the higher levels of organic

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ROY W. SIMONSON

matter in the Mollisols of the midwestern United States were responsible for their higher productivity. Given these beliefs, it logically followed that Ultisols could be made more productive if the levels of organic matter were raised. Efforts were made to raise levels of organic matter by adding large quantities of barnyard manure or other materials. Some trials lasted 20 years, at the end of which levels of organic matter in the soils had not been changed appreciably. Rates of decay or organic matter had kept pace with the larger additions. This would be expected now. Studies in recent years have shown a relatively rapid turnover of organic matter added to soils, most fractions breaking down between one year and the next (Bartholomew and Kirkham, 1960). As advances were made in chemistry and plant physiology during the nineteenth century, corresponding changes followed in the conception of soil as a medium for plant growth. Investigators learned that a number of elements were essential plant nutrients. Consequently, the views held by Tull, Wallerius, and Thaer were first replaced by what may be called the nutrient bin concept of soils. This was promoted vigorously and successfully by Liebig ( 1843) during the first half of the 19th century. One of his statements follows: “A soil may be considered a magazine of inorganic matters, which are prepared by the plant to suit the purposes destined for them in nutrition.” Liebig expanded the earlier balance sheet approach, followed when humus was considered food for plants. H e argued that fertilizer needs could be estimated by analyzing plants and soils and by striking a balance between amounts in the soil and in those in the growing plant. The soil was likened to a bank in which money must be deposited before it could be withdrawn. Similarly, plant nutrients must be added to soils if they were not to be exhausted. Withdrawals of plant nutrients must be offset by additions of fertilizers if soil fertility were to be maintained. This approach, designated a balance sheet theory of plant nutrition, was widely accepted and followed. Analyses were made of soils and of plants. The resulting data were then used to appraise the fertility of soils and to estimate fertilizer needs. A rather different version of the same basic conception, i.e., soil as a nutrient bin, was developed in the United States near the close of the nineteenth century. Whitney ( 1 892) concluded from his studies that all soils were high enough naturally in nutrients for satisfactory plant growth. Benefits from fertilizer applications were recognized, but these were believed to be due to improvements in moisture and temperature relationships, changes in physical properties of soils which made them more hospitable to roots (Whitney and Cameron, 1903). Further, it was be-

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lieved by Whitney (1892) that the texture of soil provided a direct index to moisture and temperature conditions important to plant growth. His conclusions were based in part on observations of the yield and quality of tobacco produced on soils differing in texture in Florida, Maryland, North Carolina, Pennsylvania, and Wisconsin. His conclusions also rested on observed yields of wheat, corn, and pasture on soils in different parts of Maryland west of Chesapeake Bay. These interpretations of soil-plant relationships were drawn from observations in a rather small universe and were extended to soils generally. This may have been done because it was known that most mineral soils were similar in their gross composition. Whitney may have arrived at his interpretations because he had noticed that many differences in productivity were not explainable on the basis of available chemical analyses (Whitney, 1892, 1904). Moreover, students of soil had not yet recognized the complexity of something so commonplace. T h e proposition that all soils naturally contained enough nutrients for satisfactory plant growth was debated vigorously in this country during the first decade of the present century (Whitney, 1904; Hilgard, 1904). After that first decade, however, the proposition was gradually dropped. Arguments offered in support of the proposition are not persuasive now but are of interest as indicators of the prevailing state of knowledge about soils. Thinking of the soil as a nutrient bin from which plants could draw for their needs seems to be an accurate reflection of the state of knowledge sixty years ago. The nutrient bin idea has been replaced, however, as the study of soil continued and understanding was improved. Looking at soil as a medium for plant growth, both as a substrate providing mechanical support and a s a source of essential nutrients, continues today. The conception has been elaborated much during the last fifty years. Soils are now known to be complex, as are soil-plant relationships. Thus complexity is readily evident from the discussions of soil-plant relationships io the books by Russell (1961) and Black ( 1968). T h e evolution of the idea of soil as a medium for plant growth subsequent to the turn of the century is not traced here. This is not to imply that changes have been small or unimportant. Information on the changes, however, is far easier to find than that on earlier developments. Only earlier stages in the evolution have therefore been sketched on these pages. The conception of soil as a medium for plant growth can be expected to continue into the indefinite future. Soil remains of primary importance to mankind because it will support plants that provide food and shelter. Emphasis on this function of soil is not likely to diminish: rather it may

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increase. Consequently, one test of the value of other conceptions of soils will be an assessment of what they contribute toward increased efficiency in the use of soils for production of food and fiber. c . BASICMATTEROF

THE U N I V E R S E

A conception of earth or soil as one of four basic components of all matter was put forward by Empedocles about 400 B.C. (Neuss, 1914). He held that all matter consisted of fire, water, earth, and air. Earth and soil were apparently synonymous terms. The idea that earth or soil was one basic component of all matter persisted for many centuries. How long the idea prevailed in some form is brought out by a pair of sentences in a book on soils, published in the middle of the nineteenth century. Fallou (1 862) wrote, “The basic forces, which have acted during present and past ages to destroy and modify the massive rind of our planet, are air, water, and fire. These three elements have since ancient times been in conflict with the fourth, the earth.” The belief that earth or soil was a basic component of all matter is also reflected in a current nursery rhyme about fire, water, earth, and air. Evidence that the conception of earth or soil as basic matter was used for a number of centuries is provided in a book written about sixty years before the birth of Christ (Lucretius, 60 B.c.). Not only is the idea of fire, earth, water, and air as the basic matter of the universe included in the book, but Lucretius has high praise for the wisdom of Empedocles. In the same book, Lucretius states that “earth and fire are mortal things indeed.” Thus, it seems that he thought of earth or soil as basic matter which could be changed. The kinds of changes considered possible were doubtless much different from the kinds of changes now considered parts of processes of soil formation. Passages in the book also demonstrate clearly that Lucretius thought of soil as a medium for plant growth. He writes about the improved taste of fruit grown with “fond and fostering care,” about the clearing of forests from slopes to provide more land for the plow, and about the groves of olive trees “marking the plotted landscape” (Lucretius, 60 B.c.). What was written by Lucretius demonstrates that an able mind can at the same time hold and use more than one conception of soil. In thinking about basic composition of the earth, Lucretius accepted ideas put forward several centuries earlier by another scholar. In thinking about the growing of crops, Lucretius considered soil as a medium for plant growth. Whether the shift from one conception to the other was deliberate because Lucretius believed that he was dealing with one kind of universe in one setting and with a second kind of universe in another setting is an

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intriguing question to which no answer can now be obtained. Shifts from one conception to another or from one way of thinking to another are common enough in soil classification now, however, to show that they need not be deliberate (Butler, 1958; Simonson, 1963). A single mind can hold several conceptions of the same complex object simultaneously. The use of one o r the other depends on the circumstances requiring thought. Ideas held may rest on beliefs so deeply buried in the mind that their very existence cannot be recognized without careful analyses (Mander, 1947).

D. MANTLEOF LOOSEA N D WEATHERED ROCK A new conception of soil followed the birth and development of the science of geology during the late eighteenth and early nineteenth centuries. Geology was the first science to develop field methods, and these could be applied to the study of soils. Geologists gave attention to soils because they were considered products of rock weathering. By the middle of the nineteenth century, a number of studies of soils had been made by geologists, mostly a s parts of broader investigations of geologic formations (Coffey, 19 12a). The knowledge that the bulk of weight of most soils consisted of weathered rock materials seems to have been general in the early nineteenth century. This understanding was well expressed in the report of a geological survey of Albany County, New York (Eaton and Beck, 1820), viz: “That all the earthy part of soil consists of minute fragments of rock does not require argument, or need proof, but inspection merely to determine it. We have only to place specimens under the magnifier and their rocky origin will become manifest.” The same authors began the first section of their report with the following statement: “The present, so far as it has come to our knowledge, is the first attempt yet made in this country to collect and arrange geological facts, with a direct view to the improvement of agriculture.” The field work required a period of 4 weeks, during which “all facts and specimens” were collected. Afterward, the men made certain analyses of the specimens, a s the soil samples were called. Methods are described for determining water content by loss of weight under gentle heating, amounts of vegetable and animal matter by loss on ignition, particle size distribution, soluble salts, and carbonates. Particle size distribution was determined by sedimentation after shaking soil samples in water. The proportions of silex (sand) and of alumine (finer size fractions) are given for the samples. Soluble constituents were estimated by evaporating clear liquid to dryness and weighing the residue. Amounts of carbonates were

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determined by measuring loss of weight following treatment with hydrochloric acid (Eaton and Beck, 1820). The analyses were apparently made to characterize the different kinds of soils recognized in the field work. Text of the report suggests that the kinds of soils were identified through field observations, though methods are not described. The soils of Albany County, called “alluvial formations,” were divided into geest (sedentary materials) and alluvion (transported materials). Geest was much the .more extensive in the county. For agricultural purposes, geest was subdivid.ed into granulated soil, hard pan, upland loam, upland clay, and lowland loam. Alluvion was subdivided into sandy alluvion, river all.uvion, loamy .alluvion, and clay alluvion. Descriptions of the different kinds of soils are not given except for the analytical results. Statements are made about the suitability of each kind of soil for various crops. The study in Albany County was the earliest attempt in the United States to classify soils of a definite geographic area (Coffey, 1912a). The terminology and the discussions in the report on Albany County clearly demonstrate that soil was considered all or part of the mantle of loose and weathered rock, some part or the whole of the regolith. The report by Eaton and Beck thus provides an early statement of the geologic conception of soil. More explicit statements on the conception of soil as a mantle of weathered rock appear in later publications of the nineteenth century. Ruffin (1832) argues in the first paragraph of the first chapter of his “Essay on Calcareous Manures” that it is necessary to distinguish between earths and soils in their many varieties. Later, he continues: “The earths important to agriculture, and which form nearly the whole of the known globe, are only three-silicious, aluminous, and calcareous.” Still later in this first chapter, Ruffin explains that soils are mixtures of three earths plus “a sufficiency of animal or vegetable matter, putrescent, and soluble in water.” Given this proper mixture, “. . . plants can extend their roots freely, yet be firmly supported, and derive all their needful supplies of air, water, and warmth, without being oppressed by too much of either. Such is the natural surface of almost all the habitable world.” Further on in the first chapter, Ruffin continues Soil seldom extends more than a few inches below the surface, a s on the surface only are received those natural supplies of vegetable and animal matters, which are necessary to constitute soil. . . . Below the soil is the subsoil, which is also a mixture of two or more earths, but is as barren a s the unmixed earths, because it contains very little putrescent matter, the only food for plants.

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13

Ruffin seems to have looked at soils from two points of view. He thought of soil as the mantle of weathered rock, a mixture of “earths,” and also as a medium for plant growth. He seems to have accepted the proposal that humus was taken up and used as food by plants. Soils, according to Ruffin, were mixtures of the “earths” and organic matter. Moreover, the soil was the uppermost part of the mantle of weathered rock. The deeper parts of this mantle (the regolith) were barren and should not be considered soil. Fifteen years after the book by Ruffin was first published, Hitchcock (1838) prepared a geologic map and reports on the economic geology of Massachusetts. He includes in his report an explicit statement of one conception of soil: All geologist and chemists agree in regarding soil as the result of abrasion, disintegration, and decomposition of rocks, with the addition of certain saline, vegetable, and animal substances. . . . The earthy portions always constitute by far the largest part, and, hence, if we know the composition of the rocks whence they were derived, we shall know the earthy and metallic constituents of the soils.

Later in the same report, Hitchcock continues: In general, if anyone wishes to recognize the different kinds of soil, let him look at the Geological Map that accompanies my former report, and he may conclude that the different soils cover those portions of the surface that are represented as occupied by the rock from which they were derived. There is one circumstance, however, that prevents us from considering the boundaries of rock formations as perfectly coincidental with those of soils. Diluvial action has removed nearly all loose covering of our rocks in a southerly direction, and more or less mingled the soils from different formations.

A classification of the soils of Massachusetts was given by Hitchcock ( 1838) in a report on economic geology. This classification was believed

by the author to be satisfactory for all practical purposes when used with the “common divisions” into sandy, loamy, clayey, and calcareous soils. A total of 13 major kinds of soils are listed. Four of these major kinds are subdivided into two and one into three classes. These kinds constitute “all important varieties of soils in Massachusetts.” Examples of the major kinds are sandstone soils, limestone soils, Tertiary soils, and alluvium. Approximately twenty years after Hitchcock conducted his studies of geology in Massachusetts, Hilgard (1 860) carried forward a more detailed study of the geology and agriculture of Mississippi. In his early work, Hilgard accepted the conception of soil as the surface mantle of weathered rock, though he obviously also thought of soil as a medium for plant

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growth. He ascribed the formation of soil to the action of atmospheric agencies which slowly but surely disintegrated the hardest rock. The initial processes responsible for disintegration were mechanical action of water in attrition and the transportation of materials; changes in temperature; wetting and drying; and chemical reactions of water, carbonic acid, and oxygen with minerals. Much stress was placed by Hilgard (1860) on the importance of rocks and their characteristics to the kinds of soils, as shown by this statement in the preface of his report on the study in Mississippi: The main object in a practical point of view being . . . the promotion of the interests of Agriculture and of kindred pursuits, . . . and the agricultural features being so closely dependent upon the geological, as to render the subdivisions based upon the latter almost equivalent to those which represent the former: since, moreover, the materials of the geologic formations are important, not only as imparting their character to the soils, but also as furnishing natural manures of the highest value; the study of the geological phenomena is obviously the first step toward a full understanding of the agricultural resources, both present and future.”

Hilgard prepared his report in two nearly equal parts, the first on the geology and the second on the agriculture of Mississippi. The early sections of the second part are discussions of principles of rational agriculture and of agricultural chemistry. The subsections on soil are centered on its functions in supporting plant growth. “The term soil implies the surface stratum of earthy material, a s far a s the roots of planrs reach; no matter whether it be sand, or potter’s clay, or, as in most cases, a mixture of the two.” Like Ruffin before him, Hilgard seems to have thought of soil in two ways. When thinking about its formation, he considered the soil to be a mantle of weathered rock, or the upper part of the regolith. When he was thinking about agricultural use of soil, Hilgard considered it a medium for plant growth. The two ideas are evident in the 1860 report and also in a study published thirty-two years later (Hilgard, 1892). By this later date, however, Hilgard was beginning to recognize soil differences related to distinctions in climate. Fallou (1862) offered the most colorful statement of the conception of soil as the surface mantle of loose and weathered rock: “Soil is considered to be the product of weathering, formed as the tooth of time incessantly grinds the solid covering of our planet and gradually decomposes and destroys its solid mass . . .” From his studies in Saxony, Fallou developed a classification reflecting his concept of soil as a direct function of the parent rock. The two main kinds were residual soils and sedimentary or alluvial soils. The first of these was then subdivided into granitic

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15

soils, limestone soils, sandstone soils, etc. The second main kind was subdivided into gravelly soils, marly soils, loamy soils, etc. The classification demonstrates that soil was considered a product of rock weathering and that soil characteristics were thought to be determined by the rock sources. An approach closely paralleling that of Fallou was followed by Shaler ( I 877) and his staff in Kentucky somewhat later. The first part of a brief discussion of soil in a report on physical geography and natural resources of the state reflects the conception of soil. All the Kentucky soils except the strips ofalluvial land along the banks of the rivers have been derived from the decay of the underlying rocks. They may be called soils of immediate derivation, as distinguished from the soils made up of materials that have been borne from a distance by water, or which deserve the name of soils of remote derivation. This feature of immediate derivation gives the Kentucky soil a more local character, dependent on position, than those of any state north of the Ohio.

The geography of soils of Kentucky is then described on the basis of the age and general character of the major geological formations. Major kinds are listed as soils from Cambrian limestone, soils from Silurian limestone, soils from Devonian shale, and soils from Carboniferous rocks. Several versions of the conception of soil as the mantle of loose and weathered rock at the land surface emerged during the nineteenth century. These prevailed in various forms well into the present century. For example, Fippin ( 19 I I ) proposed a soil classification system based on the mode of accumulation and the lithology of the regolith. This system is more elaborate but still much like that suggested earlier by Fallou ( 1862). The same basic approach is followed in the description of soils of the United States published in 1913 (Marbut et al., 1913). Primary emphasis is given to mode of accumulation of the regolith in the classification of soils and in the map showing their general distribution in a report issued in Iowa thirty years ago (Brown, 1936). According to Hunt (1967), geologists use the term ‘‘soil’’ to refer to the surficial layer altered by weathering. Thus, for geologists soil might include all or part of the regolith. Closely related to but not identical with the geologic concept of soil is that used by engineers (Hough, 1957), Civil engineers use the term to refer to that part of the regolith removed in excavations, used for fill materials, or providing foundations for structures. Thus, in an engineering sense, soil generally includes the whole of the regolith, the full mantle of unconcolidated materials, whether they are several feet or hundreds of feet thick. Leggett ( I 953) urged that the term “soil” be defined to cover

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the whole rather than part of the regolith. He proposed that some other term be found for that part of the regolith serving as a medium for plant growth. The conception used by civil engineers does not coincide fully with that of geologists, though the two are not far apart. Both include the unconsolidated and weathered mantle and the two may thus be identical. If the thickness of unconsolidated materials is great, as is true in the coastal plain bordering the Gulf of Mexico, more of the regolith would be considered soil by civil engineers than by geologists. Geologic beds which might be unconsolidated but had not been altered appreciably by weathering would not be included as part of a soil by geologists, whereas they could be by civil engineers. To pedologists, the upper part of the regolith is soil, as a general rule. If the depth to rock is a matter of a few inches or feet, the whole regolith may be part of the soil, as the term is used by pedologists. If the regolith is many feet thick, however, only the upper part is considered by pedologists to be soil. The lithology of the upper regolith, how long the land surface may have been stable, and to what climatic conditions it may have been exposed are recognized by pedologists as important to the kind of soil that can be formed within their use of the term. There is consequently an important relationship between the conception of the earthy weathered mantle and the conception of recognizable natural bodies paralleling rocks, flora, and fauna.

E. RECOGNITION OF SOILS AS ORGANIZED NATURAL BODIES Recognition of soils as organized natural bodies worthy of scientific study is a recent development in human history. Such recognition emerged only during the last century. The conception of soils as organized natural bodies paralleling rocks, fauna, and flora first arose in rudimentary form and has followed a long and rocky path in its development. Tracing the general path of development, partly in Russia and partly in the United States, is the purpose of this section. No effort is made to cover all developments in all countries. Relationships between the conception of soils as organized natural bodies and other conceptions are discussed later.

I . Early Development in Russia Little more than a half century ago, Coffey (1 9 12a) published a resume of soil survey work in the United States and in other countries. His resume includes the following statement:

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17

To Dokuchaiev belongs the honor of founding 3 new school of soil investigation, a school which viewed soil as a natural body anh having a definite genesis and a distinct nature of its own, and occupying an independent place in the series of formations of the earth’s crust. . . . The soil is considered a biological as well as a geological formation, and unless the material has been influenced by life in some form it must be classed as rock and not soil.

Fifteen years later, in a publication prepared for the first International Congress of Soil Science, Afanasiev (1927) attributed to Dokuchaiev a more explicit statement of this same conception: “Soil is an independent natural body which must not be mistaken for surface rocks.” The classic monograph on Russian Chernozems prepared by Dokuchaiev in 1883 argues vigorously that soils are products of extremely complex interactions of local climates, plants and animals, parent rocks, topography, and the ages of landscapes (Dokuchaiev, 1948). The idea that soils were natural objects worthy of scientific study was actively promoted by Dokuchaiev and his students (Muir, 1961). Even so, the development and spread of this idea progressed slowly. As might be expected, Dokuchaiev and his students did not begin with a full understanding of the soil profile and the relationships among horizons. The Chernozem was considered to be what is now largely called the A horizon or the mollic epipedon of such soils in the United States. This conception of Chernozem is reflected in a report published for the World’s Columbian Exposition in Chicago (Dokuchaiev, 1893). One paragraph reads as follows: It may now be considered to be clearly established that all soils, clothing the earth’s surface with a more or less thin film of half a foot to six feet in thickness, must be divided into normal, lying on the spot where they were formed and appearing as far as possible with their primitive properties: and abnormal, which are either excessively washed or even moved bodily to other situations. It is completely demonstrated that the first of these, the normal soils, are the result of the very complex interaction of the following soil-formers: ground, climate, vegetable and animal organisms, the age of the country and of the contour of the locality. In places where these variables are the same, the soils are identical; where they are different, the results of their activity cannot be the same.

During the first few decades, the Dokuchaiev school was not consistent in restricting Chernozems to what is now called the A horizon. In some instances, members included the upper part of the ‘‘subsoil’’ or a segment of what would now be called the B horizon. For the most part, however, the subsoil was considered the parent rock which retained its fundamental petrographic features (Fireman, 1901 a,b). The ideas of Dokuchaiev and his school are a marked advance over

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earlier conceptions of soil and earlier explanations of soil formation. His conception of soil seems rudimentary in the light of what has been learned since 1880. The concept of the soil profile and its related horizons, for example, was a later contribution. Even so, the contribution of Dokuchaiev and his students was of major importance. They recognized the operation of a number of factors in soil genesis. They called attention to the significance of the interactions among the factors. This significance was not grasped for many years and may not be fully appreciated even now. Dokuchaiev seems to have developed his ideas of soils and their formation in large measure through the method of geographic correlation. He observed the occurrence of some kinds of soils in given situations over and over again. He concluded therefore that the elements of the environment in each of these situations governed the nature of the soil. The studies of Russian Chernozem, interestingly enough, were in part a by-product of a program to establish a better basis for tax assessment in the Zemstvo of Nishni-Novgorod. Dokuchaiev divided the program into two parts. The first part was to establish a satisfactory classification of soils. The second part was to grade these soils according to their agricultural potentialities (Yarilov, 1927). An approach similar to that followed by Dokuchaiev and his staff was used as a basis for land classification for tax assessment in western North Dakota approximately fifty years later (Kellogg and Ableiter, 1935). In the course of the program to develop a better basis for tax assessment in the one part of Russia, Dokuchaiev and his staff developed a system of soil classification. The system seems elementary and sketchy against the background of what is now known about soils. A fair appraisal of the contribution embodied in the early Russian system of classification would be possible, however, only if a person could examine that system against the general understanding of soils during the last fourth of the nineteenth century. Insofar as it is possible to reconstruct that background now, the development of the classification system with the conception of soils it entails seems a major contribution. Indications are that Dokuchaiev did not give much time and energy to the study of soils other than Chernozems. He gave some attention to Podzols. He was apparently interested in many subjects and distributed his time and energy among all. He thus left largely to others the task of organizing and presenting the findings gained through the investigations he promoted, and in some instances started. Luckily, he had a student and follower in Sibertzev who pulled together and presented the findings of the new school of soil science. The textbook by Sibertzev (1 95 I ) ,

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completed in its first edition in 1900, was the first synthesis and full statement of the findings of the new school of pedology. Furthermore, Sibertzev gave much more attention to Podzols (Spodosols) in his own research than had Dokuchaiev. Russian pedologists prior to 1900 thought of the Podzol as what are now called the A1 and A2 horizons, especially the A2 horizon (Muir, 196 I). Below the A2 horizon was subsoil or parent material, which was not considered part of the Podzol. This parallels the understanding of Chernozems held at the same time in Russia. The A I horizon was considered to be the Chernozem and the part of the profile below that horizon was parent material. These conceptions of soils are sketchy and incomplete compared to those now held. The introduction of these conceptions, however, represents a sharp break with the past. Therein lies a major contribution to soil science of the present. Perhaps as much as two decades passed between the first studies of Chernozems in Russia by Dokuchaiev and the recognition of genetic relationships between A and B horizons of soils. Early Russian pedologists realized that substances were being lost from the A2 horizon or from the Podzol, but apparently did not think of the possibility that the substances might stop in the subsoil or underlying material (Muir, 1961). Muller (1887) of Denmark was the first to recognize the B horizon of the Podzol (Spodosol) as a layer in which downward moving substances had accumulated. He gave special attention to the cemented or partially cemented B horizons, then called Ortstein in Germany and Denmark. The layers identified as Ortstein were known to affect tree growth adversely. Such layers were also troublesome when the soils were cultivated. His studies of the Ortstein led Mueller to conclude that it was cemented by substances brought down I'rorn the overlying A2 horizon. the Podzol of Russian pedologists (Muir, I96 1). Identification of something below the A horizon as related to it and as a part of the soil considered an organized natural body was thus a contribution from Western Europe rather than Russia. The idea seems to have spread from Denmark to Germany and thence to Russia during the last years of the nineteenth century. The rocky path followed in the development and spread in Russia of the conception of soils as organized natural bodies with a genesis of their own- that soil formation entailed more than weathering and that soils were more than weathered rock mantles on the land surface-is clearly indicated in the report by Muir (196 1 ) on the Podzol and Podzolic soils. The slow acceptance is illustrated by the history of horizon designations in Russia. Use of A for an eluvial horizon, B for an illuvial horizon, and C

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for parent material was suggested by Glinka during the first decade of the twentieth century. The convention was finally adopted in the Soviet Union about 1930. The convention was then followed in profile descriptions prepared for the Second International Congress of Soil Science held in 1932. The slow progess in the development and spread of the conception of soils as organized natural bodies parallels progress in science generally. Conant ( 1 95 1) has described the work of several great scientists as case histories to show how science proceeds. All of the men followed large numbers of false leads and explored many dead ends in their investigations. Consequently, much of their work was fruitless. The experience of these men demonstrates that even able minds do not find a high road to scientific truth.

2. Spread of Conception to Other Countries The studies in Russia, which began about 1880 and continued at a brisk pace until the outbreak of World War I , were known only to a limited extent outside of that country. Few soil scientists in the United States were acquainted with the work, and the same statement would probably be valid for most other countries. More than likely, the generally held conceptions of soils as mediums for plant growth or as superficial mantles of weathered rock seemed adequate. At least three publications in English describing work done in Russia appeared in the United States and the United Kingdom during a fifteenyear period between 1893 and 1908. Reports by Dokuchaiev ( 1 893) on the Russian Steppes and on the study of soils in Russia were printed as part of an exhibit on agriculture and forestry in Russia at the World’s Columbian Exposition in Chicago. This report by Dokuchaiev attracted little attention. The publication and the display of Russian monoliths at the exposition were probably responsible for the study of soils made by Vanderford (1897) in Tennessee shortly afterward. He makes no mention in his bulletin of the exposition and the Russian exhibits. On the other hand, he followed existing Russian methods in his collection of soil monoliths. An excellent summary of three papers by Sibertzev on classification and geography of the soils of Russia appeared in the Experiment Station Record at the turn of the century (Fireman, 1901a,b), but the two-part summary must largely have escaped notice. A brief description of the Russian soil classification system was also published in England a few years later (Tulaikoff, 1908). That also seems to have had little effect; at least it failed to register on soil scientists of the day.

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The findings of the new school of soil science in Russia were known in some measure to American soil scientists. Fifty-five years ago, Coffey ( 19 12a,b) referred to the work of Dokuchaiev and his students and published a short description and critique of the approach being followed in Russia. Like the earlier publications providing information on Russian work, those prepared by Coffey in 1912 had little evident impact. A summary of the body of fact and theory developed by the new school in Russia became available in Western Europe during the first year of World War I (Glinka, 19 14). The book, published in German, was a much abbreviated version of the text by the same author (Glinka, 1931). This text was standard for university courses in soil science in Russia at the time. The short book “Die Typen der Bodenbildung” must have been known to a number of people in Western Europe and the United States, but it had little effect at first, perhaps because of Warld War I and the upheavals that followed. Either during the war years or soon thereafter, however, the book was read by Marbut ( 1928a) and influenced him deeply. Through him, the book also came to influence the course of soil science in the United States. All in all, the Russian school with its conception of soil and theories of soil formation did not come into prominence over the world prior to the First International Congress of Soil Science in 1927. A group of 12 bulletins summarizing the Russian findings to date was prepared and published for the first Congress. These bulletins and the translated book by Glinka ( 1 927) soon became standard references in the teaching of soil genesis, morphology, and classification in the United States. It seems probable that the spread of the ideas developed in Russia to other countries was also greatly facilitated by personal conversations held during the 1927 Congress. The conception of soil and the theories for its formation developed in Russia during the last fourth of the nineteenth and the first fourth of the twentieth centuries have had a profound influence on soil science everywhere in the world. As more and more of the early Russian publications are translated into English, however, it becomes evident that selective rather than wholesale adoption of the original ideas has been general practice. Interpretations and adaptations of the Russian ideas were made as those spread to other countries. The interpretations may have been deliberate o r due to incomplete understanding. The risks of misinterpretations in transferring ideas and terms from one language to another, especially through the medium of a third language, are underscored by Muir (1961), who was familiar with the Russian work and knew well the translations and interpretations into English. Furthermore, the same

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ideas may well take on different meanings against differing cultural backgrounds. The significance of the world view held by people of a given society to the prosecution of scientific investigations and to their grasp of science was highlighted recently (Odhiambo, 1967) in a plea that science be given a more prominent place in the economic development of the new nations in Africa. Partial or incomplete understanding of scientific findings transferred from one place to another in the world seems common rather than unusual. Conceptions of soil much like that developed in Russia by Dokuchaiev and his school evolved in the United States during the last part of the nineteenth century. For example, Shaler (1891) argued that man needed a larger conception of “the soil coating” than did prevail. H e continued as follows: He should clearly see that this mass of debris, which at first sight seems a mere rude mingling of unrelated materials, is in truth a well organized part of nature, which has beautifully varied and adjusted its functions with the forces which operate upon it. Although it is the realm of mediation between the inorganic and organic kingdoms, it is by the variety of its functions more nearly akin to the vital than to the lifeless part of the earth.

A similar but less complete conception was expressed by Hilgard ( 1 893) about the same time. Earlier, Hilgard ( 1 860) had recognized that many soils had “. . . a sensible change in the character of the material . . . at a moderate depth, say from 3 to 12 inches-about the same as the usual range of the plow. If, therefore, as a rule, in speaking of virgin soils, we designate as soil, the surface layer down to the point where a perceptible change takes place, and as subsoil, what lies beneath, we shall in most cases find these subdivisions to coincide with what, in a proper system of tillage, becomes the soil and subsoil in the sense of the agriculturalist.” The 1860 report by Hilgard was ahead of its day. The title and text do show that Hilgard thought of the combined “soil” and “subsoil” as the surface mantle of weathered rock in considering soil formation and as a medium for plant growth in considering agriculture. The conception of soil reflected in the 1860 report, like the later ideas offered by Dokuchaiev, should be appraised against the prevailing background of knowledge. That background cannot be reconstructed fully now. From what can be learned, it is evident that the approach of Hilgard was a marked advance in the study of soils. This advance seems to have been known and appreciated to a greater degree by the Russian school (Glinka, 1931) than it was in the United States. In his study of soils of the United States, Coffey (1912b) outlined a

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more complete and explicit conception of soil than that published by Shaler twenty years earlier. This conception is reflected in critiques of the approaches already tried in the classification of soils, including that developed in Russia. Coffey spelled out requirements for an ideal classification and suggested the setting apart of several broad soil groups on the basis of what had been learned since the American soil survey program was started in 1898. The requirements for an ideal classification, as outlined by Coffey in 1912, are still valid. Three of the broad groups of soils he proposed correspond to the Aridisols, Mollisols, and Histosols of the 7th Approximation (Soil Survey Staff, 1960). The conceptions of soil advanced and developed by Hilgard, Shaler, and Coffey departed greatly from those generally held in the United States during the last half of the nineteenth and early part of the twentieth centuries. The degree of departure must have been a factor in the lack of acceptance of the new conception of soils. Orthodoxy is important in science (Polanyi, 1963), as it is in other fields of learning. The ideas presented by the three American scientists and those offered by the Russian school were much different from the “conventional wisdom” of the times. The upshot was that the ideas failed to capture imaginations of American soil scientists as a group until the third decade of the present century. The early history of the development and spread of the conception of soils as organized natural bodies parallels the history of the growth of knowledge generally. The reluctance to accept new ideas is not peculiar to soil science. Reluctance to drop once accepted interpretations in favor of new ones seems a common phenomenon. Dewey (1958) wrote: “ I t is not easy to break away from current and established classifications and interpretations of the world.” The history of science suggests that scientists themselves may resist rather than accept new discoveries. Barber ( 1 96 I ) quotes the following statement from a letter written by Helmholtz to Faraday: “New ideas need the more time for gaining general assent the more really original they are.” The development and spread of the conception of soils as organized natural bodies thus seems to have repeated the history of new developments in science generally.

3. Later Development in the United States The eventual acceptance in this country of the conception of soils as organized natural bodies followed mainly from studies necessary in the soil survey program started in 1898. Some field studies of soils had been made earlier-for example, those in Albany County, New York (Eaton and Beck, 1820) and those of Hilgard ( 1860) in Mississippi. The network

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of field observations required in soil surveys of many areas, each a few hundred square miles in size, was more complete and the observations thus more closely spaced than any made previously. At the beginning of the soil survey program and for approximately two decades, two conceptions of soil were reflected in the classification and mapping. Under the one conception, soil was considered a medium for plant growth. Under the other, soil was considered the uppermost part of the mantle of weathered rock. No indication of the conception of soil as an organized natural body, foreshadowed in earlier publications of Shaler and Hilgard and outlined more fully by Coffey after the soil survey program had begun, is to be found in the early reports of field operations. It is also true, however, that the observations being made and the information gathered during the first years of the program clearly affected the approach and outlook of a number of scientists. The conception of soil held by Coffey ( 1912b) seems to have been an outgrowth of his experience in the study and mapping of soils in various parts of the United States. More than likely, other individuals also began to think about soils in ways that raised questions about prevailing conceptions. Soil survey field methods are described in the first Report of Field Operations, Division of Soils, U.S. Department of Agriculture (Whitney, 1900). The description shows that the conception of soil as a medium for plant growth was an important part of the framework for classification. The description follows: I n the field classification of soils all features are taken in consideration which appear in any way to influence the relation of soils to crops. The classification is based mainly upon the physical properties and condition of the soil as determined by the soil expert, but it is not based solely on this. Any chemical feature, such as deposits of marl, of highly calcareous soils, or of highly colored soils, is considered, as well as the character of the native vegetation and the condition of crops. The topography of the country is often a very safe guide in outlining the boundaries of soil conditions. For this reason is is very advisable that there should be reliable maps of the different districts to base the soil work on and that these maps should show the important topographic features. In order to make the maps of the greatest possible local value it has been decided to publish the soil maps, so far as possible, on a scale of I inch to the mile. Local variations in the character of the soil of less than one-fourth of a mile in extent are generally ignored, unless this variation constitutes a very prominent feature, such as a strip of meadow land along a stream, or unless there are a number of small areas by which a certain character is given to the district. For example, if rocky areas occur, small in each case, but extending over large areas, they should be indicated in some way upon the soil map.

The first guide for soil survey field work in the United States was published in 1902 (Bureau of Soils, 1902). One paragraph on establishing soil

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types and a second describing one soil type tell their own story about the nature of the work in progress. These paragraphs follow: In the humid portions of the country the description of a soil type is to extend to a depth of 3 feet, and in the semiarid and arid regions to a depth of 6 feet. In speaking of a type the material to these depths is meant, not the surface soil alone. . . . Sharkey Clay. Yellow, waxy clay, 0 to 6 inches; yellow, waxy clay, 6 to 40 inches. Surface suncracks and resembles buckshot land. Forested area between stream courses in Yazoo River Delta, Mississippi. Subject to overflow. With diking and drainage would form valuable cotton soil. Very fertile.

Later editions of the guides or instructions to field parties were published by the Bureau of Soils in 1903, 1904, 1906, and 1914. These are historically interesting because they provide a record of the changes in progress as the soil survey program continued. Thus, for example, the first guide (Bureau of Soils, 1902) has paragraphs on preparation and submission of expense vouchers, selection of headquarters, suggestions for hiring teams of horses, and even arrangements for laundry. By 1906 (Bureau of Soils, 1906), however, the title of the guide had been changed to “Soil Survey Field Book” and instructions were restricted to the necessary operations in the field examination and mapping of soils. The second conception of soil held by the men directing and performing soil survey work in the early days of the program is given in the second edition of the guide for mapping (Bureau of Soils, 1903). The pertinent paragraph gives instructions on the classification of soils. Repeated in the 1906 and I9 14 editions of the guide, the paragraph reads as follows: When the Norfolk sand is being deposited the conditions somewhere in the area will undoubtedly be favorable to the deposition of gravel, of silt, of fine sand, or loam, and of clay, and wherever material of these characters is encountered, presumably coming from the same source and being deposited essentially at the same time, they should be given this distinctive name so as to show their relation to one another. Knowing as we do the process of soil formation, either from the disintegration of rocks in place or the transportation by wind or water, we should expect that materials from the same source would differ in their texture. The relationship of the derived soils would be shown by the use of a common name.

Whitney ( 1 892) had concluded from his own studies that texture of soil was of the first importance to plant growth. Consequently, when the soil survey program was started under his direction, texture was a major characteristic to be observed and recorded (Whitney, 1900). Soil texture was considered to be largely but not entirely an expression of particle size distribution. Texture was a characteristic of the soil itself, one of the first to be considered systematically in soil surveys.

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Texture was to be identified and described for what was later called “the soil section.” This was a vertical section to a depth of 3 feet in humid regions and 6 feet in arid regions. The earliest descriptions were made as though vertical differences in texture did not exist within the soil section. Later, as the program continued and more observations were made, the soil section was divided into three parts, viz. the surface soil or “soil,” the subsoil, and the substratum. The surface soil, commonly referred to as the ‘‘soil,’’ was the uppermost part and extended to a depth of about 7 inches. The subsoil was the part of the section between 7 and 20 inches. The substratum constituted the remainder of the section, to a depth either of 3 or 6 feet as the case may be. Descriptions of soil in the first reports of field operations provided information on the “soil” and subsoil, as a rule. The substratum may or may not have been mentioned. Fifteen years after the soil survey program began, most descriptions provided information on all three parts of the soil section. Descriptions on the basis of these three parts remained in vogue for about forty-five years, overlapping by fifteen years introduction of the concept of the soil profile in the United States. A set of similar bodies of soil delineated on the maps in the first years of the survey program was called a soil type (Whitney, 1901). Each soil type was described briefly, and some information was provided on its usefulness. The nature of these early descriptions is indicated by that of Sharkey clay, quoted earlier in this section. The early descriptions reflect the generally held conceptions of soil. Examination of the eady soil maps and of the soils within delineated bodies brings out several items of interest. The delineated bodies of soil were large, as a rule. Relatively few mapping units were needed in an area of 100 square miles. Because the delineated bodies were large, the characteristics common to the dominant soil within such bodies were few. One soil type recognized in the first year of field work consisted of rather wet soils formed in alluvial sediments. Ranges in texture, wetness, and horizon sequences among the component kinds of soil were large. Another soil type, mapped a few years later, consisted of soils formed from marl and having a thick dark surface layer. The set of soils mapped as types in survey areas in the early part of the century are comparable to fairly broad soil associations shown on present-day county maps (Simonson, 1952a,b). A change in the concept of the soil type followed the introduction of the soil series in 1903 (Simonson, 1952a). Soil series were to consist of groups of soil types which were alike in origin but differed in texture. Insofar as can be determined now, the textural differences between soil

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types in one series were thought to hold for the entire soil section, not only for the surface layer. Soils were considered to be alike in origin if they were derived from the same rocks or in sediments derived from the same source rocks and deposited at the same time. The bases for determining similarities are clearly stated in the instructions to field parties, though statements are implicit rather than explicit. The intended relationship between the soil series and the soil type during the first few years of their use is expressed in the following statement (Bureau of Soils, 1904): Much advance has been made in the past year in this matter of correlation and in using the names of soils to bring out their relations. Several of the original types have been merged into others, or have been given new names to bring them into a uniform series, where this could be done without danger of confusion. In doing this the object has been to establish certain series in the different physiographic divisions of the United States, and we are finding that there are a few general classes of soils that are in a way related. Due caution must be observed in this matter of correlation, but a greater latitude may be taken in correlating soils of widely separated areas than at first would appear possible, from the fact that the soil of each area is fully described in all its characteristic and special features. A soil, to be correlated with a type, must conform to it in certain broad, general features, but it may differ in some unessential details. . . . Every effort has been made to group the soils into series for the purpose of bringing out more clearly the relationship of the different types, and their relative agricultural value. The grouping has also been made with a view to reduce the number of local type names. If the field men acquaint themselves with the general characteristics of these series, they will find it a great aid in placing soils of new areas. Furthermore, if any type in a series is thoroughly understood, the remaining types can be called to mind without reference to the printed description, thereby reducing the number of type descriptions necessary to be carried in the head. To complete the Norfolk series, the Susquehanna gravel has been changed to Norfolk gravel, the Windsor sand to Norfolk coarse sand, the Windsor sandy loam, encountered for the first time in 1903, to the Norfolk coarse sandy loam, and the Sassafras loam to the Norfolk silt loam. This practically perfects the series. Several of the old local names have been dropped as the true relationship of these soils to the Norfolk series has become apparent. Similar work has been done with the other principal series, so that many of them now are practically complete, having a stony loam, gravel, gravelly loam, sand, find sand, sandy loam, fine sandy loam, loam, silt loam, clay loam, and clay, of related materials.

After the soil series category was introduced in 1903, it soon became the basic set of classes in efforts to classify the soils of large areas such as states. The series also formed the basic classes for the classification of soils in counties and other survey areas. The soil type, however, continued to be the basic entity in mapping for several decades.

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The ranges in soil characteristics allowed within series were gradually narrowed as the soil survey program continued (Simonson, 1964). The rate of change is illustrated by the reduction in the range of the Miami series between I903 and 191 1. Several types of Miami soils were mapped in New York and North Dakota in 1902, after the first types were mapped in Ohio in 1900. The several types mapped between 1900 and 1903 included soils now classified as Alfisols (Gray-Brown Podzolic soils), Mollisols (Chernozems and Brunizems), and Inceptisols (Sols Bruns Acides). By 1904, the second year in which the series category was in use, the Miami series had been correlated in a number of states within a triangle formed by lines connecting Mississippi, New York, and North Dakota. Seven years later, the Miami series had been restricted to Alfisols formed in glacial drift. The Mollisols were set apart as the Carrington series. These later soils, also formed in glacial drift, had thick rather than thin dark surface layers. The Memphis series had been defined for soils similar to those of the Miami series but formed in loess rather than drift. The Marshall series also had been set apart for soils like those of the Carrington series but formed in loess rather than drift (Bonsteel, 1911). All four series were restricted geographically to what was called the glacial and loessial province extending from Maine on the northeast to Mississippi on the south and to Montana on the northwest. Within a time span of seven years, a marked narrowing had been made in permissible ranges of characteristics within soil series. The changes seemed to have followed primarily from the observations made in the field work. These observations were centered as much on crop adaptations or usefulness of soils for plants as they were on soil characteristics. Regardless of the primary focus of the observations, however, progressively more attention was being given to soil characteristics and further information about the soils themselves was being accumulated. Parallel efforts were being made at the same time in studies of soil management and soil fertility. The narrowing of permissible ranges within soil series has continued since 191 I , as the number of series recognized in the United States will show. Approximately 550 series had been defined before the outbreak of World War I (Marbut et al., 1913). By 1930, the total for the country approached 1400 and by 1938 had grown to 2000 (Shaw and Baldwin, 1938). In 1951 the list of established and tentative series numbered approximately 5500. The total had grown to 8500 by 1967. The increasing number of series has been due to a pair of factors. One is the narrowing in permissible ranges in characteristics. The other has

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been the making of soil surveys in regions with little or no previous mapping. I n the early years of the soil survey program, increased numbers of series were due primarily to the extension of mapping into new regions. During the last twenty-five years, however, the growth in numbers of series has been due more to the narrowing of ranges in characteristics than to surveys in new areas. Thus, for example, the Sassafras series, one of the first recognized, originally included soils which were distributed among 30 series by 1943 (Lyford and Quakenbush, 1956). The number has increased further since I943 and now totals almost 50 series. During its entire history in the United States, the soil series has been a category of low rank, a set of parallel classes believed to have relatively narrow ranges in important properties (Simonson, 1964). Thus, every soil series was believed to consist of a set of closely similar bodies of soil. The crossing of a divide in the development and diffusion of the idea that soils were organized natural bodies can be marked in the United States by two papei-s presented by Marbut (1921, 1922). The approach outlined in these papers had been foreshadowed by Coffey (1912b) a decade earlier and in part by Shaler ( 1 891) and Hilgard ( 1 892) three decades earlier. The conception promoted by Marbut in the 1920’s was more fully developed than those offered previously. It seems to have been a synthesis of ideas drawn from several sources. One source must have been the publications of his predecessors. Another source was certainly the book by Glinka ( 1 914). A third source was the information that had been collected through the soil survey program. A fourth source consisted of the field studies made personally by Marbut in this and other countries. As important as were the papers by Marbut (1921, 1922), equally important was the receptiveness of his audience to new ideas. Similar though less complete conceptions of soil had been published in the United States before 1921. Those conceptions got little or no hearing even a decade earlier. Perhaps enough effort had gone into the study of soil in “its natural habitat” by 1920 to make soil scientists as a group appreciate the shortcomings of existing conceptions and thus be ready to consider a new one. The extent of the departure from earlier conceptions of soils widely held by soil scientists is illustrated in the following quotation (Marbut, I92 1): What is a soil survey? It will be sufficient for our purpose to define a soil survey as an institution devoted to the study of the soil in its natural habitat. I t is concerned primarily with the determination of soil characteristics as they developed by soilmaking processes, including the work of man, the study of the significance of each,

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the isolation of the several groups of characteristics that should constitute a soil individual, the fixing of these groups by proper nomenclature, and the determination of the area and distribution of each soil unit. The existence of soil units is a prerequisite to the mapping of soils in detail. It is self-evident that the distribution and area of an object cannot he determined until that object exists in the mind of the surveyor and in nature. . .. That a soil unit must exist in the mind of the soil surveyor, formulated on the basis of tangible criteria, before such unit can be identified on the ground and its distribution determined, cannot he questioned. No recognized formulation of such unit on the basis of tangible soil characteristics had been made prior to the inauguration of soil survey work in this country. When the soil surveyor first went to the field he was confronted with the fact that he was instructed to identify objects for which he had no sufficient description. . . . The soil surveyor has had, therefore, two lines of work to perform. On him has been placed the duty of determining, through observation extended over a wide area of country and therefore over a great many different kinds of soils, what soil characteristics are. What are the features of soils and what are their relative importance? . . . In short, he has had to define the soil unit in terms of soil characteristics. He has had to create the soil unit.

Marbut stressed the soil profile and its importance in the definition of soil units and in their classification. For example, he wrote (Marbut, 1924): “The soil surveyor established the soil units or the soil individuals on the ground by personal observation. The soil units are delineated by the field men on the basis of the soil profile.” Soil types and soil series were to be defined in terms of the soil profile. Furthermore, the A, B, and C notations for horizons within profiles were introduced. Studies of the relationships between horizons in profiles also followed. These several steps required changes in the conceptions of soils that were held generally in the United States at the time. The idea that the horizons within a profile were genetically related and had evolved together implied that soil was an organized natural body. Although the spread of this conception was slow in the United States, as it had been in Russia, it was gradually accepted. Examination of papers published in scientific journals for successive decades beginning with that of 1920 to 1930 show that the conception of soil as an organized natural body was more and more widely used as the years passed. Conceptions held earlier were not necessarily replaced. Reference might be made by the same mind to one conception in some situations and to another conception in others. A degree of amalgamation of the old and new also took place as the new conception gained acceptance. During the First International Congress of Soil Science, Marbut (1927) discussed three steps in soil unit definition in the soil survey program in the United States. First was the stage in which geology formed the basis for mapping soils. Second was the stage in which soils were

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defined on the basis of climatic forces, prompted by the findings in Russia. Third was the stage of defining soils on the basis of their own properties. Part of the discussion by Marbut (1 927) of “the first stage of soil unit definition” follows: Geologists, as the science developed during the 19th century, had assumed that, since soils lie on the earth’s surface and are mainly of mineral particles, therefore soil units are equivalent to geological units and the relationship5 of soils in widely separated areas are the same as the relationships of the geological formations in those areas. . . . The soil surveyor accepted at first the geological definition of soil because he had no other basis. He had merely begun his work and had accumulated no considerable amount of soil knowledge. . . . This was the scientific inheritance transmitted by the scientific world to the soil surveyor. He accepted it as fundamentally correct. He had no reason for doing otherwise. Neither he nor any one else had examined the evidence, for no evidence was yet in existence. Had the soil surveyor continued indefinitely to accept this result without question and had he confined himself to the engineering phase of his work solely, he would have been saved much figurative sweating of blood. His inquiries, however, got him into trouble. Although he was theoretically and practically, . . . supposed not to be concerned with soil facts. . . and is still supposed to let them alone, he found it impossible to do so and has become, to an increasing degree, involved in the matter. He has done this because his necessary contact with the soil caused him to accumulate soil knowledge and to compare this with the assumed knowledge on which his determination of unit relationships was based. He gradually realized that he was placed in contact with a series of bodies about whose features as bodies nothing was known. He had been placed unwittingly in a virgin field. He realized that he was t h e j r s t real soil investigator, t h e j r s t srudenr of soil as soil.

In retrospect, the discussion by Marbut ( 1 927) of stages in soil unit definition seems a record of changes in the conception of soil rather than the evolution of soil unit definitions. The latter are governed by the conception of soil in the minds of the men constructing the definitions. Consequently, the conception of soil is of the first importance to soil unit definitions. During the third and part of the fourth decades of the twentieth century it was generally thought that all kinds of soil named as a single soil type were uniform in all important characteristics of the profile. Each body of soil represented as one delineation on a detailed map was believed to be homogeneous in all important features including texture of the surface layer (Marbut, 1928b, 1935). This view is expressed in a number of statements published between 1928 and 1935 as illustrated in this one by Marbut ( 1935): . . . Since the series name is a group name, including soils which differ in texture of the surface layer it is evident that the designation of a soil which is uniform

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throughout in all characteristics, including that of the texture of the surface horizon, must be a double designation, one part covering series characteristics, the other covering the texture of the surface horizon. For example, the expression Norfolk sandy loam, when applied to soil, connotes, in the first or geographic part of the expression, all the characteristics of that soil except the single characteristic of the texture of the surface horizon, and is applied to a group of soils, the members of which differ from one another in the texture of the surface horizon but are alike in all other features. The words sandy loam are descriptive of the texture of the surface horizon. The two terms, therefore, designate a soil unit. All areas of Norfolk sandy loam, wherever they may occur, are uniform in all respects, and such a soil unit is called, in the nomenclature of the Bureau of Chemistry and Soils, a soil type. . . . Very few if any, soil series contain individuals representing all the possible textures of the surface horizon. Most soil series in the United States, however, include more than one texture individual. In most cases two or three texture individuals will include by far the greater part of the areas of any given series.

As the mapping and associated study of soils continued, men slowly came to realize that the soil bodies represented as delineations on their maps were not uniform in all important features. More than one kind of profile could be found within a delineated soil body. Moreover, it was also gradually recognized that the mapping of soils in any practicable scale must allow some “mavericks” within delineated bodies. The great bulk of a delineated body might fit within the range of one soil type, but some part of that body would not. Two or more soil types were therefore represented by the component kinds of soil in every delineated body. Even though the major part of a delineated body did fit within the range of a single type, not all of the body would. Minor kinds of soils within delineated bodies were called mapping inclusions or simply inclusions so long as their proportiops were small. Recognizing inclusions as characteristic of delineated bodies of soil, however, is a long step in recognizing some smaller entity as basic in soil classification and mapping. Recognizing inclusions also tends to focus attention more directly on the three-dimensional nature of basic soil entities and takes some attention away from the soil profile. Several consequences have followed. First, the belief that the soil type could, on the one hand, be a set of mapped soil bodies and, on the other hand, serve as a category in a general classification scheme became untenable. Second, it became necessary to give more weight to the occurrence of basic soil entities as segments of the rind of the earth, as creatures having length, breadth, and depth. Study of the soil profile was not enough, though it remained important. Third, it became desirable to search or create a soil unit, to use Marbut’s phrase, smaller than the mapped bodies shown on detailed maps to serve as a basic entity for

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relating those mapped bodies to classes at some level in a general system. These consequences have all contributed to continuing evolution in the conception of soil. The principal current conception in the United States will be discussed in the next section of the article. The whole of this section is an attempt to trace changes in the conceptions of soil and the efforts to find or create a soil unit in the United States. During the first years of the soil survey program soils were conceived to be mediums for plant growth and surficial mantles of weathered rock. These conceptions have gradually given way to the conception of soils as organized natural bodies. Changes have been slow and evolutionary, fueled in part by the studies in Russia and in other countries. The evolution has also been fueled by efforts of men in the soil survey program to construct a working model of soil on the basis of facts gathered about soils of the country. As pointed out by Cline ( 1 961) the working model or conception of soil must accommodate facts that are known and prevailing theories of soil genesis. II. Basic Soil Entities

The nature of basic soil entities is discussed in this section. Their functions in the mapping and classification of soils are also considered. Relationships of the basic soil entities to prevailing conceptions of soil are explored because of the important reciprocal effects of the conceptions or working models of soil and the understanding of basic entities in the minds of scientists. Before possible basic entities are considered, however, a current conception of soil is outlined. That conception in turn underlies the concepts of basic soil entities. Several years ago in writing about the changing model of soil, Cline ( 1 96 1) reviewed several conceptions of soil that had been or were held in the United States. He gave primary attention to the conception outlined by Marbut (1 935) and to that in the monograph on the 7th Approximation (Soil Survey Staff, 1960). These two conceptions are separated by twenty-five years. The earlier conception stressed the place of soil as the outer layer of the earth’s crust whereas the latter stressed soil as a collection of natural bodies. Both conceptions are within a concept of soil as something at the land surface, but the two conceptions lead to obvious differences in the way of thinking about soils as objects for study. The importance of the concept of the objects under study to processes of thought is stated by Cline ( 196 1 ) as follows: Within the framework of its accumulated knowledge, every science develops a mental image of the thing with which it is concerned. This model of a science is the

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organized aggregate of accumulated facts, and laws and theories based on those facts; it is a mental picture of that which is known viewed in organized perspective through verified quantitative relationships, which we call laws, with varying degrees of distortion by virtue of theories that attempt to explain the observed relationships. The picture is not the same to all who work in the science, for it is composed of knowledge and the extensions of theory from knowledge into the unknown, and different men know, or think they know, different things.

Several conceptions of soil, each reflecting the state of knowledge in its day, have been held in the past. These conceptions were reviewed earlier in the article. It is evident from the history of the several conceptions that more than one is in use at any given time. The existence and application of several conceptions or working models is not peculiar to soil science; one need look back only a short way for the wave and corpuscular theories of the propagation of light. Changes in the working model or in the conception of the objects under study follow as knowledge itself changes, for whatever reason. Since soil first became a subject for deliberate study, the single most profound change in the conception or working model was the introduction of the idea that soil was an independent natural body with genetic horizons (Cline, 1961). Steps in the early development and spread of this conception in Russia and the United States have been discussed in previous sections. By comparison with the recognition of soils as organized natural bodies further changes are much less far-reaching though they also have importance. During the last three decades the principal modification in the conception of soils as organized natural bodies has been the growing recognition that the basic entities are three-dimensional. They consist of volumes or polyhedrons of some kind. It is true that soils form a continuum over the land surface with few sharp breaks, but soils differ from place to place (Simonson, 1957). Because of the differences from place to place the continuum can be considered a mosaic or patchwork consisting of many polyhedrons (van Wambeke, 1966). Soil mantles the land surface of the earth generally. This mantle may be looked upon as a collection of organized natural bodies that contain living matter and either have horizons or are subject to horizon differentiation. The morphology of these bodies reflects many paths of horizon differentiation. Horizonation also has a wide range, taking all segments of the mosaic as a group. Some kinds of soils have few and faint horizons, whereas others have prominent horizons. All soils share a number of characteristics. A11 are three-phase systems composed of the same major constituents. All are open systems to which substances may be added and from which substances may be lost. All

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have profiles, some with more distinct horizons than others. Local kinds of soils merge with one another as a general rule; gradations are the normal mode of change. Spatially, basic soil entities must be three-dimensional, i.e., have length, breadth, and depth. Each stands in relation to the earth much as a small piece of rind stands in relation to the whole of an orange. Conceptually, Crowther (1 953) considered soils multidimensional, meaning that many characteristics must be specified to define soils. To restate the argument, soils are as complex as they are commonplace. A. NATURE A N D DIMENSIONS OF BASICSOIL ENTITIES The major difficulty in defining soil individuals or basic entities follows from the existence of soil as a continuum. Discrete individuals comparable to those of plants and animals do not exist. The problems in defining some small volume of soil as a basic entity is thus similar to those faced in defining some basic unit for classification of bodies of rock and rock formations. As part of a discussion of basic principles of soil classification, Cline ( 1 949) offered the following definitions: “The smallest natural body that can be defined as a thing complete in itself is an individual. All the individuals of a natural phenomenon, collectively, are a population.” The definition does not provide limits for a soil individual or basic entity. What the smallest natural body of soil can most appropriately be must still be spelled out in some way. Various ways have been tried (Simonson and Gardner, 1960). The term “pedon” has been proposed as a collective noun for small basic soil entities (Soil Survey Staff, 1960; Simonson and Gardner, 1960). As a generic term, pedon would thus parallel the word “tree” as a collective noun covering oaks, pines, elms, and other kinds. The pedon is in a sense an abstraction or soil unit that is a creation of the mind, to use the terminology of Marbut ( I 92 1). According to the terminology of Knox ( I965), the pedon is an artificial rather than natural individual. Moreover, Knox (1 965) questions the desirability of trying to define either natural or artificial soil individuals. He argues that natural individuals do not exist in a universe that is not particulate. He also believes that classification and mapping of soils can proceed without defining some volume of soil as an individual. Recognition of some physical entity, some volume of soil as basic in mapping and classification does have certain advantages, at least for most people. It is imperative that mapped soil bodies be related to classes in a general system to permit the transfer of knowledge about the nature and

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behavior of soils from one place to another with reasonable assurance that the transfer will be valid. Relating the mapped bodies to classes at some level in a system will be easier for most people if they can think in terms of some physical entity rather than exclusively in terms of an abstraction. This ia a prime argument for attempting to define some small volume of soil as a basic entity. Each pedon consists of a small volume of soil that is part of the continuum mantling the land surface. Each is a tiny segment of the rind of the earth, an irregularly shaped solid or polyhedron. Each pedon begins at the surface and extends downward to include the full set of horizons or to some arbitrary depth corresponding approximately to the vertical dimensions of a set of horizons. The upper boundary is clear enough, but the lower boundary remains vague, as a rule. The perimeter is gradational from one pedon to its neighbors. Distinguishing pedons is thus like setting apart different kinds of climate. Sharp changes in climate are uncommon though differences exist over the face of the earth. Moreover, some of the differences are substantial. Similar statements apply to the soil mantle. A concept broader than, but parallel in some ways, to that of the pedon was proposed a few years earlier by Jenny ( 1 958). He suggested the term “tessera,” which was to be a small three-dimensional element consisting of soil plus vegetation. Lateral dimensions might be of the order of 1 square meter or as small at 8 x 8 inches, with the dimensions to be determined by the purpose of each study. The vertical dimension or thickness of the tessera was to be the combined height of the vegetation and depth of the soil. The proposed tessera includes not only the soil, but also the vegetation growing on that soil. Concepts of the tessera and pedon are thus related but not identical since the latter does not include vegetation. Certain requirements must be met by any definition of the pedon. It must be large enough to be observable, to be sampled, and to exhibit a full set of horizons. At the same time it should be as small as possible while meeting the above requirements. A volume of soil or a polyhedron could be very small and still be observable. With care a person could examine a vertical core no more than a few centimeters in diameter. A volume of soil large enough to be sampled with an auger can be examined, but full observation of the relationships between horizons is not possible. Consequently, some larger volume is required. Setting the minimum dimensions for the pedon can be attempted by analogy to the defining of unit cells in crystallography. The unit cell can be defined in crystals of minerals such as kaolinite and mica, though the cell is an abstraction and does not exist alone. The unit cell can be

CONCEPT OF SOIL

37

recognized by the succession and spatial arrangement of ions in the lattice. Each unit cell is like the next. The analogy between the unit cell in a crystal and the pedon in a soil body can readily be carried too far. Crystals have more regularity in structure than does the soil mantle in its morphology. A pedon must be a large enough volume of soil to include a full set of horizons and permit observation of the boundaries between them. For example, a single pedon would have to consist of a volume of soil that includes the whole set of features associated with a coarse prism in a fragipan. A single pedon would consist of a volume of soil expending from the middle of the coarse prism to the middles of the neighboring prisms. Thus, the study of horizons and their interrelationships in place largely govern possible minimum and maximum dimensions of the pedon. Small differences in the nature of horizons must be permitted within a pedon. For example, a krotovina passing through a horizon would not be the basis for identifying a second pedon. Cyclical variations are also permitted within pedons because they are essential parts of relationships between horizons in some kinds of soils. These cyclical variations are also keys to the genesis of some soils. For example, the downward tonguing of both A and B horizons is a normal feature of Spodosols (Podzols) formed in sandy materials with good drainage. Study of requirements that must be met demonstrate that the best definition now possible for the pedon is in terms of lateral dimensions. The pedon must be large enough to be sampled. It should be large enough to show the relationships between horizons. It should also be as small as practicable in order to minimize variability within the unit. The best definition of the pedon that can be offered now is not complete but represents a step toward full definition. A pedon consists of a small volume of soil which includes the full solum and the upper part of the unconsolidated parent material (or a volume of comparable size if horizons are faint), is usually less than 2 meters in depth, and has a lateral cross section that is roughly circular or hexagonal in shape and between 1 and 10 square meters in size. The smallest of these lateral dimensions is proposed for use in most soils. The larger dimensions, u p to the maximum of 10 square meters, are proposed for use where needed to cover the full amplitude of one cycle in the arrangement of horizons. Where the nature and arrangement of horizons are cyclical, the full cycle must occur within a lateral cross section that is roughly circular and is 10 square meters in size if the volume of soil under observation is to be considered one pedon. Otherwise, the volume of soil under observation is to be considered as two or more pedons (Sirnonson and Gardner, 1960).

A single pedon can usually be defined by preparing a description of a

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soil profile plus statements of the ranges in characteristics of horizons. All ranges are limited in most instances. If cyclical variations occur within a pedon, the definition requires a statement of the nature and amplitude of those variations. To return to the example of the A2 and Bz horizons of Spodosols (Podzols), the definition of a pedon would have to indicate the distribution of the horizons, for example, the frequency of tongues and the dimensions of those tongues. Possible difficulties in applying the suggested lateral dimensions for pedons in soils with cyclical variations have been suggested by Arnold (1964). He analyzed the use of the half-cycle width as the basis, with the sine curve as his model. He concluded that about 80 percent of a cycle would have to be included in each pedon if it were to cover 80 percent of the vertical horizon variability for horizons that were cyclic at linear intervals ranging from 1.3 to 4.3 meters. Another part of the effort to define basic soil entities is the proposal of polypedon as a term for a larger volume of soil than that of the pedon (Simonson, 1962). The term polypedon was proposed to replace “soil individual” as that was used in the monograph outlining the 7th Approximation (Soil Survey Staff, 1960). A polypedon consists of a group of contiguous pedons that are within the limits of one soil series (Simonson, 1962; Johnson, 1963). In other words, the set of pedons must fit within the range of one series and occur in a contiguous group to form a polypedon. For the most part, one polypedon is the most extensive in each delineated body of soil shown on a detailed map. Such bodies normally include parts of other polypedons. These parts of the second, third, and fourth polypedons constitute mapping inclusions. The relationships between a polypedon and its constituent pedons in a single body of soil are illustrated in Fig. 1. In the diagram, part of the soil body is split into pedons. All but a few of these pedons are of the same kind and represent a single polypedon. A part of one pedon, however, shown at the left edge of the diagram, is dark-colored to the bottom. That pedon and several others lack the A2 horizon characteristic of pedons constituting the dominant polypedon. The one darkened pedon along the edge plus others along the small drainageway extending into the diagram from the left side represent a mapping inclusion. They are part of a second polypedon differing from the dominant one. As soils occur naturally, parts of several polypedons are commonly inclusions within bodies of soil that can be delineated on detailed maps. To keep the illustration simple, however, a single mapping inclusion has been shown in the diagram.

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FIG. I . Schematic diagram of a body of soil that would be shown as one delineation on a detailed soil map and named as one phase of a soil series. Part of the body is split into pedons, a few of which represent a mapping inclusion or part of a polypedon different from the dominant one within the body as a whole. (Sketch by Walter M. Simonson.)

B. RELATINGTHE MAPPINGA N D

CLASSIFICATION O F SOILS

Some classification of soils is applied deliberately or otherwise by everyone who uses them. The classification may be simple or sketchy, taking few features into account. For example, it was believed by homesteaders filing their claims in north central North Dakota just prior to 1900 that land supporting clumps of silverbrush (Eleagnus argentea) was better for growing wheat than land without patches of the shrub. The single criterion used in that classification of soils was a kind of vegetation, the presence of which on a given spot could have been due to any one of several factors. More sophisticated classifications have since been developed, and these have greater value in appraising the usefulness of the soils. The combination of maps showing distribution of kinds of soils and a classification of them is most effective. The bodies of soil shown as delineations on maps are related to a classification system, usually to classes of low rank, through the process of correlation (Simonson, 1963). Existing knowledge about the nature, origin, and behavior of soils can then be brought to bear more effectively on specific tracts of land.

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Existing knowledge may be applied to further the understanding of soil genesis. More commonly, existing knowledge is applied to achieve better use of soil resources in the satisfaction of human wants. The ultimate objectives in the process of relating mapped soil bodies to a classification system are the same as those of soil science generally, i.e., to predict what will follow from the use of soils in different ways and to predict the long time effects of such’use on the soils. The making of predictions and the transfer of results of experience and research from soils of one locality to those of another are greatly facilitated by adequate classification and reliable maps. The two must be tied together to be most useful. The basic soil entities discussed in the immediately preceding section are intended to simplify the task of relating mapped bodies of soils to classes in a general system. Each class of low categoric rank in a classification system, e.g., the soil series in the United States, can be looked upon as a group of similar pedons or polypedons. All pedons belonging to one class, i.e., one soil series, are required to be alike within narrow limits in the sequence, arrangement, and character of definitive horizons except the surface layer. If horizons are few or faint, the uniformity is required in a fixed portion of the soil between depths of I0 and 40 inches (25 and 100 cm.). To state these requirements in another way, the ranges in thickness, color, texture, consistence, and the like, of individual horizons must be small or they must be cyclical within a limited lateral cross section. The soil series category is roughly comparable to that of the species in the classification of plants, animals, and minerals. The word “roughly” is used because analogies can only be illustrative for sets of natural objects such as a collection of pedons and a collection of orthoclase grains. Even so, it may be helpful in grasping the concept of a series to think of each one as a group of closely similar pedons paralleling the group of oak trees known as Quercus alba or the group of mineral grains known as orthoclase. The pedons or polypedons with the specified definitive features are members of one class, a single series, regardless of their geographic occurrence. The basis for grouping the pedons into one class is the degree of similarity among them rather than how or where they occur. Their occurrence is independent of their classification into a given series. Pedons of one series need not occur in the same delineated soil body or even in the same county. In contrast to classes such as the soil series, a mapping entity or single body of soil shown as a delineation on a map must consist of pedons or polypedons and parts of polypedons that occur together. Each delineated

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body is a bundle of contiguous pedons. The contiguous occurrence is an essential feature of mapping entities. A boundary must be drawn around each entity in order to record on a map the position of that entity on the face of the earth. Every mapping unit consists of a number of these individual entities, each of which is a segment of the soil continuum. Pedons and polypedons cannot be moved about to facilitate their classification or mapping. It is not possible, for example, to take all pedons of the Sassafras series and arrange them in a clump somewhere so that a single boundary could be placed around the whole lot. In practice, mapping entities must be defined so as to accommodate the existing distribution of pedons and polypedons in the soil continuum. In detailed surveys, mapping entities are defined insofar as practicable to consist of pedons or polypedons representing a single series. Rarely does a delineated soil body consist of one polypedon. This is illustrated by diagram in Fig. 1. Virtually all delineated soil bodies or mapping entities include pedons representing several series. Most mapping units in detailed surveys consist of delineated soil bodies in which the number of pedons of a single series, i.e., the extent of one kind of polypedon, is greater than that of any other kind. Conventions have been developed to permit the naming of mapping units -each set of soil bodies delineated on field sheets and identified by the same symbol - to show their relationships to taxonomic classes, primarily the soil series. Thus, a mapping unit recognized in a detailed survey might be named as a phase of the Palouse series. According to present conventions for naming mapping units, this means that polypedons of the Palouse series form major proportions of all the delineated bodies and that included kinds of soils are either closely similar or form small proportions of the whole. Some pedons or parts of polypedons classifiable in other series can be expected in all delineated bodies named as phases of the Palouse series. If the set of soil bodies comprising a mapping unit consists of appreciably differing pedons or polypedons and these occur in large enough proportions, the names of two or more series are used in the naming of the unit. Multiple component units may be named as complexes, undifferentiated groups, or soil associations, depending on the pattern of occurrence of the component kinds of soil. Conventions for nomenclature are outlined in the Soil Survey Manual (Soil Survey Staff, 195 1) and are not repeated here. Summarizing briefly, a taxonomic class of low categoric rank consists of a set of pedons or polypedons considered together because of their similarities. The grouping is independent of the geographic occurrence of

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the basic entities themselves. A mapping entity, on the other hand, consists of a bundle of contiguous pedons or the whole of one plus parts of several polypedons in place. Classification is a mental operation exclusively and the pedons can then be considered apart from their mode of occurrence. Mapping, however, must accommodate itself to the distribution of pedons in the soil mantle. The pedon promises to be useful in two ways. It will be of value in relating bodies of soil represented as delineations on maps to the classes of soil in a general system. Pedons will also serve as the entities studied in field observations of soil profiles and in sampling for laboratory investigations. The polypedon will be useful as a larger volume of soil becomes important. In the production of corn or in the building of a road, the results must be related to a larger volume of soil than that of a pedon. Performance of soils when used for the growing of plants, in the construction of highways, or as foundations for buildings can thus be related to polypedons much better than to pedons. Ill. Epilogue

As emphasized earlier, conceptions of soil disappear slowly and persist for a long time, even though they may be largely superseded. A11 conceptions that have been held during historical times persist to some extent now. Only one of the several discussed in this article has largely faded away, but it has not disappeared completely. Each of the other conceptions that have been discussed is held as a primary one by a share of the present population of soil scientists. The idea that fire, water, earth, and air were the basic components of all things, introduced by Empedocles about 400 B.c., is no longer seriously held, The idea did persist, however, for many centuries. Echoes of the original idea can be found in one of the present day nursery rhymes for children. The conceptions of soils as a medium for plant growth, as the surficial mantle of weathered rock, and as organized natural bodies are all in current use. In part the different conceptions are held by different people because their interests lead them to focus on different characteristics and relationships of soils. Certain characteristics and certain relationships to other objects assume major importance in the study or use of soils for different purposes. The features of consequence in the growing of cotton are not the same as those important to the design of a residential subdivision. Men interested in growing cotton will thus look at soils from a

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43

different point of view than men designing a residential subdivision. In part the holding of different conceptions of soils also reflect original differences in the education or training of men. Things once learned are are not readily dropped or displaced, as observed by John Dewey (1958). Reasons for the long history and persistence of the conception of soil as a medium for plant growth are not far to seek. The primary interest of mankind in soils rests on their capacity to support plants that provide food and fiber. This interest is common to all humanity. Production of food and fiber retains first place even in the highly industralized nations of the world, though important shifts in usefulness of soil resources have occurred during the last half century (Simonson, 1966). It is also easy to see why the conception of soils as a surficial mantle of rocks and weathered rock persists. The great bulk of most soils by weight consists of rock fragments and mineral grains. Furthermore, the accumulation of parent materials in which soils form is due to rock weathering. The weathering of rock fragments and mineral grains does not stop once evident horizons have been formed but may continue long after horizonation is distinct. Thus, the weathering of rock is involved in the formation of all soils, though weathering alone does not result in the formation of soil. The conception of soils as organized natural bodies, last to be born of the three prevalent ones, has drawn some of its substance from the two older conceptions. Thinking of soils as organized natural bodies implies that their history is of importance to their present nature. How the parts of these bodies are arranged and related, the sequence and relationships between horizons, and their degree of expression reflect the entire history of each pedon. Part of that history is the weathering of rock and the resulting character of those materials. The duration and combinations of processes in horizon differentiation also strongly affect the present nature of pedons. Thus, some characteristics of soils are related to source rock and its weathering and some are not. Some characteristics are due to the effects of living organisms in the addition of organic matter, in the transfer of substances within the system, and in the transformation of substances. Thus, the arguments offered by Dokuchaiev (1948) little less than a century ago that soils are formed through complex interactions among a number of factors remain valid. The conception of soils as organized natural bodies has the greater promise for expanding current understanding of soil genesis -of how soils were formed -and thus acquired their present character. The recognition of soils as organized natural bodies promises to be more helpful than any other conception in dealing with problems that will

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have to be faced as society becomes progressively more dependent on science and technology. The development of powerful tractors and other large machines plus the rapid accumulation of capital is encouraging profound modification of soils in some localities in the highly industrialized nations. The trend in recent years can be expected to continue. More drastic modifications of more soils can be anticipated in the future. The cost of these modifications, as for example, the ripping of duripans (hardpans) in some soils in the central valley of California, is high. The costs of normal operations in using soils for farming, forestry, highway construction, and the like are rising. Adjustment of the operations to kinds of soils and their distribution patterns will assume greater significance in the future because of the need to keep costs down and to minimize failures. Soil resources in the industrialized nations are being used more and more in the construction of highways, as foundations for homes, for the disposal of sewage, and for parks and playgrounds. These uses are expanding as population expands. The uses of soils not directly related to the production of crops, pasture, and trees will require attention to soil characteristics of little consequence in the past. Predictions of what will follow from the use of soils must be expanded from those of crop yields under specified management and of forest growth in given time spans to the probable results when pedons and polypedons are reworked in various ways. The conception of soils as organized natural bodies with certain characteristics arranged systematically should provide better bases for predictions than can be obtained otherwise. The working model of soil must serve to organize existing fact and theory so that the whole can be mobilized and applied, both in the production of plants and in the use of soil resources for other purposes.

REFERENCES Afanasiev, J . N . 1927. Russ. Ped. Invest. N o . 5 . Arnold, R. W. 1964. Soil Sci. SOC.Am. Proc. 28, 801-804. Barber, B. 1961. Science 134, 596-602. Bartholomew, W. V . , and Kirkham, D. 1960. Trans. 7th Intern. Cong. SoilSci. 2.47 1-477. Black, C. A. 1968. “Soil-Plant Relationships,” 2nd ed. Wiley, New York. Bonsteel, J. A . 191 1 . U S . Dept,Agr. Bur. Soils Cir. 32. Braidwood, R. J . 196 I . Chicago N a t . Hist. Museum Popular Ser. Anthropol. N o . 37. Braidwood, R . J., and Howe, B. 1962. I n “Courses Toward Urban Life” (R. J . Braidwood and G . R. Willey, eds.), pp. 132-146. Viking Fund Publ. Anthropol. N o . 32. Brehaut, E. 1933. “Cato the Censor on Farming.” Columbia Univ. Press, N e w York. Brown, P. E. 1936. Iowa Agr. Expt. Sta. Spec. Rept. 3.

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Bureau of Soils 1902. “Instructions to Field Parties and Descriptions of Soil Types- Field Season, 1902.” U.S. Dept. Agr., Washington, D.C. Bureau of Soils 1903. “Instructions to Field Parties and Descriptions of Soil Types.” U.S. Dept. Agr., Washington, D.C. Bureau of Soils 1904. “Instructions to Field Parties and Descriptions of Soil Types-Field Season, 1904.” U.S. Dept. Agr., Washington, D.C. Bureau of Soils 1906. “Soil Survey Field Book, Field Season, 1906.” U.S. Dept. Agr., Washington, D.C. Butler, B. E. 1958. J . Australian lnst. Agr. Sci. 24, 14-20. Cain, A. J. 1958. Proc. Linnean SOC. London 169, 144-163. Cain, A. J. 1962. Symp. SOC. Gen. Microbiol. 12, 1-13. Clark, J . G . D. 1952. “Prehistoric Europe: The Economic Basis.” Methuen, London. Cline, M. G . 1949.Soil Sci. 67, 8 1-92, Cline, M. G. 1961. Soil Sci. SOC. A m . Proc. 25, 442-446. Coe, M. D., and Flannery, K. V. 1964. Science 143, 650-654. Coffey, G . N . 1912a. Proc. Am. Soc. Agron. 3, 1 15-129. Coffey, G . N. 19 12b. U S . Depr. Agr. Bur. Soils Bull. N o . 85. Columella, Ca. 60 A.D. “Res Rustica (On Agriculture)” (Transl. from Latin by H . B. Ash). Harvard Univ. Press, Cambridge, Massashusetts, 194 1. Conant, J . B. 195 I . “On Understanding Science.” New Am. Library of World Lit., New York. Crowther, E. M. 1953. J . Soil Sci. 4, 107-122. Davy, H. 18 13. “Elements of Agricultural Chemistry.” Longmans, Hurst, Rees, Orme, & Brown, London. Dewey, J. 1958. “Experience and Nature,” p. 219. Dover, New York. Dokuchaiev, V . V. 1893. “The Russian Steppes and Study of the Soil in Russia, its Past and Present” (J. M. Crawford, ed. of Engl. Trans.). Dept. Agr. Ministry Crown Domains, St. Petersburg, Russia. Dokuchaiev, V. V. 1948. “Selected Works of V. V. Dokuchaiev, Vol. I- Russian Chernozem.” (Transl. from Russian by N. Kaner). Israel Program Sci. Transl., Jerusalem, 1967. Eaton, A. and Beck, T. R. 1820. “A Geological Survey of the County of Albany.” Agr. SOC.of Albany County, New York. Ehwald, E. 1962. Albrecht-Thaer-Arch. 6, 95- I 10. Ehwald, E. 1964. Albrecht-Thaer-Arch. 8,5-36. Evans, E. E. 1956. I n “Man’s Role in Changing the Face of the Earth” (W. A. Thomas, Jr., ed.), pp. 217-239. Univ. of Chicago Press, Chicago, Illinois. Fallou, F. A. 1862. “Pedologie oder Allgemeine und besondere Bodenkunde.” Schoenfeld, Dresden. Fippin, E. 0. 191 I . Proc. A m . Soc. Agron. 3, 76-89. Fireman, P. 1901a. Expr. Sta. Record 12, 704-712. Fireman, P. 1901b. Expt. Sta. Record 12, 807-818. Gilmour, J . S. L., and Walters, S. M. 1963. Vistas Botany 4, 1-22. Glinka, K. D. 1914. “Die Typen der Bodenbildung.” Geb. Borntraeger, Berlin. Glinka, K. D. 1927. “The Great Soil Groups of the World and their Development.” (Transl. by C. F. Marbut). Edwards, Ann Arbor, Michigan. Glinka, K. D. 1931. “Treatise on Soil Science,” 4th ed. (Transl. from Russian by A. Gourevitch). Israel Program Sci. Transl.. Jerusalem, 1966. Hilgard, E. W. 1860. “Report on the Geology and Agriculture of the State of Mississippi.” E. Barksdale, State Printer, Jackson, Mississippi.

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Hilgard, E. W. 1892. U S . Dept. Agr. Weather Bur. Bull. 3, 1-59. Hilgard, E. W. 1904. U S . Dept. Agr. O$. Expr. Sta. Bull. 143, 117-121. Hitchcock, E. W. 1838. “Report on a Re-examination of the Economical Geology of Massachusetts.” Dutton and Wentworth, State Printer, Boston, Massachusetts. Hough, B. K. 1957. “Basic Soils Engineering.” Ronald Press, New York. Hudson, H. 1844. The Prairie Farmer 4, 1-172. Hunt,C. B. 1967. “Physiography of the United States.” Freeman, San Francisco, California. Jarilow, A. 1913. Intern. Mitt. Bodenk. 3, 240-256. Jenny, H. 1958. Ecology 39,5-16. Johnson, W. M. 1963. Soil Sci. SOC.A m . Proc. 21, 212-215. Kellogg, C. E., and Ableiter, J. K. 1935. U S . Dept. Agr. Tech. Bull. 469. Knox, E. G . 1965. Soil Sci. SOC.A m . Proc. 29, 79-84. Leggett, R. F. 1953. Nature 171, 574. Liebig, J. 1843. “Chemistry in its Application to Agriculture and Physiology.” Campbell, Philadelphia, Pennsylvania, I I I pp. Lucretius, Ca. 60 B.C. “De Rerum Naturd (Of the Nature of Things)” (Engl. transl. by W. E. Leonard). Dutton, New York, 1921. Lyfwd, W. H., and Quakenbush, G. A. 1956. Soil Sci. SOC.A m . Proc. 20,397-399. MacNeish, R. S. 1964. Science 143, 531-537. Mander, A. E. 1947. “Logic for the Millions.” Phil. Libr., New York. Mangelsdorf, P. C. 1958. Science 128, 1313-1320. Marbut, C. F. 1921. SOC. Promotion Agr. Sci. 41, 116-142. Marbut, C. F. 1922. A m . Soil Surv. Workers Repr. 3, 24-32. Marbut, C. F. 1924. In “Etat de I’Etude et de Cartographie des Sols dans divers Pays d e I’Europe, Amerique du Nord, Afrique, et Asie” (G. Murgoci, ed.), pp. 2 15-225. Inst. Geol. Roumanie, Bucharest. Marbut, C. F. 1925. Ann. Assoc. A m . Geographers 15, 1-29. Marbut, C. F. 1927. Proc. 1st Iniern. Congr. Soil Sci. 4, 1-31. Marbut, C. F. 1928a. In “Soils of Cuba” (H. H. Bennett and R. V. Allison), pp. 341-354. Tropical Plant Res. Found., Washington, D.C. Marbut, C. F. 1928b. In “The Bureau of Chemistry and Soils: Its History, Activities, and Organization” (G. A. Weber), pp. 91-98. The Brookings Institution, Washington, D.C. Marbut, C. F. 1935. In “Atlas of American Agriculture” (0. E. Baker, ed.), Part Ill. U.S. Dept. Agr., Washington, D.C. Marbut, C . F., Bennett, H. H., Lapham, J . E., and Lapham, M. H. 1913. U.S. Dept. Agr. Bur. Soils Bull. N o . 96. Mitchell, J., Moss, H. C., and Clayton, J. S. 1950. Saskatchewan SoilSurv. Rept. N o . 13. Miiller, P. E. 1887. “Studien iiber die Naturlichen Humusformen.” Springer, Berlin. Muir, A. 1961.Adv.Agron. 13,1-56. Neuss, 0 . 1914. Intern. Mitt. Bodenk. 4,453-495. Olson, L.,and Eddy, H. 1943. Geograph. Rev. 33,100-109. Odhiambo, T. R. 1967. Science 158,876-88 1 . Ping-Hua Lee, M. 1921. Columbia Univ. Studies History, Economics, Public Law, 99, pp. 33-40. Polanyi, M. 1963. Science 141, 1010-1013. Raychaudhuri, S. P. 1953. Indian Council Agr. Res. Rev. Ser. N o . 4. Ruffin, E. 1832. “An Essay on Calcareous Manures.” Campbell, Petersburg, Virginia, Russell, E. W. 1961. “Soil Conditions and Plant Growth,” 9th ed. Wiley, New York. Semple, E. C. 1921. Ann. Assoc. A m . Geographers 11, 47-74.

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Shaler, N. S. 1877. “A General Account of the Commonwealth of Kentucky.” Kentucky Geol. Repts. Progr. [ N . S . ] 2.382-385. Shaler,N. S. 1891. U . S . GeoLSuwey 12th Ann. R e p / . Pt. l,pp. 219-345. Shaw, C. F., and Baldwin, M. 1938. “Bibliography of Soil Series.” Am. Soil Surv. Assoc., Washington, D.C. Sibertzev, N . M. 195 I . “Selected Works, Vol. I-Soil Science.” (Transl. from Russian by by N. Kaner). Israel Program Sci. Transl., Jerusalem, 1966. Simonson, R. W. 1952a. Soil Sci. 74, 249-257. Sirnonson, R. W. 1952b. Soil Sci. 74, 323-330. Simonson, R. W. 1957. Yearbook (US.Dept. Agr.) pp. 17-31. Simonson, R. W. 1962. Science 137, 1027-1034. Simonson. R . W. 1963. Soil Sci. 96. 23-30. Simonson, R. W. 1964. Trans. 8th Intern. Congr. Soil Sci. 5 , 17-22. Simonson, R. W. 1966. Agr. (Montreal) 23, 11-15, 21. Simonson, R. W., and Gardner, D. R. 1960. Trans. 7fh Infern. Congr. SoilSci. 4,127- I 3 1. Soil Survey Staff I95 1. US.Dept. Agr. Handbook 18. Soil Survey Staff 1960. “Soil Classification - A Comprehensive System. 7th Approximation.” U.S. Dept. Agr., Washington, D.C. Tulaikoff, N. M. 1908. J. Agr. Sci. 3, 80-85. Tull, J. 1733. “The Horse Hoing Husbandry.” Publ. by Author, London. Usher, A. P. 1923. Quart. J . Econ. 37, 295-411. Vanderford. C. F. 1897. Tennessee Agr. Expt. Sta. Bull. 10, pp. 31-139. van Warnbeke. A. 1966. Soiis Fertiiizers 29,507-5 1 0 . Varro, Ca. 35 B.C. “Rerum Rusticarum (On Agriculture).” (Engl. transl. by W. D. Hooper and H. B. Ash). Harvard Univ. Press, Cambridge, Massachusetts, 1934. Wallerius, J. G. 1761. Dissertation, Univ. of Upsala, Sweden. Walters, S. M. 1961. N e w Phytologist 60, 74-84. Whitney. M. 1892. US. Dept. Agr. Weather Bur. Bull. 4. Whitney, M. 1900. U S . Dept. Agr. Rept. 64. Whitney,M. 1901. Yearbook(U.S.Dept.Agr.), pp. 117-132. Whitney, M. 1904. US.Dept. Agr. O@ce Expt. Sta. Bull. 142, pp. 11 1-1 17. Whitney, M., and Cameron, F. K. 1903. Bur. Soils Bull. 22. Yarilov, A. A. 1927. Russ. Ped. Invest. 11. Young, T. C., Jr., and Smith, P. E. L. 1966. Science 153, 386-391.

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PEARL MILLET BREEDING AND CYTOGENETICS' G l e n n W. Burton a n d Jerrel B. Powell U S . Department of Agriculture a n d the University of Georgia Coastal Plain Experiment Station, Tifton, Georgia

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Introduction ................................................................................... A. Taxonomy ................................................................................... B. Origin and Distribution .........................................

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A. The Florets ........................................ B. Flowering and Seed Formation .............. C. Photoperiodism .............. ................................................... D. Cytoplasmic Male-Sterility ...., ........ ... ............ ... ........................ . ... . . E. Crossing ...................................................................................... 111. Cytogenetics ....._... .. A. Karyotype ....... B. Accessory Chromosomes ............................................................... C. Chiasmata Frequency and Chromosome Pairing.. ....... ......... .. ............. D. Euploidy, Aneuploidy, and Structural Chromosome Variations .... ...... ... E. Chromosome Translocations ........ ...... . . F. Interspecific Hybrids and Genome Relationships ................................ G . Linkage and Chromosome Associations of Characters ........................ H. Pearl Millet as a Test Organism ... IV. Genetics of Qualitative Characters A. Chlorophyll-Deficient Seedlings ......,.._..._.. ........._... . ........_.. ............. B. Plant Pigmentation ...................., ........................... ... ... ... ..... .. .... .... C . Hairiness ............

...............

...................................................

F. Disease and Insect Resistance ...._.......... G . Cytoplasmic Male-Sterility ................................................... H. Other Types of Sterility .......................................................... 1. Photoperiodism. ........................... J. Linkage

51 52 53 53 53 54 55 57 58 58 60 61 61 62 63 64 64 65 65 66 66 66 67 67 67 68 68 69

'Cooperative investigations at Tifton, Georgia, of the Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture, and the University of Georgia College of Agriculture Experiment Stations, Coastal Plain Station, Tifton. Journal Series Paper No. 2 12, University of Georgia College of Agriculture Experiment Stations.

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GLENN W . BURTON AND JERREL B. POWELL

........................................ B.

Seed Size ..................................................

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........................................................ .............. ..................................... ......................................

69 69 70 70 71 72 72 72 75 85 86 87

I. Introduction

Pearl millet, Pennisetum typhoides, a robust annual bunchgrass, occupies more than 45 million acres of the earth’s surface and occurs in every continent of the world. Although best adapted to the tropics, pearl millet also does well in hot areas of the temperate zone. It will grow and mature seed on sandy or rocky soils too acid, too dry, and too infertile for sorghum or corn. Yet it has great yield potential and, given a favorable environment, will equal or surpass corn and sorghum in forage production. Although the grain-production potential of pearl millet has not been well established, there is some evidence to suggest that it may also compare favorably with sorghum and corn as a grain crop. Pearl millet has many uses. In the southeastern United States, it serves as a forage crop. It is free of cyanide at all stages of growth, and when properly managed generally surpasses other warm-season grasses in quality. Steers grazing Gahi-1 pearl millet in Georgia have gained over 2 lb. per day and have produced over 500 lb. of liveweight gain per acre per year. High yields of top-quality silage have been obtained from bootstage Gahi-1 millet supplemented, when ensiled, with citrus pulp or ground snap corn. Pearl millet is used primarily as a grain crop in Africa and India. In areas where it serves as the principal grain crop, it is usually preferred to other cereals and commands a premium at the market place. Here many people consider it an unusually good food for the winter months. These people also believe it is superior to other cereals for pregnant women. A chemical analysis of a number of Indian foods (Aykroyd et al., 1963) shows dehusked pearl millet seeds to be higher than rice, wheat, or maize in fat and minerals (particularly calcium and iron) and similar in other principal constituents. This analysis also shows the content and balance

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of essential amino acids in pearl millet to be equal, or superior, to other adapted cereals. The stalks of pearl millet left after the grain is harvested are used in Africa to build windbreaks, shelters, screen walls, and fences. In Africa and India, where fuel is short, we have seen people cooking food over tiny fires fed with pearl millet stalks. Fodder left in the fields in these countries, although poor in quality, is usually consumed by cattle and goats during the winter months. Pearl millet has many characteristics that make it an excellent tool for cytogenetic and breeding research. Yet it has been used very little for such studies. The secondary economic importance of the crop, its somewhat restricted area of use and the failure of geneticists to appreciate its potential as a research organism help to explain why it has been overlooked. It is the purpose of this review to bring together widely scattered bits of information pertaining to the breeding and cytogenetics of pearl millet. I n an attempt to make the treatment as complete as possible, we have, in a number of instances, included unpublished information. It is our hope that this review will improve breeding procedures and hasten the genetic improvement of a grass that can contribute greatly to the needs of men who live in the warmer regions of the world. A. TAXONOMY Pearl millet received its descriptive genus name, Pennisetum, from a combination of two Latin words, “penna,” feather, and “seta,” bristle, that alludes to the plumose bristles of some species. We used the species name, Pennisetum glaucum (L.)R. Br., as preferred by Hitchcock and Chase (1 95 1) until we were convinced that it was a synonym for Pennisetum typhoides (Burm.) Stapf et C. E. Hubbard, used by most millet workers outside the United States. We have now joined them in calling pearl millet Pennisetum typhoides. Pearl millet has had many different scientific names. Hitchcock and Chase ( 195 1) list 14 of these as synonyms for P . glaucum. In addition to such species names as spicatum and typhoideum, it has been given the genus names of Panicum, Holcus, Setaria, Penicillaria, Chamaeraphis, and Chaetochloa. Meredith ( 1 954) lists four allied species -P. americanum, P . nigritarum, P. echinurus, and P. albicauda - that, in our opinion, should be considered varieties of P . typhoides. Pearl millet has also had many common names. Until recently, “cattail” superseded the name “pearl” in the southern United States. In India, “Bajra” is the preferred name. In Senegal, “Souna” and “Sanio” are the

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GLENN W. BURTON AND JERREL B. POWELL

common names applied to millets similar to Gero and Maiwa in Nigeria. Babala, Nyoloti, Cumbu, and Raa are but a few of the other common names associated with this grass. Pearl millet is an extremely variable species that is usually described as a robust annual bunchgrass with culms as much as 2 to 3 meters tall. Yet in adverse environments, plants may consist of one culm less than 0.5 meter in height, whereas spring-planted, short-day, photoperiodsensitive plants in a good environment may have many culms and reach a height of more than 5 meters. Culms may be simple or branched, slender or stout, and smooth or hairy. Leaf sheaths, collars, and blades may also be smooth or hairy. The inflorescence (head) is a false spike that may range from 5 to more than 1.50 cm. in length. The involucre, borne on a stalk up to 15 mm. long, consists of a cluster of bristles that are usually inconspicuous in mature heads but may exceed the spikelet length by several centimeters to make the head resemble the bushy tail of an angry cat. The spikelets, 4 to 7 mm. long in each involucre, range from 1 to 5 in number, but usually occur in pairs with a sessile male floret and a shortly pedicelled bisexual floret. Grains vary greatly in size and color, protrude from the lemma and palea at maturity, and are usually freed from the glumes when threshed.

B.

ORIGIN A N D

DISTRIBUTION

There is good reason to believe that pearl millet originated in Central Africa. Krishnaswamy ( 1 962) states that pearl millet, “though under cultivation in India from time immemorial, is considered by botanists to have been introduced into India from Africa.” Vavilov ( 1949-50) expressed the opinion that the primary center of origin for pearl millet is Africa, particularly the region of Abyssinia and Sudan. Certainly, the greatest diversity of morphological types that we have seen in pearl millet has been associated with introductions from Central Africa. The large number of related species that grow wild only in Africa also support the concept that pearl millet originated there. There is also cytogenetic evidence to support an African origin of pearl millet. Muntzing ( I 958) observed that accessory chromosomes occur more frequently in primitive than selected commercial varieties and suggested that their occurrence might be used to indicate a crop’s center of origin. Pantulu (1960) reported that of all the pearl millet varieties that he had examined, only those originating in the Sudan carried accessory chromosomes. We found accessory chromosomes in an inbred line from a seed lot collected in Nigeria in 1953 (Powell and Burton, 1966a). More recently, we have found accessory chromosomes in two other seed lots from Nigeria.

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53

Although grown to some extent in all continents in the world, the greatest acreage of pearl millet is found in India, where it occupies some 27 million acres. Most of the crop in India is grown on the dry lands of the western half of the country. Where irrigation is possible, pearl millet is usually replaced by rice, sorghum, corn, or sugarcane. In Africa, as in India, pearl millet is grown in the hot, sandy areas too dry for other crops. Although pearl millet assumes its greatest importance on the southern periphery of the Sahara, it may be found growing (often in tiny patches) in most of the African nations. The United States, with an estimated million acres of pearl millet grown for forage, probably makes North America rank third as a grower of this crop. Although pearl millet could be grown in much of the United States, most of the present acreage is in the Gulf States of the Southeast. Except in hot, dry summers, sorghum will usually produce more forage than pearl millet in the central and northern parts of the United States. Although grown as a grazing crop in the hot regions of South America and Australia, pearl millet is not likely to assume great importance there until the economics of beef production make it profitable to grow annual grazing crops. II. Reproduction

A. THE FLORETS The shortly pedicelled bisexual florets of pearl millet have a single pistil with two feathery stylar branches and three anthers enclosed between the lemma and palea. The stylar branches are usually colorless, but in one of our genetic stocks they develop a red color when exposed to the sun. The anthers, with variable shades of yellow, red, and purple, have a tuft of brushlike, fine hair at their apex that makes them conspicuously penicillate. The sessile male florets, borne below the bisexual ones, have three normal anthers but no female organs.

B. FLOWERING A N D SEEDFORMATION In pearl millet, a protogynous species, the stylar branches are first exserted from florets in the tip third of the head. By the third day, most of the stylar branches on heads less than 25 cm. long will have emerged. Longer heads usually require more days for complete exsertion of all stylar branches. Although flowering usually occurs after the heads emerge from the boot, in some genotypes, the styles are exserted before

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head emergence. In portions of the head where anthesis also takes place in the boot, seed set is very poor. Generally, the first anthers emerge (from the first florets to exsert stylar branches) at least 1 day after most of the stylar branches on the head have appeared. Anthers in the sessile male florets emerge 2-3 days after the anthers in the bisexual florets. Thus, most heads will shed pollen for a period of 4 to 6 days. During the summer at Tifton, Georgia, when the nights are warm, anthesis occurs around the clock. In the fall, with cool nights, anthesis is delayed until one or more hours after sunrise and occurs first on the east side of the heads. Stigmas remain receptive for several days following emergence. The stylar branches continue to elongate after emergence until they are pollinated or die. Heads of cytoplasmic male-sterile Tift 23A, pollinated 2 or 3 days before anthesis, set more seed than those pollinated later (Burton, 1966b). Since many styles were not exserted 2 to 3 days before anthesis, pollen either penetrated the opening of the apex of the lemma and palea to effect fertilization or remained viable at the stylar opening of the glumes until the stigmas emerged. The latter is quite probable, since Cooper and Burton (1 965) found that pearl millet pollen, stored in glassine bags at 80°F for 1, 2, and 3 days, was 59, 10, and 3 percent as effective as fresh pollen. Mature, full-sized seeds (caryopses) may be harvested from pearl millet 3 to 4 weeks after anthesis. Although we have been able in our laboratory to grow plants from seeds harvested only 10 days after anthesis, the viability of such immature seeds has been low. C. PHOTOPERIODISM Photoperiodism, the growth response of plants to definite light and dark periods, occurs in pearl millet. Forty out of 290 introductions from Nigeria and Upper Volta exhibited photoperiodism by failing to flower when day lengths exceeded 12 hours (Burton, 1965~).These 40 shortday-sensitive lines reached anthesis in November, whether planted in May or August. When planted in December or January in the greenhouse at Tifton, Georgia, or in the field in Puerto Rico (Barnes and Burton, 1966), they flowered in about the same number of days as day-neutral lines. The other 250 introductions, like most of the pearl millet lines in our collection, were day neutral and flowered 77 to 87 days after spring planting. Introductions from Senegal and Nigeria bearing the names “Sanio” and “Maiwa,” respectively, have been short-day sensitive at Tifton, Georgia. In Africa, these millets are planted in the summer to mature

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seed in the fall. There the planting date, determined by summer rains, varies considerably from year to year. The photoperiod sensitivity of these millets ensures a late-maturing crop for winter grain regardless of planting date. Pearl millet is characterized by an insensitive juvenile period during which photoperiod has no effect on subsequent flowering. The juvenile period appears to be 5 to 6 weeks long for Tift 2 3 A (Burton, 1966a), but probably varies with genotype and may be longer for short-day-sensitive millets. Short-day-sensitive millets, planted at Tifton February 3, 1967, 6 weeks before March 16, when days become longer than 12 hours, failed to flower and were still producing leaf primordia in July. Photoperiodism may be used to improve the forage qualities of pearl millet. We have evidence, for example, to indicate that short-day-sensitive, late-maturing millets will be superior to early millets in leafiness, seasonal distribution of forage produced, and ease of management. Such millets, planted in the seed-producing areas of the U.S., grow very tall and fail to mature seed before frost. If planted in mid-September in the southern tip of Texas, however, they should mature seed at heights well suited to combine harvesting before the usual mid- to late-December frosts. D. CYTOPLASMIC MALE-STERILITY Our thirty years of developing inbred lines from pearl millet introductions have produced many male-sterile plants. When pollinated, most of these steriles have set seed that gave rise to male-fertile F1 hybrids, suggesting genetic - rather than cytoplasmic - sterility. Our few attempts to classify the fertility of Fr plants from such hybrids failed to give good genetic ratios and were not published. In India, Kajjari and Patil ( I 956) described a male-sterile pearl millet that they hoped would be of the cytoplasmic type, a hope not established in the literature. In the winter of 1955-1956, the first cytoplasmic male-sterile pearl millets were observed in the greenhouse at Tifton, Georgia (Burton, 1958a). These plants had been developed by pollinating two male-sterile Fz plants (out of some 2000) from the hybrid, 556 X 23, with Tift 23 pollen. The greenhouse-grown plants with the pedigree (556 X 23) X 23 shed no pollen and set no seed when selfed. When pollinated with Tift 23 pollen, they set seed that again gave rise to male-sterile plants. This proved that one of our best inbreds, Tift 23, would be an excellent sterility maintainer in this cytoplasm. Several additional backcrosses developed a male-sterile Tift 2 3 A that made possible the development of an Indian hybrid millet, HB-1, capable of yielding nearly twice as much grain as adapted, open-pollinated varieties (Athwal, 1965). An increase of Tift

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GLENN W. BURTON A N D JERREL B. POWELL

23A and its maintainer, Tift 23B, permitted their official release to all millet breeders (Burton, 1965a). When Tift 18, a Gahi-1 inbred, proved to be a good sterility maintainer in Tift 23A sterile cytoplasm, Tift 18A was developed through a repeated backcrossing program (Burton, 1965b) that used Tift 18 as the recurrent male parent. Two additional sources of cytoplasmic male-sterility, L66A and L67A, developed at the Punjab Agricultural University, Ludhiana, India, show some interesting relationships to the cytoplasm in Tift 23A (Burton and Athwal, 1967). Tift 23B, maintainer for Tift 23A, is a good fertility restorer for L66A and L67A. Good fertility restorers for Tift 23A, such as T239 and L4, are excellent sterility maintainers for L66A and L67A. L66B, maintainer for L66A, restores fertility in L67A, whereas L67B is a partial fertility restorer for L66A. The discovery of two new sources of sterile cytoplasm called for a system of nomenclature. Although we proposed (Burton and Athwal, 1967) “Sl,” “SP,” and “S3” as names for Tift 23A, L66A, and L67A cytoplasms, we are now suggesting that the letter, A, be substituted for the letter, S, because S with a subscript is used to describe the number of generations of inbreeding. The numerical subscripts we proposed can be assigned to the letters, A, B, and R, used to designate the cytoplasmic male-sterile line, its sterility maintainer, and its fertility restorer, respectively. Thus, Tift 23&, Tift 23B1, and Tift 239R1 would indicate the three functions in A1 (Tift 23A) cytoplasm. Likewise, Tift 239A2, Tift 239B2, and Tift 23R2 would describe the roles played by Tift inbreds 239 and 23 when Tift 239 has been introduced into AP(L66A) cytoplasm. In the summer of 1956, male-sterile plants of the hybrid (556 X 23) x 23 were hybridized with 4 1 well-established inbred lines representing a rather wide array of germ plasm. An examination of the Fl’s of these hybrids revealed that 6 of the male parents were fertility maintainers, 8 were partial fertility restorers, and 27 were good fertility restorers (Burton, 1958a). More recent investigations with many more male lines would suggest that sterility maintainers, partial and reasonably good fertility restorers occur abundantly, but lines capable of complete fertility restoration in all sterile x fertile hybrid plants are scarce. Fertility restoration, essential where grain is sought, will not be needed where pearl millet is used as a forage plant. Sterility in the F1 forage hybrid will prevent the farmer from harvesting seed and will save him the yield losses resulting when F2 generations are grown. Sterility will also help to maintain the hybrid in a vegetative condition and increase its longevity.

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E. CROSSING

1 . Natural Crossing Pearl millet is a highly cross-pollinated crop, as evidenced by the plant-to-plant variability that characterizes open-pollinated varieties. The heterozygous nature of plants chosen at random from open-pollinated varieties (easily demonstrated in the variable selfed progeny of such plants) is further proof that self-pollination for several consecutive generations does not occur. Although protogyny in pearl millet would seem to favor complete crosspollination, several factors may prevent its realization. Most pearl millet plants, unless seeded at very heavy rates, produce several culms that reach anthesis on different dates. On such plants pollen from the first culm to flower is more likely to pollinate unpollinated heads (because of its closeness) than is pollen from other plants. Unless some selfincompatibility mechanism (yet to be demonstrated in pearl millet) is present, some of the florets on these late-flowering heads will be selfpollinated. Also the plants in most open-pollinated varieties flower over a period of several weeks. Thus both the earliest and latest plants to flower are usually few in number and are separated widely enough apart to favor some self-pollination. Although insects may occasionally effect cross-pollination in pearl millet (Leuck and Burton, 1966), wind is unquestionably the major crosspollinating agent. Our research indicates that practically all pollen movement is in the direction of the wind. Thus, plants on the outside row of the windward side of a field are more subject to self-fertilization than those in the interior. 2. Controlled Pollination Pearl millet may be self-pollinated by enclosing heads in pollen-proof bags prior to style emergence. Only occasionally have heads been found with a great enough time lapse between style emergence and pollen shed to give a poor seed set when selfed in this way. To reduce the likelihood of outcrossing, we try to enclose pearl millet heads in bags while most of the head is still in the boot. To facilitate head exsertion and bagging, we usually remove the top two leaf blades and a part of the sheath on the top leaf. We then place the selfing bag over the head and fasten it in place below the head with a gem clip or a stapling plier. A number of different selfing and crossing bags have been used on pearl

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GLENN W. BURTON A N D JERREL B. POWELL

millet. Krishnaswamy (1 962) reports that pearl millet breeders in India first selfed millet with bags made of fine muslin cloth, but they soon found these bags unsatisfactory because pollen passed through the cloth. Many Indian breeders now use a bag made of “butter paper” that “serves well.” The pearl millet selfing bag must exclude pollen and protect the seed from insect and bird damage. Bags made of 40-lb. kraft paper meet these requirements. Putting an insecticide dust, such as DDT, into the bag before it is placed over the head gives good insect control. Generally, kraft paper bags will protect the seed from bird damage long enough for the seed to mature. These bags deteriorate in the weather with time and are likely to be torn off by rain storms and birds if left more than 5 or 6 weeks. For our controlled pollinations of pearl millet, we use both a glassine and a brown paper bag. The brown paper bag, 3 X 14 inches in size, is made of special, water-repellent, 40-lb. kraft paper, sealed with waterproof adhesive. Soaking these bags in bundles of 1000 (as they come packed from the factory) in a 1 % solution of aldrin in mineral spirits (a volatile petroleum product) and then allowing the mineral spirits to evaporate leaves enough of the insecticide in the paper to control seedeating insects. Glassine bags are used to enclose heads that will serve as females in hybridizing pearl millet. These bags are made of 27-lb., bleached, glassine paper sealed with waterproof glue and are 3 X 14 inches in size. When most of the stylar branches are exserted (a condition that can be seen without removing the bag), the head is ready to pollinate. Pollen is collected in 3 X 14-inch kraft paper bags fastened with a paper clip on pollen-shedding heads of the male parent at least one day before the cross is to be made. The pedigree is written on the pollen-collecting bag with a soft lead pencil; the glassine bag is removed from the female head: the pollen bag is placed over it; and is agitated to pollinate all stigmas. The bag is then fastened around the peduncle with a paper clip or staple to keep the bag in place until harvest. Hybrids may be made at any time of the day, but those made at midday generally set the least amount of seed per inch of head (Cooper and Burton, 1965). 111. Cytogenetics

A. KARYOTYPE The cytogenetics of pearl millet, somewhat neglected until recently, is beginning to receive the attention that this crop deserves. General review articles, which include references to pearl millet, are those of

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Carnahan and Hill ( 196 1 ) and Krishnaswamy ( I 962), as well as a bibliography of the world literature on millets (George Washington University, 1967). Knowledge of the karyotype of lines within species can give some indication of chromosome diversity and possible barriers to recombination. In pearl millet, the reports on karyotype have been divergent, yet our experience in breeding this species has uncovered no significant barriers to recombination. Avdulov ( 193 1 ) examined Penicillariu spicata Willd. (-Permiseturn typhoides) and presented a drawing of the 14 chromosomes. The shortest chromosome pair was the satellited pair and was approximately 66% as long as the longest chromosome. The satellite was located on the short arm of the shortest chromosome. Centromeres of the nonsatellited chromosomes were median to submedian. On the basis of pachytene analysis, Pantulu ( 1 958) grouped the seven pairs of chromosomes into four categories: (1) the two longest having median centromeres, (2) two somewhat shorter with median to submedian centromeres, (3) two medium-sized with submedian centromeres, and (4) the nucleolus-organizing short-chromosome pair. Pantulu (1960) clearly indicated the presence of a satellite on the shortest arm of the shortest chromosome in the complement. Later (Pantulu, 1967) another reference was made to the satellited short arm in a line carrying a B chromosome. Most lines of pearl millet have one pair of nucleolus-organizing chromosomes (Pantulu, 1960), although exceptions have been noted. Two chromosomes commonly associated with a single nucleolus in an inbred line were derived from seed collected in Nigeria (Powell and Burton, 1966a). Although the karyotype of this line has not been examined, the nucleolar-organizing region is probably associated with a second pair of satellited chromosomes. T h e karyotype studies of pearl millet, cited by Krishnaswamy ( 1 962), revealed chromosomes with median and submedian centromeres and two nucleolar chromosomes. Two reports of ideograms in pearl millet differed considerably from earlier work. Al-Fakhry et al. ( 1 964) noted a satellite on the long arm of the fourth-longest chromosome in the complement. Gill and Gupta (1966) located the satellite on the short arm of the longest chromosome in the complement. They also reported that the longest chromosome was 2.69 times longer than the shortest. We have examined five inbred lines for karyotype and all have karyotypes similar to that reported by Avdulov (1931). In these lines, the satellite was unmistakenly located on the shortest arm of the shortest chromosome in the set. Centromeres were median to submedian and the

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GLENN W. BURTON A N D JERREL B. POWELL

shortest chromosome was approximately two-thirds the length of the longest. Except for the report of Gill and Gupta (1 966), there are few indications of gross assymmetry in the karyotype of pearl millet.

B. ACCESSORY CHROMOSOMES Pantulu ( I 960) first recognized the presence of accessory chromosomes in pearl millet. He examined both root-tip cells and microsporocytes and found from one to three accessories in root cells. His report indicates, however, that all microsporocytes scored for pairing frequencies contained three accessories. He noted that these accessories were about one-half the size of the shortest A chromosomes, tended to be heterochromatic on one end, and located the centromere subterminally. Accessories have now been discovered in several other stocks. In one line (Powell and Burton, 1966a), the accessories had the peculiar property of organizing nucleoli. These accessories, unlike those studied by Pantulu (1960), also varied in number from one to five from plant to plant, head to head, and spikelet to spikelet, as well as from microsporocyte to microsporocyte. A later report by Pantulu (1 967) described a stock with three to five accessories in the microsporocytes. The accessories in our stock ranged in size from approximately 1.00 to 0.25 times the length of the shortest A chromosome. Two other stocks containing accessory chromosomes were recently discovered in Nigerian plant introductions. The accessory chromosomes in these Nigerian stocks did not organize nucleoli, and one arm of the accessory tended to stain deeply. Thus, their behavior more nearly fits the description of those studied by Pantulu (1 960). All the accessory chromosomes discovered to date in pearl millet originated in Africa. Those described by Pantulu (1960) were found in an introduction from Sudan. All three lines more recently studied (Powell and Burton, 1966a) are from Nigeria. Two independent observations of very small, dotlike chromosome fragments (Powell and Burton, 1 9 6 6 ~ Pantulu, ; 1967) in pearl millet, which are accessory to the normal complement, are of much interest. Both reports are alike in that the experimental material in which the fragments were found was treated with mutagenic agents. Even though very small (length less than the width of an A chromosome), these particles have centromere activity and regularly pass anaphase I of meiosis. Anaphase 11, however, is less regular, and considerable numbers are lost at this stage because of exclusion from the nucleus. Although the evidence to date suggests that these fragments arise from A chromosomes broken

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at the centromere, additional observations on their behavior and distribution in mutagen-treated and control populations are needed. c.

CHIASMATA

FREQUENCY AND

CHROMOSOME PAIRING

Little is known about the frequency and location of chiasmata in pearl millet. Chiasmata occurring in paired chromosomes of microsporocytes terminalize early in pearl millet, giving the chromosomes a very characteristic oval shape at late diakinesis and metaphase I. Most chiasmata appear to occur in the terminal one-third of the chromosome arms. The short satellited arm is left open in some inbred lines. The degree of chromosome pairing and chiasmata terminalization was reported for inbred Tift 23B (Powell and Taylorson, 1967). Of 1600 chromosome pairs examined, 59.3% had closed bivalents, 34% had open bivalents, and approximately 6.8% were unpaired at metaphase I . This inbred is lower in chiasmata frequency than a number of other inbreds which have been examined. Inbred 26, which is one of four inbreds making up Gahi-1 (Burton, 1962), has a much higher chiasmata frequency than Tift 23B. Rangasami (1935) studied meiosis in pearl millet and observed microsporocytes with three chiasmata per bivalent. Two were regularly terminal and the other interstitial, terminalizing by diakinesis. Frequencies of chiasmata per cell or variations between chromosomes were not presented. Megasporogenesis was observed and seven closed bivalents were regularly present.

D. EUPLOIDY, ANEUPLOIDY, AND STRUCTURAL CHROMOSOME VARIATIONS Autotetraploids have been repeatedly induced in pearl millet (Krishnaswamy et al., 1950; and Gill el al., 1966) and have been studied rather thoroughly (Raman et al., 1962). These 28-chromosome plants generally are less fertile than the diploids, exhibit some gigas characteristics, and at meiosis form multiple chromosome associations. Autotriploids are easily obtained by crossing the tetraploid forms with the diploids. We have observed spontaneous autotriploids, which presumably resulted from the fertilization of unreduced female gametes. They were highly sterile. A clear case of multiploidy with more than 100 chromosomes aligned as bivalents at metaphase I in microsporocytes has been observed in our mutagen-treated pearl millet. The inheritance of this abnormal behavior could not be studied, as it has in some other crops, because we did not recover the trait in the F2 progeny. Desynapsis has been reported at least twice (Krishnaswamy et al.,

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1949; Patil and Vohra, 1962) in pearl millet, and other unreported cases are known. Frequently, desynaptic plants give rise to trisomic offspring and, therefore, are very useful in assembling tester stocks for genetic linkage tests. Several trisomic plants that occurred in different ways have been described (Krishnaswamy, 1962; Pantulu, 1967). One of these arose from a desynaptic background, another originated from an ionizing radiation treatment, and still another (in our material) occurred spontaneously. Other structurally altered chromosome types present in pearl millet include isochromosomes and paracentric inversions. Pantulu ( 1967) found evidence for both types. Krishnaswamy ( 1 962) cited a case of an inversion which did not survive. We likewise have observed both aberrant types, but have been unable to isolate and establish genetic stocks of these aberrations.

E. CHROMOSOME TRANSLOCATIONS The contribution of reciprocal chromosome translocations to the evolution of the pearl millet karyotype is poorly understood. We know of only two cases of spontaneously occurring chromosome translocations. Pantulu (1 958) discovered a chromosome interchange in “Gahi- 1 ,” a first-generation synthetic consisting of approximately 75% of chance hybrid seed and 25% of selfed and sibbed seed of the four inbred parents (Burton, 1962). Our studies have further revealed that one of these inbreds, No. 13 (a domestic selection) was responsible for the structural rearrangement, which gave rise in hybrids to the ring of four chromosomes at meiosis. Since the inbred was heterozygous for the structural rearrangement, both the standard and nonstandard chromosome arrangements (designated “T 13”) have been isolated. Another natural-occurring chromosome translocation in pearl millet has been isolated, and its relationship to the nonstandard arrangement of inbred 13 has been established. The second interchange was found among late-maturing plant introductions from Upper Volta in Africa. Crosses of this line with T13 produce hybrids with rings of six chromosomes, indicating that both stocks have one translocated chromosome in common. Pollen abortion was approximately 33% for the structural chromosome rearrangement involving four chromosomes and 52% for those involving six chromosomes. Pantulu (1958) reported 40% sterile pollen in plants with the ring of four chromosome configuration. Some directed orientation of chromosomes at metaphase I evidently increases pollen fertility. Pearl millet is more like barley and tomatoes than like corn and sorghum with regard to apparent directed chromosome orientation.

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Krishnaswamy ( 1962) noted sterile plants in selfed lines, which later were found to be due to a chromosome translocation. Whether these types were spontaneous or were found in X-rayed material was not stated. A number of investigators have observed chromosome translocations in pearl millet treated with mutagenic agents (Krishnaswamy and Ayyangar, 1941, 1942; Burton and Powell, 1966; Pantulu, 1967). Rings of four chromosomes at meiosis are the most frequently observed configuration of interchanged chromosomes. However, multiple translocations up to rings of eight chromosomes (Pantulu, 1967) have been observed.

F. INTERSPECIFIC HYBRIDSAND GENOME RELATIONSHIPS Of the many species which make up the genus Pennisetum, only P . purpureum Schumach. is closely related to P. typhoides. Both species have a basic chromosome number of seven. Attempts to hybridize either of these (Penicillaria section) species with those outside this section that have a different basic chromosome number have generally been unsuccessful. Hrishi (1952) made an intensive study of the cytotaxonomic relationship of six species representing the sections Gymnothrix, Eupennisetum, and Brevioalvula and found no evidence that any were closely related to the Penicillaria group. Three exceptions of hybrids accomplished between groups differing in base chromosome numbers have been reported. All hybrids were made with considerable effort and located by efficient screening methods. Gildenhuys and Brix (1961) reported on a single hybrid plant arising from a P. typhoides cross with P. dubium. Only a 14-chromosomegamete was received from P . dubium (2 n = 66) to give the 21-chromosome hybrid. No genome pairing was observed. Another wide cross between P. typhoides and P . squamulatum Fres. was reported by Patil et al. (1 96 1). This hybrid, having 41 chromosomes, originated from a functional unreduced female gamete of P. typhoides. N o intergenome pairing was reported. Three-way interspecific crosses between P. squamulatum, P. typhoides, and P . purpureum (Rangasamy and Ponnaiya, 1963; Menon and Krishnaswamy, 1963; Menon and Devasahayan, 1964) indicate some segments in the P . squamulatum genome are common with the A and B genome in P . purpureum. Patil and Sing (1964) were able to cross P . typhoides with P . orientale L. C. Rich., a diploid with 18 chromosomes. In this hybrid, pairing between one o r two chromosomes of the different species was easily identified because the P. orientale chromosomes were about one-half the size of the P . typhoides chromosomes. Some homology between the genomes of the two species was suggested. The genome relationships between P. typhoides and P . purpureum

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were established in 195 1 (Krishnaswamy, 1962). P . typhoides carries a genome designated A and P . purpureum carries two genomes designated A and B . The common genome in the two species and the genome of unknown origin give rise to seven bivalents and seven univalents at meiosis in the triploid interspecific hybrid (Krishnaswamy and Raman, 1954; Raman and Krishnaswami, 1960). The amphidiploid, induced by colchicine treatment of .the triploid interspecific hybrid, has been studied rather thoroughly (Krishnaswamy, 1962). This 42-chromosome hexaploid is fertile (Krishnaswamy and Raman, 1953; Gildenhuys and Brix, 1964) and forms mostly 21 bivalents at meiosis (Krishnaswamy and Raman, 1954; Khan and Rahman, 1963). Gildenhuys and Brix (1964) reported, however, that the amphidiploid was not stable for somatic chromosome number and when backcrossed to P . typhoides, exhibited various degrees of incompatibility. Embryo and endosperm abortion after fertilization was found to be independent of the direction in which the diploid X polyploid cross was made (Glidenhuys and Brix, 1965). This amphidiploid showed no significant gigas characteristics when compared with the triploid interspecific hybrid. The backcrosses of the amphidiploid (used as a pollen source) to P . purpureurn resulted in progeny with some sterile plants. These were reported (Krishnaswamy and Raman, 1954) to be monosomic, having only 24 chromosomes. Backcrosses of the amphidiploid (pollen source) to P . typhoides gave progeny with higher chromosome numbers than predicted (Gildenhuys and Brix, 1964). A selective advantage for the functioning of unreduced female gametes was shown.

G . LINKAGE A N D CHROMOSOME ASSOCIATIONS OF CHARACTERS

No linkage tester stocks of pearl millet are currently available because easily classified genetic traits have not been linked. Recently work has been initiated in several laboratories to locate genetic traits on the seven chromosome pairs in pearl millet. H. PEARL MILLETA S A TESTORGANISM Pearl millet should be an excellent organism for research directed toward the solution of unanswered questions of genetic and cytogenetic significance. For investigations in the area of genetic recombination, combining ability, and chromosome behavior, pearl millet would seem to be unexcelled. It is a diploid with a low basic chromosome number of seven. The chromosomes are large and the meiotic stages are excellent for detailed study. A vigorous spaced plant from a single zygote can produce up to one billion male gametes and more than 35,000 recombin-

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ants (caryopses). If a method could be developed to select the polIen grains carrying genetic mistakes, resolutions near those obtained in microorganisms might be possible. Although naturally cross-pollinated, pearl millet may be easily maintained in a highly inbred condition. Its bisexual flowers permit selfpollination merely by enclosing heads in paper bags. Yet, its protogynous flowering habit makes emasculation unnecessary for controlled hybridizations that usually supply more than 1000 hybrid seeds per pollination. At least three generations can be produced each year. Pearl millet is equal to corn and sorghum in genetic diversity. Little is known concerning the inheritance of its many plant colors and shapes. The fact that none of this genetic diversity has been mapped on chromosomes probably is the greatest factor limiting its use as a test organism for genetic research. Pearl millet is particularly well suited for research directed toward an understanding of heterosis. The best hybrids can produce twice as much grain as adapted, open-pollinated varieties. Heterosis may also manifest itself in greatly increased size and yield of the whole plant. Finally, and of no small significance so far as we are concerned, is the fact that pearl millet is an important economic crop. Thus, basic and applied research can be conducted simultaneously and their fruits can be used immediately to meet the needs of man. IV. Genetics of Qualitative Characters

A. CHLOROPHYLL-DEFICIENT SEEDLINGS Lethal chlorophyll deficiencies of many kinds occur in pearl millet. Since seedlings homozygous for these recessive genes usually live only a few days, they may be overlooked until an inbreeding program is initiated. It was in this way that Ayyangar and Hariharan (1935) first observed and reported an albino seedling character controlled by a single recessive gene. Later, Kadam et al. ( I 940) reported both mono- and digenic ratios for pearl millet chlorophyll-deficient seedling characters named “albino,” ‘‘yellow,” “virescent yellow,” “golden yellow,” and “zebra.” More recently, Burton and Powell (1965) described six monogenic, naturally occurring, chlorophyll-deficient seedling characters and suggested a much-needed system for their nomenclature. Several investigators have noted the occurrence of mutagen-induced chlorophyll-deficient mutations in pearl millet. Krishnaswamy and Ayyangar ( 1942) described albino and pale yellow lethal seedling types

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giving mono- and digenic ratios in progenies from pearl millet treated with X-rays. Chandola et al. (1963) found seven types of chlorophylldeficient mutants in the pearl millet varieties, RSK, RSJ, and Nokha, after exposure to four different levels of gamma irradiation. Burton and Powell (1 966) observed that treating pearl millet seeds with thermal neutrons or ethyl methane sulfonate (EMS) increased the frequency with which chlorophyll-deficient seedlings occurred in selfed progeny of pearl millet up to five times the untreated checks. In their studies with 10 inbreds, the most effective EMS treatment induced over eight times more mutants in one inbred than in another. Not all chlorophyll-deficient seedlings are lethal. Krishnaswamy ( 1 962) described a monogenic, pale green seedling character that enabled the homozygous recessive to grow into a weak adult plant. We also have stocks with light green chlorophyll, which can be maintained in a homozygous condition.

B. PLANTPIGMENTATION Pearl millet plants show a wide range of pigmentation. In our genetic stocks are lines with golden, green, red, and purple pigmentation in most of the plant parts. The nodes and sheaths of other lines may be purple or sun-red on plants otherwise green in color. Although styles are generally colorless, we have one line with sun-red styles. Another color in these stocks includes various shades of yellow, red, and purple. Our stocks also contain lines with green plants and yellow, red, or purple bristles. Athwal et al. (1 966) describe a number of similar genetic stocks. Genetic studies at Tifton, Georgia, although not complete, indicate that green is dominant over golden and recessive to red and purple. Fz ratios of crosses involving these characters suggest that each may be conditioned by a single gene.

C. HAIRINESS Pearl millet plants may (1) be completely smooth, (2) have only hairy nodes, (3) have hairy nodes and leaves, or (4)have hairy nodes, leaves, and stems. Although all the genetic relationships between stocks selected for these types have not been worked out, we have always found that smooth plant parts are dominant over hairy parts and Fz populations generally give 3 : 1 smooth :hairy ratios. D. BRISTLES

Krishnaswamy (1 962) described a cross between a very bristly variety of pearl millet and a nonbristly variety that gave a very bristly F1genera-

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tion, and an Fz generation with plants of many grades and lengths of bristles. When he grouped all bristled Fz plants together, the bristled :nonbristled ratio was 846: 262. In our material, a cross of a long-bristled inbred with a nonbristled inbred gave all bristled F1plants, but the bristles were about half as long and less dense than the bristled parent. In the Fz generation, three-fourths of the plants were bristled, indicating that a single gene controlled bristle extension. However, the frequency distribution of the bristle lengths gave a normal distribution curve, suggesting that bristle length may be conditioned by several genes. Since bristle density varies greatly among lines, bristle density should be subjected to genetic study. E. SEED

Athwal er ul. (1 966) described five pearl millet seed stocks with colors of light slate, deep slate, pearly amber, deep yellow, and purple. In addition to these, we have seen white and brown-seeded types and have noted that yellow seed color is dominant over slate (also described as bluish green). This may be expressed as a xenia effect. Krishnaswamy ( I 962) reports that the “genetics of the grain color is said to have been determined as due to three factors.” The first yellow-endospermed pearl millet was discovered in a field of Dauro millet growing in the foothills of the Jos Plateau in Nigeria (Curtis et al., 1966). This millet contained 0.9 to 2.0 ppm. of carotene, compared to 0.6 and 4.0 ppm. of carotene in yellow-endospermed sorghum and corn, respectively. The inheritance of this trait, that appears to be dominant over the slate-colored seeds of Tift 23, is currently being studied.

F. DISEASEA N D

INSECT

RESISTANCE

Pearl millet breeders around the world are concerned with increasing the resistance of improved varieties to disease and insects. Lines in the World Collection free of rust, smut, green ear disease, leafspot, ergot, marasmea, and midge suggest that genes for resistance to these organisms are available. In India, F1hybrids with Tift 23A have shown a high degree of resistance to the green ear disease, caused by Sclerosporu graminicola. At Tifton, several inbred lines of pearl millet have shown resistance in the seedling stage to the fall armyworm, Spodopreru frugiperda. However, the genetics of these traits have not been established.

G. CYTOPLASMIC MALE-STERILITY Cytoplasmic male-sterility in pearl millet apparently results from the interaction of a recessive gene, ms, in the homozygous condition with a

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sterile cytoplasm designated as “B-cytoplasm” by Menon (1 959). Burton and Athwal (1 967) described three different sources of sterile cytoplasm S1, Sz, and S, with corresponding rns,, msS, and rnss genes that maintain male-sterility when homozygous recessive. Genotypes with normal cytoplasm and the suitable rns rns gene pair make good maintainers for their male-sterile counterparts. Menon ( 1 959) reported that the dominant M s gene in the homo- or heterozygous condition restored complete fertility to his male-sterile genotype. In our experience, complete fertility restoration has been difficult to achieve. Although we believe that a dominant M s gene in either the homo- or heterozygous condition generally restores fertility to genotypes sterilized by its corresponding rns gene, modifying factors and environment may also operate to prevent complete fertility restoration (Burton and Athwal, 1967).

H.

OTHER

TYPESOF

STERILITY

Krishnaswamy (1 962) described a number of kinds of sterility in pearl millet under the headings of male-sterility, complete sterility, partial sterility, headless plants, female sterility, gappiness in panicles, and tip sterility. Since he later described “sterility owing to meiotic disturbances,” he apparently considered the former to be genetically controlled in plants with reasonably normal meiosis. A case of genetic male-sterility and the gappiness reported by Krishnaswamy in panicles were controlled by single recessive genes, but genetic ratios for tip sterility were closer to a 9:7 than a 3: 1 ratio. Inheritance models for the other forms of sterility were not supplied. Burton (1952) observed a wide range in the self-fertility of inbred lines of pearl millet. Semi-self-sterility, resulting in seed set of about half as many seeds per inch of head when selfed as when topcrossed, was a highly heritable character based on progeny studied, just as was complete self-fertility. Genetic models to explain the type of inheritance involved were not developed. 1. PHOTOPERIODISM Photoperiodism in pearl millet is a heritable character. Studies of the parents, F1 and F2 generations of hybrids between one late (short day) and two early (day neutral) inbreds suggested that photoperiodism is controlled by several genes that exert an additive effect with little dominance (Burton, I95 1). Bilquez ( 1 963) reported that one gene, acting without dominance, controlled photoperiodism in a day-neutral X short-day hybrid. More recent studies of the parents, F1 and F2 generations be-

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tween one short-day inbred and four day-neutral inbreds confirm our earlier report, namely, that several genes acting additively without dominance condition photoperiodism in pearl millet (Burton, 1966a). Additional support for this hypothesis may be found in a group of inbreds developed from these crosses. When planted at Tifton, Georgia, about May 1, different groups of these inbreds will flower each week from midJuly until November.

J . LINKAGE Although we and several other investigators have studied hybrids between lines carrying a number of different genetic markers, sufficient data have not been collected to establish good linkage relationships. V. Genetics of Quantitative Characters

A. SPACED PLANTCHARACTERS

To ascertain the inheritance of eight spaced plant characters, Burton ( 1 95 1) measured 100- to 200-plant populations of several pearl millet

inbreds and their Fl’s interplanted with 700- to 1900-plant Fzpopulations of each cross. In this study, heterosis was manifested in the inheritance of plant yield, plant height, head length, culm diameter, and internode length. Few culms were dominant over many culms in one hybrid, but little evidence of dominance was expressed in the inheritance of leaf width and number. The minimum number of genes conditioning these characters, estimated by Wright’s formula, ranged from two to eight. Ahluwalia et al. ( 1962) studied combining-ability effects for tillering, plant height, and yield per tiller in a set of diallel crosses between six inbred parents and found evidence for full dominance, overdominance, and partial dominance, respectively, for these three characters. Conspicuous epistatic effects were demonstrated for each character. Gupta and Athwal ( 1 966b) studied the genetic variability (genetic variance X lOO)/(phenotypic variance) in a collection of 156 lines of pearl millet from India, Africa, and America. For the total collection, they obtained genetic variability values of 58, 33, 59, 38, 55, 41, and 56% for green fodder yield, leaf size, leaf number, stem thickness, plant height, tiller number, and earing date, respectively. Jain et al. ( 1 96 1) showed that the length and girth of the pearl millet spike were largely governed by additive genetic factors in a set of diallel crosses involving six inbred lines. Only partial dominance and no significant heterosis or epistasis were detected.

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Burton and Fortson ( 1 966) studied the inheritance of dwarfness in pearl millet by analyzing height measurements of over 39,000 parent, F1, Fz, and backcross plants of hybrids involving five dwarf and four normal inbreds. F1 hybrids between four of these dwarfs were as tall as normal X dwarf hybrids, proving that these dwarfs carried different recessive genes for dwarfness. One dwarf X dwarf hybrid was little taller than its taller dwarf parent, suggesting that both carried the same or similar dwarf genes. Most dwarf X normal F1 hybrids were significantly taller (up to 37 percent) than their normal parents. Inheritance of dwarfness in normal X D1 or Dz inbreds appeared to be conditioned largely by one or two recessive genes. When transferred to a near-isogenic background, dwarfness in D, and D2was found to be controlled by single but different recessive genes, dl and d2, respectively. Near-normal Fa distribution curves and minimum gene-number estimates indicated that dwarfness in DS, D4, and D5 was controlled by more than two recessive genes. B. SEEDSIZE

Seed size is a highly variable character in pearl millet. Gupta and Athwal(1966a) examined a number of grain characters in 30 to 40 lines or hybrids from three geographic areas in 1964. Seed size, the most variable of all characters they measured, gave genetic variability values of 98,67, 77, and 54% for Indian varieties, African varieties, Indian inbreds, and American inbreds, respectively. Burton (1952) showed that seed size (weight of 100 seeds) a heritable character, decreases with inbreeding. Studying the immediate effect of gametic relationship on seed size, he found that 30 S1 lines when topcrossed produced 9.5% heavier seeds than when selfed. Sixty S3 lines (from the 30 S, lines) developed 18.9% heavier seeds when topcrossed than when selfed. The immediate gametic effect on seed size ranged from zero in some hybrids to more than 40% in others. Progeny studies proved that immediate gametic effects on seed size is heritable.

C. FORAGE YIELD 1 . Genetic Variance Component Studies In a study of the genetic variance components for forage yield in 18 sets of pearl millet diallel hybrids, the nonadditive components ranged from 7 to 106% and averaged 56% of the total genetic variance (Burton, 1950). Thus, a rapid advance in forage yield would require the development of commercial F1 hybrids. The ability of the F1 hybrid mixture, Gahi-I, to give three times the advance in forage yield obtained from the synthetic Starr millet added support to this conclusion.

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2. Heterosis Heterosis for forage yield in pearl millet is a well-established phenomenon. Over a four-year period, Gahi-1 (a mixture of six F1 hybrids between four inbred lines) yielded 52 and 29% more dry matter than the open-pollinated common and Starr varieties (Burton, 1962). In one of our three-year, forage-yield trials, 106 F1hybrids yielded 6 1% more than their inbred parents. In another three-year comparison, the six possible F1 hybrids from the four Gahi- 1 inbred lines produced 7 1% more forage than their inbred parents. Three of our pearl millet experiments show that, on the average, heterosis for forage yield in pearl millet closely parallels the heterozygosis of the material tested (Burton, 1968). 3 . Epistasis

Epistasis for forage yield occurs in pearl millet. In one experiment in which we used five or six inbred testers to detect epistasis by the Bauman method ( 1959), epistatic deviations were observed in 16 of 33 two-year tests involving the six possible sets from selected pearl millet inbreds 13, 18, 23, and 26. Parents, testers, and environment influenced the amount of epistasis observed. In another two-year, forage-production test of the parents, FI’s, and Fz’s of a nine-inbred diallel, five of our Fz hybrids demonstrated the presence of epistasis when they deviated significantly from expected yields, calculated from the linear model (PI PZ 2F1)/4.

+ +

D. GRAINYIELD

Gupta and Athwal (1966a) studied grain yield of 148 lines of pearl millet in 1964 and found highly significant differences among them. An average genetic-variability value of 35% was obtained for grain yield in this material. Durand ( 196 1 ) reported that heterosis for grain yield was exhibited by 9 out of 10 pure-line stocks crossed with tester strain 165. Ahluwalia and Patnaik ( 1 963) measured significant amounts of heterosis for grain yield in 6 out of 19 F1 hybrids between 9 inbred lines and 2 open-pollinated testers. These produced from 33 to 70% more grain than their best parent. Rachie ( I966a,b) reported grain-yield increases over the open-pollinated check varieties ranging from 75 to 100% for four F, hybrids between Tift 18A, Tift 23A, or LlOlA and selected Indian inbred lines tested in 7 to 20 different locations in India. Athwal (1966) described 16 trials laid out in all the important pearl

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millet-growing states in India in which three F1 hybrids-Tift 23A X BIL-3B, lOlA x BIL-3B, and Tift 23A x BIL-1 -yielded, on theaverage, 100, 89, and 68% more grain than the checks.

E. CORRELATIONS AMONG CHARACTERS In the F2 population of a single pearl millet hybrid, correlation coefficients of +0.69**, +0.3 l**, +0.26**, and +0.26** between plant yield and stem number, stem diameter, plant height, and leaf width were found (Burton, 195 1). Guptaand Athwal(1966b) obtained correlation coefficients of+0.34**, +0.41**, +0.07, +0.17*, +0.20*, and +0.16 between green plant yield and leaf size, leaf number, stem thickness, plant height, tiller number, and earing date, respectively, when they combined all measurements from 158 pearl millet lines of diverse origin. Other correlation coefficients that these workers obtained among other pearl millet fodder characters, although sometimes statistically significant, were too small to indicate close relationship. In a study of the combined measurements of 148 lines from India, Africa, and America, Gupta and Athwal ( 1 966a) found tiller number to be the only character noticeably related to grain yield, r=+0.41**. Their correlation coefficients between grain yield and other characters in this material, namely, grain density, grain hardness, grain size, plant height, and earing date were+O. 14,+0.16*,+0.10,+0.11 ,and+. 14,respectively. These correlations indicate that none of the characters considered (other than yield) would be a good selection index for yield of forage or grain if taken alone. Of greater importance perhaps is the evidence that a number of plant characters may be altered by selection without materially affecting forage or grain yield. VI. Breeding

A. GENERAL OBJECTIVES 1 . Forage Production

Although the ultimate objective of any forage breeding program will be to increase yields of animal products, such as meat or milk, it is not enough to set increased forage yield as the only objective. Making pearl millet better adapted to the environment in which it must be used should increase forage yields. But if the better-adapted variety does no more than increase dry-matter yields a few percent, it may contribute very little to the livestock industry. Since animals that consume forage crops eat about the same amount of

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forage each day, it is important that forages to be grazed have a uniform growth rate over a long period of time. If the forage is to be ensiled or otherwise processed, the seasonal distribution pattern becomes less important. For processing, rapid terminal growth that will give a high yield of relatively young forage is probably better than the slower sustained growth desirable in the pasture plant. Forage plants must be dependable, and annuals like pearl millet, that must be established from seed each year, need certain traits not always necessary in perennials. The seeds should be able to germinate in cold soils to permit early spring planting. The young seedlings should be resistant to disease and insect pests and should be able to grow fast enough to get ahead of the weeds. Drought resistance a t all stages of the plant’s life is highly important. Although pearl millet possesses a good measure of these important characteristics, the tremendous variability in pearl millet should permit improvement if effective selection pressure for these dependability characters can be imposed. Annual forage crops, such as pearl millet, must be grown on cultivated land. Such land is often costly and must be planted to high-income crops. In order to compete successfully with cereal crops for such land, annual forages must frequently be fertilized and/or irrigated to give the high yields required. Although pearl millet has a high yield potential and is able to use water and fertilizer efficiently, we believe these traits can be improved by breeding. Many animal scientists believe that pearl millet supplies better quality forage than warm-season perennial grasses. Even so, we believe the quality of pearl millet can be improved by breeding and that improved quality should be one of the major objectives of the pearl millet forage breeder. One obvious way to improve quality is to increase leaf percentage. This can be most easily done by introducing a dwarf gene to shorten stem internodes and reduce stem percentage. T o ascertain the quality benefits to be achieved, we compared in 1967 near-isogenic tall and dwarf forms of Tift 23 pearl millet. Cut at the boot stage with a forage harvester, these tall and dwarf millets yielded 3.2 and 2.5 tons of dry matter per acre and the artificially dehydrated forage fed to dairy heifers contained 5 3 . 6 and 80.6% of leaves, respectively. Dry-matter digestibility values for the tall and dwarf forms were 55.8 and 59.7%, respectively, and the heifers receiving the dwarf forage ate 20% more per day and gained 50% faster than heifers consuming forage from the tall type. Thus, breeding dwarf varieties may be one way to improve quality, particularly if the forage is to be fed to animals in the boot stage. We have much experimental evidence to prove that short-day, photo-

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period-sensitive pearl millets remain vegetative longer, give a more uniform seasonal distribution of forage, and are easier to manage when grazed than early-maturing types. As a consequence, late maturity has become one of our objectives for improving the quality of pearl millet. Although we have only begun to test the digestibility of pearl millet genotypes, preliminary investigations (Burton et al., 1964) and the breeding of the more digestible Coastcross- 1 bermudagrass (Burton et al., 1967) have convinced us that the digestibility and, hence, the quality of pearl millet can be improved by breeding. Tomorrow’s pearl millet forage breeders will, no doubt, let digestibility and other quality factors yet to be discovered rank high among the characters sought in the genetic improvement of this important grass. 2 . Grain Production Many of the objectives for the genetic improvement of pearl millet for grain production are identical with those set for forage and will not be repeated. Whereas short-day photoperiod sensitivity and late maturity seem to be desirable objectives for forage production, photoperiod insensitivity and early maturity will generally be sought where grain is desired. These traits will permit the successful growth of pearl millet as a catch crop where the favorable moisture regime is short. They will also allow farmers to grow several crops a year whenever the temperature and moisture conditions are favorable. Leafiness, a highly desirable trait in forage varieties, will be less important in the varieties used for grain. In fact, there is evidence in rice and other crops to suggest that maximum grain yields may be achieved with pearl millet varieties that have seven or eight short, stiff, erect leaves per culm. Dwarfs will replace tall types, as they have in grain sorghum, if harvesting is mechanized. Even with hand harvesting, dwarf types will be used in pearl millet, as in wheat, rice, and sorghum, to permit the application of fertilizer, irrigation, and other practices to maximize grain yields. The ability to develop many grain-producing, lodge-resistant tillers, characters known to be related to high grain yield (Gupta and Athwal, 1966a), will be sought in future grain varieties. Where the fodder is used for livestock feed, after the grain is harvested, attention will be given to improving the acceptability and digestibility of the mature plant with the grain removed. Grain losses due to birds may be reduced by breeding bristled varieties. Athwal and Luthra ( 1 964) reported that bird damage could be cut 10 to

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25% by growing the bristled variety, S-350. Dowker (1963) observed, however, that under severe bird attack, the Hedgehog variety with many long, stiff bristles gave bird protection, but other bristled varieties were susceptible. Ahluwalia and Shankar ( I 964) stated that bristles reduce the extent of bird damage and the amount of pollen washed away by rainfall during flowering. Although we found bristles over 0.5 inch in length tended to deter bird damage in Fe populations segregating for bristle development, we also noted that even those heads having bristles an inch in length did not escape some loss of seed due to birds. It is possible that this reduction in seed lost to birds will be offset by losses due to poor cross-pollination and damage from insects (protected by such long, heavy bristles). Certainly, if long bristles will give bird protection without reducing yields of harvestable grain, they will be added to future grain varieties. But more research is needed to establish the merits of the bristled-head character. Improved grain quality will be a major objective of breeders concerned with improving pearl millet. This will include adding the yellow-endosperm character to increase vitamin A content and improving the quantity and quality of the protein in the grain. Finally, these changes must (if possible) be made without altering those color and textural characteristics that make the grain acceptable to the consumer. If increased grain size and the pearly, amber endosperm, sought by some breeders, will make pearl millet grain more attractive to those who eat it, these characters will be added to future varieties. Since tomorrow’s grain varieties are likely to be F1 hybrids produced on cytoplasmic male-sterile stocks, fertility must be completely restored for maximum yields. Thus, males selected for these hybrids must carry genes to restore fertility, as well as genes for other important characteristic s. B. BREEDINGMETHODS 1 . Collection and Maintenance of Germ Plasm The success of any plant breeding program is dependent to a very large degree on the genetic diversity of the germ plasm available. Recognizing this truth, plant breeders have long sought diverse germ plasm wherever it could be found. Only recently, however, has it been possible to gather together large “world” collections of economically important crops. In 1959, four agencies-the Indian Council of Agricultural Research, the Indian Agricultural Research Institute, the State Department of

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Agriculture, and the Rockefeller Foundation - initiated a cooperative project to make a systematic field collection of maize, sorghum, penniseturn, and the millets (Rachie, 1966a). From this three-year effort came 754 pearl millets to which were added 1360 inbreds or varieties from millet breeders around the world. Ahluwalia el al. (1963) examined over 1700 samples from this collection and noted variability for pest and disease susceptibility, stalk thickness and height, tillering, pigmentation, pubescence, spike characters, and maturity. Later the world collection was planted and evaluated at four locations, representing abundant and sparse rainfall, in northern and western India (Murty, 1966). Data collected on 20 characters revealed a wide range of variability, but only a very few lines from Africa were free of rust, smut, or the green ear disease. Murty reported that “alleles for early flowering (55 days) were found in the East African collections and some of the lines from Ghana. Late-flowering types (80 days or more) were observed mostly in the West African material, particularly from Mali and Senegal. Dwarf lines in unselected material came from Bechuanaland and Northern Rhodesia. Bold-seeded types were mostly from Kenya, Mali, Sudan and Senegal. Bristle length was the largest in the Bechuanaland and N yasaland collections.” How should the plant breeder handle such a large collection of diverse germ plasm in a species such as pearl millet that is highly crosspollinated? Well-established inbred lines can be maintained by continued selfing. Varieties can only be maintained, however, if propagated in isolation in sufficient quantity to retain all genes. To maintain such a large number of varieties in this way is practically impossible. Varieties carrying genes of recognized value (such as resistance to a specific disease) may be selfed or sibbed with selection imposed to retain the character desired. Varieties with similar traits, such as maturity or height, may be grouped and increased in isolation to make a gene pool. Such gene pools, properly managed, can break linkage blocks and allow for gene interchange between the germ plasms pooled. These pools tend to obscure, however, specific traits observed in the original varieties to the extent that much time may be required to recover them. The ideal procedure for handling world collections of cross-pollinated species would seem to be the collection of enough seed of each variety (perhaps 1 lb. of pearl millet) to permit evaluation, long-time storage, and the development of germ plasm pools. Although this would be difficult and sometimes impossible, we believe the advantages of this procedure would far offset its added cost.

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2. Mass Selection A glance at the pearl millets collected in Africa reveals that indigenous man has developed a great many different varieties. Many of these, developed without the aid of trained plant breeders, are the product of mass selection. Certainly, the diverse, highly cross-pollinated pearl millet lends itself well to such manipulation. Krishnaswamy ( 1 962) states that “mass selection seems to have been the method tried during the earliest attempts at improvement of this crop.” Criteria for selection were “well-filled panicles, compactness, fairly good length of ear, heaviness of grain, and uniformity in ripening as far as possible.” Following these procedures, farmers were able to achieve a “very high degree of uniformity with respect to duration and panicle characters.” The first use of systematic breeding techniques with pearl millet, carried out at a number of locations in India, consisted of the following five steps: ( 1 ) collection and observation of many varieties in small plots; (2) selection of best plants in a population: ( 3 ) study of the progeny of a single plant; (4)comparison of the yield of individual plants either alone or in compact family blocks; ( 5 ) evaluation of the best selections in local and district trials before general distribution. Krishnaswamy ( 1962) reported that from this effort came a number of high-yieldingvarieties, such as Co. I , 2, and 3 for Madras; AKP 1,2, and 3 for Andhra: and A 1/3 and T55 for Punjab. Pearl millet improvement in Niger (Nabos, I966), Nigeria (Abifarin, 1966), and Senegal (Etasse, 1966) has paralleled the early work in India. Varieties collected from local cultivators, with or without further mass selection, have been tested for grain yield, and the best have been named and released for farm use. Mass selection is an effective method of improving characters with high heritability and should be used to improve such characters in pearl millet. Unless the performance of the individual plant and its progeny are correlated, however, selection on a single-plant basis cannot result in improved performance. Although pearl millet grain yields have been improved by mass selection, the increases have been small and the progress has been slow. Although significant differences in the forage yield of United States pearl millet varieties have been observed, these differences have not been great. Since the heritability for forage yield on a single-plant basis is usually very low, improvement of forage yield by mass selection is likely to be slow.

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The availability of commercially usable cytoplasmic male-sterile lines of pearl millet and the outstanding gain in grain and forage yield exhibited by the best hybrids will likely restrict the use of mass selection as a yield-improving method to those areas where cultivators cannot produce or purchase hybrid seed. 3 . Synthetic Varieties

Synthetic varieties are usually developed in cross-pollinated crops by pooling together two or more sources of germ plasm that have certain desired characters in common. Diversity, aside from the desired traits, is usually sought in order to increase the levels of heterozygosity and yield. The history of the development of Starr millet (Burton and DeVane, 195 I ) will serve as an example of how synthetics may be developed. Farmers criticized the common pearl (cattail) millet, grown for grazing in the United States in the 1930’s, by pointing out that it was stemmy and matured too quickly. In an effort to overcome these faults, a short, leafy, late-maturing selection was crossed with a broadleafed, highly palatable, early-maturing common inbred in 1944. In the following year, a number of medium-tall, leafy, late-maturing plants in a large Fz population of this cross were selfed. Selfed seeds of a number of FBprogenies of these plants that bred reasonably true for the desired characters were pooled and increased in isolation to form Starr millet. In clipping trials, Starr millet, that flowered about one month later than common, produced I5 to 20% more forage and gave a better seasonal distribution than the common check. Since steers grazing Starr millet made better daily gains (2.2 Ib. per day) than those on common millet (1.7 Ib. per day), Starr was also assumed to be superior to common millet in quality. Recently, we developed a short-day, photoperiod-sensitive late synthetic variety, Tiflate, by combining the germ plasm of 54 late introductions from Nigeria and Upper Volta. These accessions failed to flower in field plantings and were phenotypically similar. Seed from these introductions planted in one section of a greenhouse at Tifton, Georgia, gave rise to plants from which we harvested cross-pollinated seed in the winter of 1963- 1964 (Burton, 196%). This seed was further increased in Puerto Rico the following winter to supply seed for field evaluation (Barnes and Burton, 1966). In highly cross-pollinated crops like pearl millet, the first synthetic generation developed from inbred lines carries a high proportion of singlecrosses and usually yields more than later generations. Thus, yield data from such synthetic varieties of pearl millet have little significance unless

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the varieties have been stabilized by growing them in isolation for several generations. Continued top performance of synthetic varieties is dependent upon eliminating, insofar as is possible, the selfing of individual plants. This could best be accomplished in pearl millet if seed fields were planted at seeding rates so heavy that each plant would produce only one head. However, the seeding rates required to achieve this end and the effect of such rates on seed size and yield have not been established. Where several heads are produced on one plant (as is usually the case), the first head to emerge is frequently shedding pollen as the latest-developing heads are exserting styles. Since the first head to emerge is usually a little taller than later heads, conditions for self-pollinating late-emerging heads on a several-culmed plant are excellent. Producing seed of synthetic varieties in regions with good winds and few obstructions such as trees should help to keep pearl millet synthetic varieties near their maximum yield potential. Introducing cytoplasmic male-sterility into synthetic varieties by bulking hybrids of like maturity that carry one or two different sterile cytoplasms would also help to reduce the amount of inbreeding. 4 . Chance Hybrids

From 194 I to 1946, Burton ( 1 948) compared the forage yields of six mechanical mixtures of pearl millet seed, ranging from 100% F1 hybrid seed to 100% parent inbred seed. When seeded at the conventional rate of 10 Ib. of seed per acre in 30-inch rows, a 50-50 mixture of hybridparent seed yielded as well as 100% hybrid seed. When seeded at 2 1/2and 5-lb. rates, however, yields of the 50-50 mixture fell halfway between the pure hybrid and pure parent yields. The superior performance of the 50-50 mixture at the heaviest seeding rate was believed to be due to the more vigorous hybrid seedlings eliminating most of the weaker inbreds early in their life cycle. These findings caused Burton to suggest that commercial hybrid seed of pearl millet might be produced by harvesting all seed from a field planted to a mechanical mixture of two or more inbred lines. He pointed out that in such a program, a “mixture of four self-fertile lines might be expected to give approximately 75% of hybrid seed and 25% selfed or sibbed seed, whereas a mixture of two such lines could only be expected to give 50% of hybrid seed” (Burton, 1948). A forage yield evaluation of the singlecrosses produced from many inbreds crossed in diallel combination led to the isolation of four inbred lines- 13, 18,23, and 26-thatgavegood hybrids in all possiblecombina-

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tions. In repeated yield trials, seed harvested from a field planted to a mixture of equal numbers of pure, live seeds of these four inbreds yielded as well as doublecross hybrid seed from the same inbreds and produced 50 percent more forage than the common checks. Further study revealed that the chance-hybrid seed contained about 75% of hybrid seed as expected. Named “Gahi (Georgia hybrid) -1,” this chance hybrid was released to the public in 1958 (Burton, 1958b). Since its release, the National Foundation Seed Project has increased the Gahi inbreds and prepared the seed mixture used by commercial seedsmen to produce Gahi- I pearl millet seed. By substituting four dwarf inbreds-D1, D2, D3, and D4-for Gahi-1 lines, we were able to produce seed of Gahi-2 that could easily be harvested with conventional combines (Burton and Fortson, 1966). Because the dwarfs were different, their six possible hybrids were as tall as hybrids between normal lines. When grown for forage, the tall Gahi-2 hybrids crowded out the dwarf selfs and sibs and gave yields comparable to 100% tall hybrid seed. Although Gahi-2 usually has produced less forage (about 5%, on the average) than Gahi-I, we believe it would yield as well if its parent lines were dwarf forms of the Gahi-1 parents. Athwal(l966) reports that “More than two decades ago, the work was taken up in India to develop high grain-yielding hybrids. The usual method for the production of hybrid seed was to grow the parental lines in mixture and let them cross among themselves. The resultant seed was expected to contain about 40% hybrid seed if the two parents flowered about the same time. The hybrids developed in this manner were released for cultivation in Madras (Rao et al., 195 1) and Maharashtra (Chaven et al., 1955). However, the superiority of the hybrids over the openpollinated varieties ranged from 10 to 25% only. These hybrids had a narrow range of adaptability and failed to perform better than the openpollinated varieties in other states.”

5 . Cytoplasmic Male-Sterile Hybrids The discovery of cytoplasmic male-sterility in pearl millet and the development and release of two distinctly different usable male-sterile lines made possible the production of commercial singlecrosses (Burton, 1948, 1965a,b). Although two additional sources of male-sterile cytoplasm have been discovered (Burton and Athwal, 1967), most of the work to date has been carried out with the (A1) source, the first sterile cytoplasm released.

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The remarkable speed with which Indian pearl millet breeders were able to develop very high grain-yielding hybrids using our male-sterile lines makes one of the most outstanding plant breeding success stories of all time (Rachie, 1966a,b; Athwal, 1966). In early 1962, we supplied K. 0. Rachie, coordinator for the All-India Pearl Millet Breeding Program, with seed of Tift 23A and Tift 23B. This seed, sent to Coimbatore, enabled Indian breeders during the winter of 1962- 1963 to make many hybrids on Tift 23A, using locally derived and relatively early-generation lines as pollinator parents. Similar hybrids were also produced from March, 1963 plantings by other Indian breeders at Delhi, Jamnagar, and Ludhiana. Up to 256 of these hybrids were tested in the monsoon season (July sowings) of 1963 at six locations in the major pearl millet-growing regions of India and at five other locations in intermediate trials. In these trials, the best 5 % of the hybrids produced 103% more grain than the local check varieties. Unfortunately, none of the pollinators used in the first hybrids restored fertility to all their hybrid offspring (Rachie, 1966a). To improve fertility restoration, some 1900 selfed plants from lines giving the best fertility restoration were immediately testcrossed on Tift 23A at Coimbatore and Jamnagar during the winter of 1963-1964. By immediately planting these testcrosses in observational rows, it was possible to evaluate their fertility status and select several good fertility-restoring pollinators in time to make hybrid seed for testing during and immediately following the 1964 monsoon season. The best of these hybrids, tested at 27 locations (representing the major pearl millet-growing areas of India), yielded about twice as much grain and 9% more forage than the local checks. Additional advantages of the hybrids over the open-pollinated checks were greater uniformity (less bird damage), better forage quality, and a much higher level of resistance to the green ear disease. A description of the release and seed increase of HB-1, the first hybrid from this program, follows (Rachie, 1966a): “The All-India Millets Workers Seminar, held at Delhi February 13-14, 1965, unanimously decided to release the hybrid, Tift 23A X BIL-3B, developed by Dr. D. S. Athwal of the Punjab Agricultural University at Ludhiana, and tested cooperatively at 20 locations, ranging from 11” to 3 1” N. latitude in India. The Central Varieties Release Cornmittee, constituted by the Indian Council of Agricultural Research, considered the recommendation of the conference and officially released this hybrid on March 12 as ‘Hybrid Bajra Number 1 (HB-I).”’ By immediately planting seed-production fields to the first parental stocks released,

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eight seed growers were able to produce over 10,000 Ib. of HB-1 seed in time for July, 1965 sowings. HB-1 hybrid seed was produced on 4000 acres in 1966. The success of the F1hybrid in India indicates that future improvement of pearl millet for grain production will most certainly be directed toward developing better hybrids. Generally, those methods found successful in improving maize hybrids should apply. A few specific suggestions follow (Athwal, 1966; Burton, 1966b; Rachie, 1966a). Introducing the d2 gene used to dwarf Tift 23A and B into superior males, such as BIL-3B, through a backcrossing program should quickly make possible the production of dwarf forms of such highly successful hybrids as HB-I. The dwarf Tift 23DA and 23DB lines, near-isogenic with Tift 23A and Tift 23B, released in 1967, will facilitate the development of these dwarf hybrids (Burton, 1967). The release in 1968 of Tift 239DAz and Tift 239DBz in (Az)cytoplasm (dwarfed with the d2 gene) offers many breeding possibilities. Since Tift 23DB will restore fertility to Tift 239DAr and Tift 239DB2 will restore fertility to Tift 23DA, many lines rejected for lack of fertility-restorer genes for (A1) cytoplasm may carry fertility-restorer genes for ( A r )cytoplasm. These two cytoplasmic male-sterile systems may also be used as previously suggested to increase the probability of cross-fertilization in synthetic varieties. Where complete fertility restoration is required, time and effort can be saved by testing for fertility restoration early in the inbreeding program. Nonrestorers may either be eliminated or if promising, may be crossed with fertility restorers to introduce the major gene for fertility restoration. A bulk-self method that we have used for a number of years is recommended as an economical and effective way of maintaining diversity while moving heterozygous varieties toward a state of homozygosity. This method consists of bagging single heads on 10 to 15 good plants of each line. These heads are tied together when harvested and are threshed as a single unit. A sample of this bulked seed is used to plant the next generation. At the F5 generation, the bulked material is evaluated for yield on one or more testers. Further selection within the best bulks will immediately give highly uniform inbred lines. This procedure involves very little effort and places selection pressure on seed yield per head and self-fertility. Since it is possible to grow three generations per year either with or without greenhouse facilities in most of the pearl millet-growing areas of the world, heterozygous varieties can, thus, be rapidly advanced to a stabilized homozygous state before measuring their performance in testcrosses.

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Hybrids used for forage need not have the uniformity of those used for grain. Recognizing this fact, we are developing a late-maturing (short-day, photoperiod-sensitive) male population that may be used to produce the commercial hybrid, Tift 23A X late synthetic. Selected plants from our late synthetic are selfed and are also used as males on Tift 23A. These testcrosses are evaluated in 9 X 9 lattice-square yield trials from May I until September 15. Selfed seed of the top 20 males in each trial are planted in 8-foot rows in the greenhouse about September 25. As these flower in December each group of 20 are intercrossed in two 10-unit diallels to give a total of 180 diallel crosses. These crosses, planted in the greenhouse in January in 2-foot plots and thinned to four plants per plot, are selfed and are again crossed on Tift 23A. Seed from these crosses are mature and ready to plant in the field by May 1. Two 25 X 30-foot greenhouse sections without benches have been adequate for the winter plantings. The procedure allows us to complete one cycle of improvement each year. Forage-yield studies, comparing hybrids made with males from the first and second cycles, show a 10% increase in forage yield. Our objective in this study is to build up in the late population the frequency of genes that combine with Tift 23A to give forage yields. The preliminary yield data suggest that this objective is being realized. Preliminary observations by W. R. Cowley, Superintendent, Lower Rio Grande Valley Research and Extension Center, Weslaco, Texas, suggest that a September 15 to 25 planting of Tift 23A and the late synthetic in the Brownsville area of Texas will bring both lines into flower at about the right time to produce F, hybrid seed. A similar breeding procedure, using a male population of the same maturity date as Tift 23A, has not proceeded far enough to indicate the advance in forage yield that may be expected.

6 . Species Hybrids Most interspecific hybrids of pearl millet are partially o r completely sterile, and are not likely to be useful in improving grain yields. Certain combinations between Pennisetum species, particularly P . typhoides X P . purpureum, exhibit heterosis for forage yield and may become important as forage crops. The first controlled P. purpureum x P . glaucum ( P . typhoides) hybrids were made in the fall of 1941 (Burton, 1944). The cross was easily made and 134 F, hybrids were grown to maturity the following year. Of these, 49 were chlorophyll deficient and were about one-tenth the size of the green plants. The remaining 85 hybrids were green and varied greatly in size and growth habit. A number of these hybrids checked cytologically contained the expected 2 1 chromosomes and were self-sterile. Forty per-

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cent of the hybrids winterkilled, and most of the survivors in the spring were less vigorous than the P . purpureurn parent. (At Tifton, P . typhoides always fails to survive the winter when left in the field.) Five of the best hybrids showed considerable economic promise and were sent to several tropical countries for evaluation. Although these hybrids grew well and could have been propagated by stem cuttings (as is P . purpureum), they flowered profusely over a long period of time and were not released. The use of a short-day, photoperiod-sensitive P . typhoides parent instead of the early, day-neutral variety used in making these hybrids should overcome this profuse, long-season flowering habit. Patil ( 1963) described two vegetatively propagated P . purpureurn X P. typhoides hybrids named “Pusa Napier 1” and “2,” that gave high forage yields and offered promise for winter forage in India. Napiergrass, Pennisetum purpureum, has great yield potential and has frequently outyielded all grass species in tropical climates. However, the common practice of propagating this bunchgrass by the slow and laborious method of planting stem or crown cuttings has greatly restricted its use. If napiergrass or similar hybrids could be propagated with pearl millet-like seeds, its use would probably be increased many fold. In an attempt to produce millet X napier hybrids, we crossed Tift 23A pearl millet with pollen from ‘Merkeron’ napiergrass in the greenhouse in the fall of 1964 (Powell and Burton, 1966b). The seed set on Tift 23A was excellent and when planted in the field in yield trials, the hybrid seed so produced gave annual forage yields equal to those from Gahi-1 pearl millet. Although the hybrid grew much slower in the spring, it outyielded Gahi- 1 from midseason on. In more tropical climates, where it would not winterkill, this or similar hybrids could behave as a perennial and might perform as well, or better, than napiergrass. A report from Ibadan, Nigeria indicated that Tift 23A X Merkeron flowered too early and too much to be a good forage crop at that latitude. Substituting short-day, cytoplasmic male-sterile lines for Tift 23A should solve this problem. Since napiergrass usually flowers in November, commercial seed production would be possible only in areas frost-free until late December. The cytoplasmic male-sterile Tift 23A or other available cytoplasmic male-steriles could be seeded between rows of the perennial napiergrass to produce F1 hybrid seed. The perennial habit of napiergrass should reduce seed-production costs by making it unnecessary to reestablish the male planting each year. Preliminary observations by Dr. A. E. Kretschmer, Jr., Indian River Field Laboratory, Fort Pierce, Florida, suggest that a planting of Tift 23A, made about September 15, will come into flower at the right time to be pollinated by Merkeron pollen at his latitude.

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C. GENOTYPE EVALUATION 1 . Forage Production A plant breeder’s success in varietal improvement is determinec in no small measure by the precision of the screening devices used to select superior plants. Characters with low heritabilities, such as yield, are particularly vulnerable to environmental effects. Soil heterogeneity may often alter performance to the extent that superior varieties will be discarded. This problem becomes more intense as the number of varieties to be tested in field plots is increased. The dearth of literature concerned with these problems makes it seem desirable to describe some of the procedures and techniques that have helped us to measure effectively the forage yields of thousands of plots each year. The first requirement for a good yield trial is good seek. We like to harvest pearl millet seed about 5 weeks after anthesis and hang it in a dry section of a greenhouse for drying. There it is kept until time will permit threshing-usually 1 to 3 months. Seed, threshed carefully to avoid damage, is then stored at 40°F. in airtight containers or in a dehumidified room. Yield trials are located in fields that carry the least amount of soil heterogeneity. These fields are planted to a summer legume, such as velvet beans, every other year to reduce soil pests, improve growing conditions, and make the soil more uniform. We have found that single rod-row plots with 3-fOOt alleys between and at the ends of the rows give yields that correlate well with yields under farm culture. By using the 9 X 9 lattice-square design, which permits the evaluation of 8 1 entries in one test, we have been able to obtain an average C.V. of 6.6% for annual forage yield and an average relative efficiency of 197% (Burton and Fortson, 1965). Our computer program (available on request) for analyzing data from such experimental designs, makes their use highly desirable for precision testing. Fertilizer is applied in rows prior to planting with a standard farm fertilizer applicator set to ridge the soil over the band of fertilizer. After a rain, the seed are planted with a machine that levels the ridge of soil ahead of the planter. This procedure destroys weeds, gives a smooth, level soil surface to permit precision planting at a uniform depth, and makes moist soil available to extend the planting period several days after a rain. Seed to plant a plot at a rate of about 10 Ib. per acre is measured into prenurnbered, 2 1/4 x 3 1/2-inch kraft packets. These packets are then arranged in order by rows according to field plan. Special cone-type, seed

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distributors, attached to two standard disk-type planters, give uniform seed distribution and enable a tractor driver and two men to plant over 700 plots per hour in fields where 45 to 50 plots can be arranged end to end. Spraying with an insecticide, such as chlordane, behind the planter helps to ensure against stand loss by insects. A power-takeoff forage chopper, such as the New Holland 606, is used to harvest the plots three or four times during the growing season. A trailer, pulled behind the chopper, funnels the chopped forage into standard 30-gallon plastic garbage containers. Four men on the trailer fill the containers, adjust the weighing device, record the weight, and empty the containers into a hopper that meters the chopped forage onto the soil between the rows. With this equipment, a tractor driver and four men can obtain green yields from three plots per minute. Similar genotypes, cut at the forage stage (usually 3 to 5 feet tall) have not differed significantly in dry-matter content. Thus, taking occasional forage samples to give a dry-matter factor for converting green weights to dry weights has been adequate in such trials. Skips in stand, if 18 inches or greater in length, are recorded. These skip measurements are used with suitable formulas (developed from experiments designed to measure skip effects on yield) to adjust yields on plots where stands are not good. 2. Grain Production

Many of the procedures and techniques used for establishing forage yield trials should also apply to grain-production tests. Seeding rates will, no doubt, need to be altered. Particular attention must be given to control of birds and insects that destroy grain. If these pests cannot be completely controlled, it may be necessary to calculate seed yields from a head count per plot and seed yields obtained from a few heads protected by insecticide-treated bags. Where singlecrosses are being tested and head size is quite uniform, the average grain yield of a few protected heads multiplied by the number of heads per plot may give acceptable grain yields.

E. CONCLUSION Pearl millet has great economic potential for forage and grain production. Its unusual diversity, flexibility, and responsiveness suggest that its improvement as a useful crop for man has only begun. At the moment, no crop seems better able to supply the major food requirement for man and beast in the dry, infertile lands of the tropics. Few organisms of economic worth are so well suited to basic cytogenetic and plant breed-

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ing research. Only the limits imposed by man’s imagination and industry will determine the ultimate role of this crop in the affairs of men. REFERENCES Abifarin, A. 0. 1966. Sols. Africains 11, 139- 142. Ahluwalia, M., and Patnaik, M. C. 1963. IndianJ. Genet. Plant Breeding 23,34-38. Ahluwalia, M.,and Shankar, K. 1964. Sci. Cult. (Calcutta) 30,340-341. Ahluwalia, M . , Shankar, K., Jain, S. K., and Joshi, A. B. 1962. Indian J . Gene?. Plant Breeding 22,45-53. Ahluwalia, M., Shankar, K., and Rachie, K. 0. 1963. Curr. Sci. (India) 32,32 1-322. Al-Fakhry, A. K., Sarvella, P., and Grogan, C. 0. 1964.J . Hered. 55,57-60. Athwal, D. S. 1965. Indian Farming 15,6-7. Athwal, D. S. 1966. IndianJ. Genet. Plant Breeding 26A,73-85. Athwal, D. S.,and Luthra. R. C. 1964. Indian Farming 14, 14. Athwal, D. S., Gill, G. S., and Gill, B. S. 1966. J . Res. tudhiana 3,122-1 3 I . Avdulov, N . P. 1931. Bull. Appl. Botany Genet. Plant Breeding Suppl. (Leningrad) 43, 428 p. (In Russian). Aykroyd, W. R., Gopalan, C., and Balasubramanian, S. C. 1963. Indian Council Med. Res. (6th Rev. ed.). Ayyangar, G. N . R., and Hariharan, P. V . 1935. Madras Agr. J . 23,394-397. Barnes, D. K., and Burton, G. W. 1966. Crop. Sci. 6,2 12-2 13. Baurnan, L. F. 1959.Agron. J. 51,53 1-534. Bilquez, A. F. 1963.Agr. Trop. (Bogota) 12,1249-1253. Burton, G. W. 1944.J . Hered. 35,226-232. Burton, G . W . 1948. J . A m . Soc. Agron. 40,908-9 15. Burton, G. W. 1950. Agron. J . 51,479-48 I . Burton, G. W. I95 I . Agrott. J . 43,409-4 17. Burton, G. W. 1952.Agron. J . 44,424-427. Burton, G . W. 1958a.Agron. J . 50,230. Burton, G. W. 1958b. Progressive Farmer, April, pp. 138-139. Burton, G. W. 1962. Crop. Sci. 2,356. Burton, G . W. 1965a. Crops Soils 17,19. Burton,G. W. 1965b. CropsSoils 18,19. Burton, G. W. 1 9 6 5 ~Crop . Sci. 5,333-335. Burton, G. W. I966a. Proc. fOth Intern. Grassland Congr., Helsinki, p p . 720-723. Finnish Grassland Assoc., Helsinki. Burt0n.G. W. 1966b.AfricanSoils 11,39-42. Burton, G. W. 1967. Georgia Agr. Res. 9,6. Burton, G . W. 1968. Crop Sci. 8,229-230. Burton, G . W., and Athwal, D. S. 1967. Crop Sci. 7,209-2 I 1. Burton,G. W..and DeVane, E. H. 195 I . Southern Seedsman, March, 17-18. Burton, G. W., and Fortson, J . C. 1965. Crop Sci. 5,595. Burton, G . W.. and Fortson, J. C . 1966. Crop Sci. 669-72. Burton, G . W., Knox, F. E., and Beardsley, D. W. 1964.Agron. J; 56,160- 16 I . Burton, G. W., and Powell, J. B. 1965. Crop Sci. 5 , l - 3 . Burton, G . W., and Powell, J . B. 1966.Crop Sci. 6, 180- 182. Burton, G. W., Hart, R. H., and Lowrey, R. S. 1967. Crop Sci. 7,329-332. Carnahan, H. L., and Hill, Helen D. I96 I . Botan. Rev. 27, I- 162. Chandola, R. P., Bhatnagar,M. P.,andTotuka, I . 1963.Curr. Sci. (India) 32,179-180.

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GLENN W. BURTON AND JERREL B. POWELL

Chavan, V. M., Patil, J . A.,and Chaudhari, B. B. 1955. Poona Agr. Coll. M a g . 46,148-150. Cooper, R. B., and Burton, G. W. 1965. Crop Sci. 5,18-20. Curtis, D. L., Burton, G. W., and Webster, 0.J. 1966. Crop Sci. 6,300-30 I . Dowker, B. D. 1963. E. African Agr. ForestryJ. 29,52-57. Durand, Y. 196 I , Agron. Trop. (Nogent-Sur-Marne),16,255-258. Etasse, C . 1966. African Soils 11,269-275. George Washington University. 1967. “The Millets; A Bibliography of the World Literature Covering the Years 1930-1963,” Biol. Sci. Commun. Proj., 154 pp. Scarecrow Press, Metuchen, New Jersey. Gildenhuys, P.,and Brix, K. 1961.Zuechter 31,125-127. Gildenhuys, P., and Brix, K. 1964. Heredity 19,533-542. Gildenhuys, P., and Brix, K. 1965. Ann. Botany 49,709-7 15. Gill, B. S., and Gupta, A. K. 1966. J. Res. Ludhiana 3 , l 18-1 2 I . Gill, B. S., Sraon, H. S., and Minocha, J. L. 1966.5. Res. Ludhiana 3,260-263. Gupta, V. P.,and Athwal, D. S. 1966a. J . Res.Ludhiana 3,l 1 1 - 1 17. Gupta, V. P., and Athwal, D. S. 1966b.J. Res. Ludhiana 3,379-383. Hitchcock, A. S., and Chase, Agnes. 195 I . US.Dept. Agr. Misc. Publ. 200,727,933. Hrishi. N. J. 1952. Genetica 26,280-356. Jain, S. K., Ahluwalia, M., Shankar, K., and Joshi, A. B. 1961. Indian J. Genet. Plant Breeding 21, 175- 184. Kadam, B. S., Patel, S. M., and Kulkami, R. K. 1940. J . Hered. 31,201-207. Kajiari, N. B., and Patil, J. A . 1956. Indian J . Genet. Plant Breed. 16,146. Khan, M-UD-D., and Rahman, H-UR. 1963. West Pakistan J . Agr. Res. 1,61-65. Krishnaswamy, N . 1962. Indian Council Agr. Res., Cereal Crop Ser. 11. Krishnaswamy, N., and Ayyangar, G . N. R. I94 I . J . fndian Botan. Soc. 20, I I 1 - 1 17. Krishnaswamy, N . , and Ayyangar, G. N. R. 1942. Proc. Indian Acad. Sci. 16,l-9. Krishnaswamy, N., and Raman, V. S. 1953. Cytologia (Tokyo) 18,305-33 1. Krishnaswamy, N., and Raman, V. S. 1954. Genetica 27,253-272. Krishnaswamy, N., Raman, V. S., and Menon, P. M. 1949. Proc. Indian Acad. Sci. 30, 195-206. Krishnaswamy, N., Raman, V. S., and Nair, N. H . 1950. Curr. Sci. (India) 19,252-253. Leuck, D. B., and Burton, G. W. 1966. J. Econ. Entomol. 59, 1308-1309. Menon, P. M. 1959. Curr. Sci. (India) 28,165-167. Menon, P. M., and Devasahayan, P. 1964. Madras Agr. J. 51,70. Menon, P. M., and Krishnaswami, S. 1963. Madras Agr. J. 50(6), 241-244. Meredith, D. 1954. “The Grasses and Pastures of South Africa,” p. 448. Cape Times Ltd., Parow, C. P., South Africa. Muntzing, A. 1958. Trans. Bose Res. Inst. (Calcutta) 21,l-15. Murty, B. R. 1966.African Soils 11,456-462. Nabos, J. 1966.African Soils 11,365-381. Pannikkar, M. R. 1962. Indian Farming 11,13,30. Pantulu, J. V. 1958. Curr. Sci. (India) 27,497-498. Pantulu, J. V. 1960. Curr. Sci. (India) 29,28-29. Pantu1u.J. V. 1967. Nature 213,101-102. Patil, B. D. 1963. fndian Farming 12,20and 23. Patil, B. D., and Sing, A. 1964. Curr. Sci. (India) 33,255. Patil, B. D., and Vohra, S. K. 1962. Curr. Sci. (India) 31,345-346. Patil, B. D., Hardas, M. W., and Joshi, A. B. I96 I . Nature 189,4 19-420. Powell, J. B., and Burton, G. W. 1966a. Crop Sci. 6,13 1- 134.

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Powell, J. B., and Burton, G. W. 1966b. Crop Sci. 6,378-379. . Sci. 6,590-593. Powell, J. B.,and Burton, G. W. 1 9 6 6 ~Crop Powell, J. B., and Taylorson, R. B. 1967. Crop Sci. 7,670-672. Rachie, K. 0. I966a. Africun Soils 11,429-439. Rachie, K. 0. 1966b. Span 9,49-53. Rao, P. K., Nambiar, A. K., and Menon, P. M. 1951. A4adrusAgr.J. 38,95-100. Raman, V. S., and Krishnaswarni, D. 1960.J . Indian Bot. Soc. 39,382-385. Raman, V. S.. Krishnaswarni, D., and Nair, M. K. 1962. J. Indian Bot. SOC. 41,268-270. Rangasami, K. 1935. J . Indian Bot. Soc. 14,125- 13 I . Rangasamy, S. R. L a n d Ponnaiya, 6. W. X. 1963. A4adrusAgr.J. 50,239-241. Vavilov, N. 1. 1949-50. C h o n . Boran. 13,l-6.

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MOVEMENT OF NUTRIENTS TO PLANT ROOTS S. R. Olsen a n d W. D. Kemper United States Department of Agriculture, Colorado State University Agricultural Experiment Station, Fort Collins, Colorado

I. 11.

Introduction ....................................................................................... lnterdiffusion of Ions of Like Charge ................................................... A. Anions .......................................................................................

..........................

................. A. General Theory and Equations ...................................................... B. Effects of Soil Properties on Salt Diffusion ......................

91 92 92 I10 I20 I20 129 I30 130 I33

B. Solutions for Transient-State Conditions .......................................... C. Comparison of Diffusion and Convection as Mechanisms for Bringing Nutrients to the Root Surface 13.5 V. Role of the Plant _ _ .......................................... ................. 142 A . Anatomical Features of Roots ........................................................ 142 B. The Plant Root as a Sink for Nutrients ................................. 143 C. Relation between Water Transport and Ion Transport in Plants 14.5 D. The Relation between Ion Uptake and Concentration I45 E. Root Systems as Related to Diffusion .............................................. 146 F. Distances from the Root to Which Nutrients Are Extracted ... I48 G . Effect of Water Content of Soil on the Absorption Mechanism of the Root ................................................................. 149 References ......................................................................................... I49

I. Introduction

Adequacy of a plant nutrient is usually determined by the concentration of the nutrient at the plant root surface. Because a surface is, by definition, infinitely thin, the quantity of a nutrient contained in a surface is practically negligible. Consequently, plant roots remove significant amount of nutrient from this surface only if the nutrient can move readily from the interior of the soil to this surface. Although nutritional adequacy has historically been characterized most often by the amount of the nutrient in the soil, the rate at which the 91

92

S. R. OLSEN AND W. D. KEMPER

nutrient can move to the surface is an equally important factor. In general, this movement will occur either by diffusion (net movement of the nutrient by thermal motion resulting from the existence of a concentration gradient) or by convection (conveyance of the nutrient to the surface as a result of motion of the solution in which the nutrient resides). The pioneering concepts of Jenny and Overstreet (1939) on contact exchange and the principles expressed by Bray (1954) on mobility of nutrients have stimulated others (e.g., Schofield and Graham-Bryce, 1960; Barber, 1962; Low, 1962; Olsen et at., 1962) to quantitatively evaluate rates at which nutrient ions diffuse in soils. Gardner (1965) published an excellent review on the movement of nitrogen in soil. More data have now accumulated on movement of nutrients in general and more quantitative analytical treatments of ion movement to plant roots have been proposed. An important area of recent study has been use of available knowledge of soil chemistry (i.e., exchange reactions, adsorption isotherms, chelation, etc.) along with the physical processes of diffusion and convection in the development of equations to describe movement of nutrients to roots. In this review we will attempt logically to outline the present knowledge of the subject rather than attempt to review, interpret, and evaluate each individual publication. II. lnterdiffusion of Ions of Like Charge

A. ANIONS

1. General Theory and Equations a . Dejnition of Components of the General Equations. Fick's law for steady-state diffusion is given as

A Q = -DA AC At

Ax

where A Q is an amount (grams; moles) diffusing in time A t t is the time (seconds) D is the diffusivity or diffusion coefficient (cm.2 set.-') A is the cross-sectional area (cm.2) C is the concentration (g. ~ m . or - ~moles ~ m . - ~ ) x is the distance in the direction of net movement of ions o r molecules (centimeters) When ions or molecules diffuse through water in soil or other porous

MOVEMENT OF NUTRIENTS TO ROOTS

93

media, several geometric, physical, and chemical properties of the system must be considered. Porter et al. ( 1 960) discussed the major effects ~ Eq. (l), where L of the geometric factors and introduced ( J ~ / L 8, )into is the macroscopic distance between two points, L , is the actual distance through which the ions diffuse, and 8 is the volumetric moisture content or fraction of the porosity through which ions may move. The fraction, LIL,, appears twice because it has two effects on diffusion, viz. dCldx is less because of the longer path, and the microscopic pathway is narrower than A 13 by the factor LIL, because the path is at an angle to the macroscopic pathway. The viscosity of the first few molecular layers of water on clay mineral surfaces is greater than that of bulk water (Kemper et al., 1964). Diffusivity of ions is inversely proportional to the viscosity of water, so the factor, a , can be included in Eq. ( I ) to account for this effect, where a is the relative mobility or fluidity of the water. When the available pathway for diffusion is through a sequence of large to small to large pores in soil, negative adsorption which excludes relatively more anions from the small pores, tends to restrict movement of the anions. The factor y can be included in Eq. (1) as a measure of this phenomenon. If the pore width is uniform y will be unity, and y will be smaller than unity if pore width varies (Porter et al., 1960; Van Schaik and Kemper, 1966). As modified, Eq. (1) is

' Q - - D ( L / L , ) ~ A o a y AC At Ax

--

If we define D , a s the diffusivity o r diffusion coefficient of the nutrient in solution of the soil or porous media where

and q = Q / A , then a s At and Ax approach zero, Eq. (2) may be expressed as

where C is the concentration of the soil solution. If one wishes to express the concentration in terms of the concentration C , , the quantity per unit volume of soil, C , = C 8, and D , = DJO. Transient conditions are more common in soils than the steady-state

94

S. R. OLSEN AND W. D. KEMPER

conditions discussed above. Equations for transient conditions may be derived from Fick’s first law and the equation of continuity. The latter is simply the statement that the rate at which a substance accumulates in a volume element, v, (e.g., for one-dimensional flow, v = 1 x 1 X d x ) is equal to the rate at which a substance enters minus the rate at which the substance leaves. In equation form for one-dimensional flow, this may be expressed as

substitution of Eq. (6) into Eq. ( 5 ) and dividing both sides by Odn, gives

Eq. (7) applies for anions or molecules dissolved completely in the soil solution such as C1- or NOs-, where D,/O is assumed to be constant b. Dirusion plus Reaction. Some species of ions or molecules are present in both the adsorbed and solution states in the soil. Because diffusion moves ions into or out of a volume element rather slowly, local equilibrium between the solution and adsorbed states can often be assumed (Olsen and Watanabe, 1966). The quantitative relationship between the two states can be expressed by absorption isotherms obtained from equilibrium studies. To obtain Eq. ( 5 ) in terms of the variables C, t, and x, q ’ must be expressed in terms of C,or q ’ =f(C), wheref(C) is some function of the concentration. Since aq’lat =-

a,4d E,then __

and by substituting Eq. (8) into Eq. ( 5 ) ,

A quantitative relation between the solid and liquid phases can be expressed by an adsorption isotherm,

95

MOVEMENT O F NUTRIENTS TO ROOTS

C‘

=

S

+ 8C + constant

(10)

where S is the amount of diffusible ion adsorbed by the solid phase in grams per cubic centimeter of soil, and C ’ is the total amount of diffusible ion (solid plus solution) per cubic centimeter of soil. When S = bC k (where b and k are constants) and C’ is plotted against C , the slope of the line equals ( b O ) , or it is the capacity factor. Therefore, for this interaction case, q’ for Eq. (6) becomes q’ = C ( b 8 ) v constant. Since q’ =f(C), then

+

+

+

+

arf(c)]iac= ( b +

(1 1)

and by substituting Eq. ( I 1) into Eq. (9)

+

If C ’ is plotted against C t l ,then S = b ’ C , k ’ , and the slope of the line equals (b’ I ) and substitution into Eq. (5) gives,

+

Thus, 6 ’ = bI8. When b + 8 , it is important to note that in one case the slope (b’ I ) depends on the value of 8, whereas in the other case the slope ( b 0 ) is nearly independent of 8. Equation (4) may be expressed in terms of C ’ , the concentration of diffusible ion or substance per cubic centimeter of soil,

+ +

By proceeding in an analogous manner from Eq. (5) to Eq. ( I 2), where q ’ = C ’ d x , Eq. (15) may be derived,

D ’ is the diffusion coefficient of the diffusible or mobile ions per cubic centimeter of soil. D’ will have the same numerical value as D,l(b 8 ) or D,/(b‘ 1). Diffusivity values obtained from transient experiments and mathematical solutions of Eqs. (12), (13), or (15) are generally labeled as an apparent diffusion coefficient and are really D J ( b O ) , or D,/(b’ I). Under certain conditions indicated later it is necessary to

+

+

+

+

96

S. R. OLSEN AND W. D. KEMPER

+

determine values for ( b f 0 ) or (b’ I ) in order to use D ’ , D , or D , . In many published solutions for the transient state (e.g., Carslaw and Jaeger, 1959), both D,, and ( b 0 ) must be known because these solutions are expressed in terms equivalent to both D , and D , / ( b 0). Equations ( 1 2), (1 3), and (1 5) are identical to the equations for heat flow (Carslaw and Jaeger, 1959), and solutions for many boundary values for Eq. ( 12) and its two- and three-dimensional counterparts have been pub1i shed. If the total amount of diffusible nutrient per cubic centimeter of soil (C ’) is an adequate measure of the concentration, many of Carslaw and Jaeger’s equations may be reduced to forms where D , / ( b 0) is the only “diffusion coefficient” necessary. However, when concentration of the nutrient per cubic centimeter of soil solution (C) is the chief factor affecting uptake and chemical reactions in the soil, C ’ is not a satisfactory concentration parameter. c. Boundary Conditions and Available Solutions. Equations of Carslaw and Jaeger (1959, pp. 335-34 1 ) describing the flow of heat have been readily adapted for the solution of problems in diffusion. This adaptation requires that K = D,,, V = C and k = K / p c or D,,/(b 0 ) where Kis the thermal conductivity, D , is the diffusion coefficient of the nutrient in solution for the soil or porous media, k is the thermal diffusivity, pc is the volumetric heat capacity and ( b 0 ) is the volumetric “ion capacity” as defined previously, V is the temperature, and C is the concentration in the soil solution. These substitutions apply to Eq. (12), but a similar adaptation may be made for Eq. ( I 3) by using the corresponding terms. Further, when S = 0, viz. for CI- or NOJ-, both b and b ‘ will be zero and they disappear from Eqs. (12) and ( 1 3), or Eq. (7) will then apply and a corresponding equation based on C and D o . Equation ( 15) can be used for two boundary conditions, viz., assuming a at the root surface and assuming a constant rate constant concentration C,. of uptake. When D ’ = D , / ( b O), D ’ can be substituted for K and k, and C’ for C. These substitutions apply for calculating the flux to a root [Eq. ( 1 S)]. If information is desired about the concentration distribution from the root surface into the surrounding soil [Eqs. ( I 9) and (2 l)] and the adsorption isotherm follows Eq. (lo), then values for C,, and C,. corresponding to CO’and C,.’ must be taken from the linear portion of the istherm. For the boundary condition, assuming that the flux is proportional to the concentration at the root surface, values must be known for ( b + 0 ) and C if D ’is to be used (Nye, 1966b). Another disadvantage of Eq. (15) is that differences between soils in their P-supplying power (Olsen and Watanabe, 1963) would be more difficult to explain on the

+

+

+

+

+

+

MOVEMENT OF NUTRIENTS TO ROOTS

97

basis of variations in D ' , since D ' for the three soils was essentially constant. Four different boundary conditions have been described for diffusive flow of ions to plant roots. Three boundary conditions adapted from equations for heat flow are presented in some detail and curves of dimensionless variables are shown to solve various problems in diffusion. These assumptions were made: (1) a unit of root surface at time equal to zero in an infinite medium at some initial concentration C = C o ; (2) cylindrical symmetry with respect to the center of the root cylinder which acts as a finite sink; (3) the roots are spaced so that there is no interaction between adjacent roots; and (4)the diffusion coefficient is constant at a given value of 6 and bulk density of soil. I . Rate of uptake is proportional to the concentration in solution at the root surface. Initially, C = C o , a < r < m , t = 0, where a is the radius of the root and Y is the distance from the center of the root. When t > 0, Carslaw and Jaeger ( 1959) describe this condition as,

=her where C , . is the concentration at time I, and h is a proportionality constant. The rate of uptake is related to D , , h, and C , . ,

dt

=D

, h C,.

where dqldt is the flux, F , across a unit area of root surface. When dqldt or F can be evaluated or estimated at t = 0 when C ,-= C o ,h may be evaluated. When Eq. (12) is solved subject to boundary conditions, Eqs. (16) and (1 7), a series of curves for different values of ah are found as in Fig. 1, where D,t/Ba2 appears on the abscissa and C,./C, on the ordinate, and B = ( b 0). These boundary conditions are essentially the same as those developed by Bouldin ( 1 96 1 ) for nutrient solutions,

+

F = D [ S ]r

= (I

=MC

where D is the diffusion coefficient in bulk solution and M is a constant

98

S. R. OLSEN AND W. D. KEMPER

of proportionality between rate of uptake and concentration in solution. Thus, h is equivalent to MID in the model developed by Bouldin (1961). The limitations of these boundary conditions will be indicated later. The use of Fig. 1 requires values for C , D,,, D,/B and h (where h = M / D , ) . 10

09

oa 07

06

0 V

\ro

5

V 04

03

02

01

1

2

3

4

5

6

7

Dpt/Ba

FIG.1. Change of the average concentration ratio at the root surface with Q t / B a 2 ,for use with Eq. (15). Numbers on the curves are values of ah. (Adapted from Carslaw and Jaeger, 1959, Fig. 43.)

The units of M are cm. sec-' when the uptake or flux is expressed in grams per cm2 sec-' and the concentration is in grams per cubic centimeter. Values of M must be determined in a well-stirred nutrient solution. Nye (1966b) has developed a similar model and has discussed its various uses in describing nutrient absorption by roots.

99

MOVEMENT OF NUTRIENTS TO ROOTS

2. At the root surface, Y = a, the concentration, C,, when t > 0, is constant, and C = Co when t = 0. Subject to these boundary conditions, the flux at the surface is given by Fig. 2 which was adapted from Fig. 42 of Carslaw and Jaeger ( 1 959,

I

-2

-1

0 1 loglo Dpt/ Ba2

I

2

3

FIG.2 . Variation of the flux parameter with D,,t/Bn2 by assuming a constant concentration at the root surface, t > 0. (Adapted from Carslaw and Jaeger, 1959, Fig. 42.) For ,, C , . ) ~ T . unit length of cylinder the ordinate is ( d q / d t ) /D , ( C -

p. 338). For short times, i.e., when T < 1.0, the flux at the surface may be calculated from Eq. ( I 8),

where T = D p where B Ba‘ ’ ~

= (b

+ 13)and b

Values of C , . as a function of

Y

2

13.

for various values of time may be

S. R. OLSEN A N D W . D. KEMPER

100

obtained from Fig. 3 adapted from Fig. 4 1 of Carslaw and Jaeger (1 959, p. 337) or from Eq. (19), when DptlBa2< 0.02

r-a

ierfc

ir - a )

4a 1/2

2

r:3/2

m

r-a (9a2 - 2ar - 7 r 2 )D,,t/B i2erfc 4 m 2B -

I

1

2

I

I

I

3 4 5 6

I

I

I

+

+

...I

I

810

20

40

60

r/a

FIG.3 . Values of C as a function of r for various values of D,,t/Ba2by assuming a constant concentration at the root surface when t > 0. (Adapted from Carslaw and Jaeger, 1959, Fig. 4 1 .)

Equation ( 1 8) has been integrated with respect to time (Olsen et al., 1962) to give the total amount of P absorbed by the root in time, t,

where Q is the amount in grams per square centimeter of root area. Equa-

M O V E M E N T OF NUTRIENTS T O ROOTS

101

tion (20) is reasonably accurate only when T < 1.0. For large and small values of T , a graphical integration has been made of the area under the curve in Fig. 42 from Carslaw and Jaeger ( 1 959). A plot of the dimensionless variables is shown in Figs. 4 and 5. These figures may be used to solve problems when T is large, i.e., for large values of I or to calculate Q for root hairs or roots with very small diameters. 3. The rate of uptake, dqldt, is constant. When Eq. (12) is solved for this boundary condition, two useful relationships have been obtained as shown in Fig. 6 and Eq. (21). The plot of dimensionless variables in Fig. 6 may be used to calculate C , as a function of time.

Equation (2 1) may be used to calculate C , .as a function of distance from the root surface for a given time.

T FIG.4. The average uptake parameter for small values of T , where T = D,t/Ba', for use with Eq. (20).

1

10

T

1000

100

FIG.5 . The average uptake parameter for large values of T , where T = D,t/Ba2. l.E

/.i

16

< 0-

14

12 10

0" 0.8 I

v

0 " 0.6 0.4

I

0.01

0.1

1.0

10

Dpt p a 2 FIG.6. The average flow rate parameter evaluated at the root surface assuming a constant rate of uptake per unit surface area as a function of D,,r/Ba2.(Data taken from Table I of Olsen et a/., 1962.)

103

MOVEMENT OF NUTRIENTS TO ROOTS

4. Concentration at the root surface decreases exponentially with time. Lewis and Quirk ( 1 965a,b) described this boundary condition whlch they applied to the flow of phosphorus to roots of wheat. A general solution of the equation for diffusion was not given and they solved the equation by numerical methods using a computer (Lewis, 1963). d. Discussion of Boundary Conditions and Assumptions. Each boundary condition has merits and limitations. The concentration at the root surface cannot be verified experimentally by known methods. The choice of a boundary condition will depend, therefore, on what information is wanted and on how accurately the calculated values predict the actual behavior of the root-soil system. The diffusion coefficient is assumed to be constant for a given volumetric moisture content and bulk density of soil. Variations in concentration of the solution could have a small effect on D,. Paul (1965) observed a constant diffusivity of C1- in Mallee sand as the concentration of C1varied from 0.01 to 0.028 M . Graham-Bryce (1963) found a 170-fold increase in diffusivity of 1- in Lower Greensand soil as the concentration of KI increased from 0.01 to 1.0 M . He attributed this effect, in part, to variations in thickness of the diffuse double layer which caused a greater exclusion of I - from narrow pores in the soil with the lower concentration. The concentrations used by Graham-Bryce (1 963) are unusually high for soils. Van Schaik and Kemper ( 1 966) measured the diffusivity of CI - in steady-state and transient systems with concentration of CI - varying between 0.00 1 and 0.15 M ,and the effect on D,, was small. Lewis and Quirk ( 1 962) reported a large variation in the apparent diffusivity of P in Enfield soil as the concentration of P in solution increased. When the apparent diffusivity was corrected for the potentially diffusible ions in the solid phase, however, the diffusivity of P was essentially independent of concentration (Lewis and Quirk, I965a). For nonlinear isotherms likely to be observed in soils at very low or very high concentrations of P in solution, the value of ( b 0 ) in Eq. (12) will vary and the apparent diffusivity, D,/b 0 , or D , itself may vary with concentration of P in solution. The first boundary condition of uptake proportional to the concentration at the surface has desirable features because it combines diffusive processes with a property of the roots, i.e., the proportionality constant, M , relating rate of uptake to concentration in solution. A well-stirred solution is most appropriate for determining M , in which case M equals h. When the flux is linearly related to the concentration, M will be constant but this condition usually occurs for a limited range of concen-

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S. R. OLSEN A N D W. D. KEMPER

tration. Over a wider range of concentration, M tends to be smaller as the concentration increases. Bouldin (1961) suggests selecting a value of M after 4 hours of absorption, in case M changes with concentration. The best method for determining M is to use roots comparable in age, size, and content of element, and to measure uptake from well-stirred solutions of composition and pH similar to those expected in the soil solution. Values of M could not be used reliably in this model if they were determined over a range of concentration where the mechanism of uptake was saturated, i.e., insensitive to changes in concentration. Additional details about this model have been discussed in Section V,D. The constant surface concentration boundary condition predicts an unrealistic high flux for the initial periods of uptake, but a reasonable picture for the later stages of absorption, i.e., a gradual decrease in flux with time. The equations for these boundary conditions have been solved so that Q may be calculated for various periods of time or for a large range of values of T . Thus, the curves in Figs. 4 and 5 are useful in evaluating the role of root hairs. The constant uptake rate boundary condition provides a more realistic description of the uptake in the initial stages than the constant surface concentration conditiim. The equation predicts negative values of concentration of P at a root surface, however, for long periods of time, i.e., greater than 10 days, which is physically impossible (Olsen et al., 1962). Olsen and Watanabe ( 1 966) have shown that excised roots of corn (Zea mays L . ) behaved according to this boundary condition for at least 36 hours in soil. In general, the actual boundary conditions for uptake by roots will be somewhere between the last two conditions described. Actual conditions will probably approach a condition of constant rate of uptake in the initial stages when the ability of the plant to move P or other ions through its cells is the limiting rate process. In the later stages of absorption, actual conditions may approach the condition of a low constant concentration at the root surface, with the rate of arrival of P or other ions at the root surface determining the rate of uptake. The first boundary condition will also approach the actual condition provided M is constant, or when M is known for the period of uptake.

2. Efect of Soil and Ion Properties a. Capacity Factor. The manner of adapting the equations on flow of heat to solve problems in diffusion has been indicated previously and a capacity factor was defined. This term is intended to be analogous to

105

MOVEMENT OF NUTRIENTS TO ROOTS

“heat capacity,” meaning the quantity of heat required to raise the temperature of a body one degree. Thus, the “capacity factor” from Eq. (10) is the slope of the line, or ( b 0 ) , and it is the amount (grams) of diffusible ion per cubic centimeter of soil required to increase the solution concentration by 1 unit (grams per milliliter of the soil solution). Olsen et al. ( I 962) measured the capacity factor for P in three soils by plotting labile P, the amount of P that undergoes isotopic dilution with 32 P in a 24-hour reaction, against concentration of P in the soil solution. The relationship was linear for an important range of concentration, indicating that the capacity factor (the slope of the line, i.e., b 0) was a constant. As more fertilizer P was added to these soils, slope of the line decreased conforming more nearly to a Freundlich isotherm, and finite difference computer solutions would be necessary to solve Eq. (12). For acid soils showing much smaller concentrations of P, the slope ( b 0 ) may be more variable as concentration of P in solution changes. However, values of ( b 0 ) have not been reported for acid soils in this low solution concentration range. When D, is to be measured in soil by a transient-state method involving self-diffusion of an isotope, the appropriate measure of the capacity factor is the slope of the line passing from the origin to the point representing C ‘ and C , as indicated by Nye (1966b). When movement of nutrient to a root is to be measured, the appropriate capacity factor is ( b 0 ) from Eq. (10) for the linear portion of the isotherm. Labile P is used to determine the capacity factor because it measures a fraction of the solid phase P that will readily transfer into the solution phase when concentration of the solution phase is reduced. A problem with this use of labile P is that the amount “exchanged,” determined by the usual methods, increases gradually with a longer reaction time, so the value obtained for 24 hours is somewhat arbitrary. The validity of Eq. (12) depends on the assumption that the rate of transfer of solid phase P to the solution is rapid in comparison with the rate of diffusion. The 31 P exchanging with 32P during long reaction periods is less likely to fit this requirement. Olsen et al. ( I 965) obtained evidence that the suggested use of labile P leads to reasonable experimental results. They measured D, by steadystate and transient-state methods, using Eqs. (2) and (121, respectively. The transient method requires an estimate of the capacity factor to obtain a value for D , . Values of D , on the same soil agreed reasonably well. This result implies that the value of the capacity factor estimated from labile P is reasonably accurate and that release of P from the solid phase

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106

S. R. OLSEN AND W. D. KEMPER

was rapid compared with diffusion. Apparently values of (LILe)' a y measured for diffusion of one anion in a soil are good estimates of these values for other anions of equal valence. For instance, values of ( I ~ I L , ) ~ a y obtained using CI- by Porter et al. (1960) may be combined with reported (Moreno, 1957; Salvinien et al., 1954) values of D for P in solution ( 5 X crn.2/sec.) to obtain values of D, for P practically identical to those measured by steady-state measurements on the same soil by Olsen et al. ( I 965). A capacity factor will need to be measured in all cases of anion diffusion in soil where the anion is adsorbed or reacts to form a solid phase. With the exception of P, methods have not been developed to measure a capacity factor for other anions. Such methods should be based on principles similar to those described for P, although isotopic dilution methods are not necessarily required. Ideally, the capacity factor should reflect the chemical properties of a given soil toward the anion and it should be a measure of the protentially diffusible anions in a given volume of soil when a unit change of concentration occurs in the soil solution. b. Digusion Coe@cient. (i) Moisture content. In a given soil, D , increases with a rise in 8, but in a nonlinear manner (Olsen et al., 1965) for ions adsorbed by the solid phase. Such a relationship follows from Eq. (3), because 8 is approximately linearly related to (L/Le)2a y as shown by Porter et al. ( 1960), i.e. (LIL,)' a y a I3 b, where a and b are constants characteristic of a given soil. Thus, the rate of diffusive movement of ions will be a hyperbolic function of the moisture content in Eq. (2) provided ACIAx remains constant as 8 changes. A relationship between D , and 8 for P in two soils is shown in Fig. 7. When a nutrient in solution is not adsorbed by the solid phase, (viz. CI- or NOs-) and the total amount of the nutrient per cubic centimeter of soil is maintained constant, the concentration of ions in solution is inversely proportional to 0. In this case the product 8 AC/Ax in Eq. (2) will be constant and D,, will be a linear function of 0. The effects of 8 on D , are both physical and chemical. As 8 decreases in a given soil, the cross-sectional area available for diffusion becomes smaller, the path length increases, and the viscosity and negative adsorption terms in Eq. (2) become more important as the water films decrease in thickness. When diffusion is compared in various soils, at equal moisture suctions D , increases with increasing content of clay in the soils provided the clays have similar water-holding properties (Olsen et al., 1965). The effect of 6 on D , for soils containing mostly kaolinite would be different probably from the effect in soils having montmorillonitic clays.

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MOVEMENT OF NUTRIENTS T O ROOTS

The curves of Porter et al. (1960) relating a y (L/LeI2to 0 extrapolate to zero at finite values of 0 , indicating that the water phase became practically discontinuous. The major portion of the water content in these soils at this point could be accounted for by a monomolecular layer of water on the mineral surfaces. 16

TUCUMCARI F So L

I

14

12

I

I

10

-

I

al

' " 8

/ / /

I

N .

5

r9

I

I I

V

I

I

I

/

6

X a

n

4

'APISHAPA SI CI

L

2

I

C

01

1

02

I

I

I

I

03

04

0.5

0.6

VOLUMETRIC MOISTURE CONTENT

0

FIG.7 . Self-diffusion coefficient of phosphorus in soils as a function of volumetric moisture content.

( i i ) Tortuosity factor. Porter et al. (1960) indicated that this factor ( L I J ~ goes , ) ~ as low as 0.15 at 1 bar suction and that it may go to 0.04 at lower water contents. In oriented clay (0 = 0.6 I ) Van Schaik and Kemper (1966) estimated ( L / L , ) zat 0.50. In comparing soils of differing texture, an important point is that (LIL.)' increases apparently as content of clay rises at a constant water suction.

108

S. R. OLSEN A N D W. D. KEMPER

(iii) Viscosity of water. The viscosity of water increases with decreasing distance from the clay surface (Kemper et al., 1964), and this effect on diffusion is accounted for by a in Eq. (2), where a is the relative mobility or fluidity of the water. Van Schaik and Kemper ( I 966) evaluated a from CI- diffusion in Na-clay at moisture contents equivalent to approximately 2.5 and 5 molecular layers of water on each surface, viz. 8 = 0.6 1 and 8 = 0.75, respectively. Values of a were 0.50 for 8 = 0.61 and 0.65 for 0 = 0.75. In this treatment a is a weighted relative fluidity because viscosity changes with distance from the clay surface. Similar values of a were found in a Ca-bentonite system for comparable values of 8. In soil Porter et al. ( 1 960) estimated a as about 0.8 at 0.33 bar water suction. (iv) Negative adsorption. Negative adsorption of free electrolyte in soil or clay causes a greater exclusion of anions from small pores and from narrow films of water connecting large pores. Consequently, heterogeneity of pore size reduces anion movement. This factor, y in Eq. ( 2 ) , is most important for anions because the cations exchange with a mobile fraction of the adsorbed cations and effectively bypass this restriction (Van Schaik et al., 1966). Van Schaik and Kemper ( 1 966) estimated y to to be about 0.5 for CI- diffusion in Na- or Ca-saturated bentonite. The influence of negative adsorption in Ca-clay was expected to be smaller than in the Na-clay, but a greater heterogeneity of pore size in the Caclay apparently balanced the greater negative adsorption in the Na-clay. Olsen et al. (1965) observed that measured values of D, for P were slightly lower than calculated values of D, estimated from C1- diffusion and Eq. (3). The calculations were based on the assumption that the effect of the factors a y (LIL,)!8 on diffusion of CI- was the same as on diffusion of P. In reality, over half of the P was present as HP04*- ions. Negative adsorption will be greater for divalent anions which could account for the lower values of D ,found by diffision of 32 P. In addition, the values for D , obtained by the transient-state method (Olsen et al., 1965) should be about 20% larger because in these transient measures of D, using an isotope in one block of soil diffusing into another block, the appropriate capacity factor is the isotopic dilution capacity factor C ' / C , whereas they used aC'laC = b 8. These capacity factors were not identical because for their systems C ' = bC 8C constant. Graham-Bryce ( 1 963) measured the diffusivity of I - in Lower Greensand soil with SrIz and KI at different concentrations. The diffusion coefficient was 3.9 X set.-' for 0.01 M K1, 6.6 x set.-' for M KI, and 3.3 X lopRern.:! set.-' for 0.01 M SrL. He attributed these differences to variations in thickness of the diffuse double layer, viz. 30, 3, and 15 A,,respectively, for the above solutions, and the re-

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M O V EM EN T OF N U TR I E N T S TO ROOTS

109

sulting differences in partial exclusion of I- from narrow pores in the soil. The diffusion coefficient for the 0.01 M KI appears to be unusually low for this soil with 8 = 0.25. Possibly the soil adsorbed I - which would cause a low apparent diffusivity. In contrast with Graham-Bryce (1963), Van Schaik and Kemper (1966) found a constant D , for Cl in Na-bentonite and Ca-bentonite as the salt concentration varied from 0.0038 N to 0. I5 N . In their systems the y factor for negative adsorption was about 0.5, which is much larger than the same factor estimated from the data of Graham-Bryce ( 1963). (v) Properties and Concentration of counterdiffusing ions. Whenever possible with soil, self-diffusion coefficients have been measured using isotopes because of their convenience and accuracy. As discussed by Low ( 1962), codiffusion (salt) and counterdiffusion operate generally in soil-plant systems. The participating ions have a mutual diffusion coefficient that depends on the individual diffusion coefficients and on their respective concentrations as shown by Eq. (22)

In this equation D is the diffusion coefficient, C the ion concentration, 2 the valence of the ions; the subscripts I and 2 represent, respectively, the given ion and the codiffusing or counterdiffusing ion (Jost, 1952). This equation indicates that the diffusion coefficient of the ion present in low concentration will largely control the mutual diffusion coefficient. Nye ( 1966a) has indicated that the appropriate average diffusion coefficient for the diffusion of an ion to a sink should be approximately equal, in most cases, to the self-diffusion coefficient of the ion measured at the concentration of the bulk soil. Diffusion of one ion species is changed if an appreciable electrical diffusion potential is caused by differential diffusion rates of other ions. Since there are generally many mobile cations in the adsorbed phase and these ions shift readily to “buffer” diffusion potentials, these potentials remain small in soils with appreciable exchange capacity. All the counterdiffusing ions expected to participate in diffusion of P have diffusion coefficients greater than P, viz. D X los is: C1, 2.03; N O a , 1.92; H C 0 3 , 1.20; and SO4, 1.08 (Parsons, 1959). None of these ions would be expected to decrease the diffusion coefficient of H2P04or HPOi2- ions. The mutual interdiffusion coefficient from Eq. (22) is a function of the

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S. R. OLSEN A N D W. D. KEMPER

concentration of the ions. Simple solutions of Eq. (12) require that D l r p / ( b 0 ) is a constant, where D I 2 ,refers to a porous system. There may be important ranges of concentration where the variation of D I 2 ,for anions is sufficiently small that an average value can be used for an approximate solution. The nonexact nature of methods for measuring diffusion coefficients in soil would appear to justify the use of average codiffusion or counterdiffusion coefficients, estimated from self-diffusion coefficients, in solving problems of ion movement to plant roots.

+

B. CATIONS Diffusion of cations differs from that of anions mainly in that cations may participate in diffusive movement while they are adsorbed on clay minerals. Anions either are not adsorbed or, when they are adsorbed, the adsorption forces are usually so strong and the adsorption sites are so far apart that practically no exchange takes place between anion adsorption sites without the anions going through the energy level characteristic of the equilibrium solution. Cation exchange sites are often sufficiently close together that weakly held cations (participating in the diffuse layer) can trade positions without attaining the high energies characteristic of the equilibrium solution. This mutual exhange of positions of adsorbed cations contributes to the overall cation interdiffusion. 1 . Contribution of Adsorbed Ions to Diffusion (Steady-State Case) The following derivation shows how the contribution of adsorbed ions may be included in an equation based on Fick’s first law. According to Jost (1952, p. 240), the most general equation for diffusion in the x direction is

where qs is the quantity passing unit cross sectional area, t is time in seconds, D,, C,, and p are the diffusion coefficient, concentration, and chemical potential of the diffusing component, R is the gas constant, T the absolute temperature, and x the distance coordinate. By assuming that Eq. (23) applies to the cations in solution, a similar equation may be applied to the adsorhed or “held” ions which are designated by the subscript a,

where C , is the average concentration of the adsorbed ions, g is the time

MOVEMENT OF NUTRIENTS TO ROOTS

111

average fraction of the adsorbed ions participating in diffusion (comparable to the time average fraction in the diffuse double layer) and D , is the average diffusion coefficient of these mobile adsorbed cations. The total diffusive flow may be obtained by adding Eqs. (23) and (241, and by accepting d p / d x = ( d p / d C o ) ( d C o / d x(where ), Co is the equilibrium solution concentration)

By substituting the following identities into Eq. (25), C,7= ( C s / C o )Co, dCo/Co= d In Co and d p = RT d(ln ao) where a = activity Eq. (26) is obtained

In cases where the activity coefficient does not change appreciably (i.e., interdiffusion of similar cations) d In ao/d In Co = I , and

The equilibrium solution concentration has been chosen as the concentration variable in this development. Some authors have defined their concentration variable to include the adsorbed cations, but such a variable has no well-defined chemical significance and it will be difficult to apply in systems where simultaneous chemical reaction and diffusion occurs. Since C in Eq. (27) is defined as the equilibrium solution concentration this equation applies exactly to diffusion in a plug of soil, the two ends of which are maintained at equilibrium with solutions of given concentrations. The quotient C,/Co is a factor accounting for a lower free electrolyte concentration due to negative adsorption of free electrolyte from the vicinity of negatively charged mineral surfaces, and C,/Co may be considered as a positive adsorption factor accounting for a higher cation concentration in the region of negative potential adjacent to mineral surfaces. 2. Transient State Case and the Capacity Factor To simplify the mathematical manipulations, the term in square brackets in Eq. (27) may be designated as a porous system diffusion coefficient, D,, and Eq. (27) becomes

S. R. OLSEN A N D W. D. KEMPER

112

The rate of accumulation dq’/dt in an element of unit cross section and of thickness dx is

where q ’ = l 2 8 dx (C, 4 CJ, and

Since a q ’ l a t = (aq’/aCo)(aC,/at),division of Eq. (29) by Eq. (30) yields

or as dx

-

0

Crank (1956, pp. 219-257) gives several graphical solutions of Eq. (32) in which his concentration dependent ‘‘diffusion coefficient” is really the whole term in square brackets in Eq. (32). Another method of solving Eq. (32) is the Boltzmann transformation used by Gardner and Mayhugh ( 1 958) and others in treating water flow through unsaturated soils. Unfortunately the application of meaningful boundary conditions to the transformed equation is very difficult for the cylindrical flow case, which is of prime importance in the consideration of uptake of nutrients by plant roots. Fortunately there are many cases (and important limited concentration ranges in other cases) where all the terms in the square brackets of Eq. (32) are relatively constant, and for these cases it may be written as

MOVEMENT OF NUTRIENTS TO ROOTS

113

where the numerator in the square bracket is the diffusion coefficient and the denominator is the capacity, B, of the soil-water system for the diffusing component. This ionic capacity, B , is the units of the diffusing component that would accumulate per cubic centimeter of soil as the concentration in the equilibrium solution is raised by one unit. The analogous differential equation for heat transfer is

where V represents temperature, K is thermal conductivity, pc is the volumetric heat capacity, and k = K / p c is the thermal diffusivity. a. Boundary Conditions and Available Solutions. Carslaw and Jaeger ( 1959) present many important solutions for the heat transfer equation which are readily adapted to the diffusion case by accepting the following analogies: Ctr= V , ( C , / C , ) D , + g ( C , , / C O ) D ,= K , I3 [(aC,/aCo) + (aC,,/aC,,)]= pc, and k = K / p c = D,/B. These adaptations have been made in Eqs. ( 1 8) through (2 I ) and Figs. 4, 5 , and 6, which, subject to the respective boundary conditions, are all reasonably accurate solutions for the case of interdiffusing cations as long as D,/B is reasonably constant throughout the range of concentrations considered. Note that whenever I3 occurs in Eqs. ( 1 8) to (2 1 ) and (30) to (33), it represents a capacity factor and should be replaced by B when ions interacting with the soil are considered. 3. Efect of Soil and Ion Properties on the Capacity Factor ( B )

As indicated in Eq. (33) the capacity factor

Negative adsorption of free electrolyte from solution near mineral surfaces is accounted for by aC,/aCo being less than 1.0, while the adsorptive capacity of the mineral surface is accounted for by aC,/aC,. a. Effect of Water Content 0 . As soil water content decreases, B tends to decrease. This is particularly true (on a percentage basis) if there is little adsorption of the diffusing component (i.e., aC,/aCois very small), in which case B is proportional to 0 . However, a reduction in I3 may also cause greater negative adsorption, which would cause aC,/aC,, to decrease. The amount of cation species adsorbed per cubic centimeter of soil is relatively independent of water content. Consequently the product OC,

114

S. R. OLSEN A N D W. D. KEMPER

should be relatively constant, and a decrease in water content will be accompanied by an increase in the value of C,,. It also follows that O(aC,/aC,) will be largely independent of variations in 8. When solution concentrations are small and there are many more ions in the adsorbed phase than the solution phase, aC,/aC, + aC,/aC,, and then the capacity factor is essentially independent of water content. 6 . Eflect of Exchange Capacity on B . In most soils, when the solution concentration is in the normal range (< 0.1 N ) most of the capacity of a unit of soil for an ion species is in the adsorbed or exchange phase. Under these conditions the capacity factor is usually proportional to the exchange capacity of the soil. An example of the effect of texture on B is given by Olsen et al. ( 1 962) for P diffusion. A larger surface area of the soil generally causes the capacity factor B to increase for both cations and anions. c. Efect of Other Species of Ions in the System on B . The amount of an ion species that will be adsorbed when the solution concentration of that ion increases by one unit (i.e., aC,,/aC,) will depend on the forces with which it is adsorbed compared to the forces with which competing ion species are adsorbed. For instance in Fig. 8 the fraction ( ~ 2 , ~ ) of the N , - = FRACTION* OF SOLN. CATIONS WHICH

ARE THE COMPLEMENTARY ION

- 04 - 0.6

TOTAL CONCENTRATION 0.027N IN EACH CASE

- 0.8

No= FRACTION' OF SOLN. CATIONS WHICH ARE Ca"

FIG. 8. Relations between adsorbed ions and ions in solution for suspensions of clay in mixed solutions. *All fractions are in equivalentslequivalents.Data points from Melsted and Bray ( 1947).

MOVEMENT OF NUTRIENTS TO ROOTS

115

exchange capacity filled with Ca2+is plotted as a function of the fraction ( n o ) of the solution ions that are C a 2 +when the complementary ions are Mg2+,K + , or Na+. In each case, for the Ca",

where E.C. is the volumetric exchange capacity of the soil (gravimetric exchange capacity times the bulk density), and COTis the combined concentration of all cations. In each case an,/ano is not constant as the fraction of Ca2+ in the solution phase changes from 0 to 1.0. When Mg2+ was the counterdiffusing ion an,/ano was approximately constant and its value would be usable if precise results were not necessary. Since most cultivated soils contain 80% or more of exchangeable Ca2+only the upper portions of these curves are pertinent to most cultivated soils. In this range the curves, even for Na+ and K + as the complementary ions, can be represented with reasonable accuracy by straight lines, and the slope may be used in Eq. (36) to obtain the major component of the capacity factor. As the total concentration of cations in the equilibrium solution increases the values of an,/ano may increase as indicated in the upper portion of Fig. 9. This increase of ano/ano with increasing COTwill partially compensate the effect of COTin the denominator on the right side of Eq. (36). 4 . Efect of Soil and lon Properties on the Diffusion Coeficient a . Effects of Water Content, Changes in Viscosity, Negative Adsorption, and Tortuosity. In dilute bulk solution, the size of the ion, its degree of hydration, and the normal fluidity of water determine its diffusivity. Additional factors tending to reduce its apparent diffusivity in clays and soils are reduction of the cross sectional area available for flow (O), decreased fluidity of the water ( a ) ,tortuosity of the flow path ( J ! , / Land ~)~ electrostatic restriction ( y ). These factors were discussed in more detail in the preceding section. The diffusion coefficients for the adsorbed ( D a ) , and solution ( D s )cations may be expressed in terms of the diffusion coefficient in bulk solution as follows

and

S. R. OLSEN AND W. D. KEMPER

116

Adsorbed cations are held near the mineral surfaces, while free electrolyte (or solution) cations are pushed away from the surface. The zone of appreciable reduction in fluidity is within the first several molecular FRACTION*OF SOLUTION CATIONS WHICH

ARE Ca** (=No for Ca”)

FIG.9. Relations between adsorbed ions and ions in solution in clay suspensions as the total concentration varies. *All fractions are in equivalents/equivalents. (Data from Figs. 22 and 27 of USDA Handbook No. 60, “Diagnosis and Improvement of Saline Soils.”

layers of water adsorbed on mineral surfaces (Kemper et al., 1964). Consequently the factors reducing fluidity, aU and as,are not equal and are defined by the equations

where C,’ and C,‘are the concentrations at any plane at distance y from the clay, of the adsorbed and solution cations, respectively, a is the relative fluidity of the water at that plane, and 7 is the average pore are the average (across the pore) concentrations. In width. C, and soils at suctions less than 1 bar, the value of aswill generally be close to 1.0, while a, may be considerably less. Electrostatic restriction reduces the probability that anions or cations

c,

MOVEMENT OF NUTRIENTS TO ROOTS

I17

in solution will move from one “capillary puddle” of water, through thin capillary films on or between negatively charged particles to another “capillary puddle.” On the other hand, mobile adsorbed cations are not appreciably restricted by electrostatic restriction due to such variation in the flow path. Moreover, since they are constantly interchanging with free electrolyte cations, they offer a medium through which all cations can largely avoid the electrostatic restriction. Consequently the value of y for adsorbed cations should be unity and for solution cations should be near unity. b. Effect of lon Size and Charge. The mobile portion of the adsorbed ions depends largely on the species of cation involved and the source and density of negative charge on the clay. Smaller ions are more strongly hydrated, and consequently a larger portion of them keep at least one molecule of water between them and the clay surface. This greater distance reduces the coulombic forces of attraction between the ion and the source of negative charge and a larger fraction of these smaller ions are free to move (Shainberg and Kemper, 1966a,b). Most clay mineral surfaces consist mainly of oxygen. The negative charge of this oxygen is largely balanced by the positive charges of the A13+, Si4+,etc., within the clay lattice. However, in cases where the energy of interaction is strongly dependent on the distance between the participating ions, atoms, or molecules, the positive charges are so far from the surface that their effect does not balance that of the negative oxygens. The energy of polarization (the case of most interest involves the attraction of an induced dipole by the oxygen “ions”) is inversely proportional to the fourth power of the distance between the centers of the oxygen and the adsorbed cation or water molecule (Bolt et a f . , 1967). Consequently the polarization effects of charges more than a molecular diameter from the ion being polarized are small and the negative charges of the surface oxygen “ions” are dominant in causing the polarization energy. The polarizability of a molecule or ion is approximately proportional to its volume. Consequently Li is less polarized by the surface oxygen than H 2 0 , and the difference in energy of polarization tends to hold H 2 0 next to the oxygen surface and push Li’ away. On the other hand Cs+, being a large ion, is polarized much more than water, and the C s + is bound tightly to the oxygen surface and is less mobile as a result of this polarization energy. c. Effect o f s o u r c e and Density of Charge. If the source of the negative charge is in the octahedral rather than the tetrahedral layer, the coulombic forces should be smaller, the cations will be adsorbed more loosely, and a larger portion of them will be mobile. If the charge density

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S. R. OLSEN AND W. D. KEMPER

of a mineral surface is high, the exchange spots are close together and their electrostatic fields overlap. Interchange of adsorbed cations occurs more readily and frequently when these negative fields overlap. Some ions, such as H + adsorbed on broken-edge bonds will have negligible mobility because of strong coordinate type bonding which hold the ions in the immediate vicinity of the source of negative charge. d . Effect of Properties of Counterdi$using Ions. ( i ) Solution phase. When two species of cations interdiffuse in solution and there is no osmotic movement of water, the interdiffusion coefficient D12,is a function of the diffisivities, D , and D 2 ;concentrations C1 and C 2 ; and valences Z , and Z 2 as shown in Eq. (22). In the derivation of this equation it is assumed that the only interaction between diffusing ions is through the electrical potential that builds up as one species of ion tends to diffuse faster than the other. In such a binary system the self-difision coefficient of the ion present in the smallest quantity tends to dominate the interdiffusion coefficient. To verify this surprising conclusion, assume C1 < Cz in Eq. (22). (ii) Adsorbed phase. If a large portion of the diffusion takes place among ions in the adsorbed phase, additional interactions not accounted for in Eq. (22) may occur. When a strongly adsorbed (and highly immobile) ion such as Zn2+occupies most of the exchange spots, with Ca2+being the relatively mobile minority ion, then most of the nearest heighbors of a given Ca2+will be Zn2+.Since the clay holds Zn2+ ions strongly, only rarely will the forces of thermal buffeting be sufficiently large to displace the Zn2+ ions from their respective exchange spots. A displaced Ca2+ ion will generally return to the same exchange spot if that spot remains empty and other negative spots do not become available. Thus, generally, ions that are strongly adsorbed and immobilized on the exchange complex will cause a reduction in the movement of other more mobile adsorbed ions. Until more is known of the effects of particular adsorbed ions on the mobility of other ions, the interdiffusion coefficients should be determined experimentally in ionic environments similar to those expected in the field conditions. Normally this will involve a largely Ca2+ saturated system.

5 . Effects of Chelating Agents and Ion Pair Formation Hodgson et al. (1967) showed that much more zinc would diffuse through an agar gel to a constantly renewed solution devoid of Zn2+ from a ZnCOj surface if citrate (a chelating agent for Zn'+) was present in the solution. They reasoned that citrate diffused through the agar to

MOVEMENT OF NUTRIENTS TO ROOTS

119

the ZnCOs surface, where citrate formed a complex ion with Zn2+which increased the total zinc (complexed plus ionic) in solution at the ZnCOs surface. The higher concentration at the ZnCOs surface provided a larger concentration gradient across the agar gel, and in accord with Eq. ( I ) , more total Zn2+moved through the agar gel. This same effect probably occurs as plant roots exude citrate and other complexing agents which diffuse away from the root through the soil, bring zinc, iron, etc., into solution in increased amounts, and these complexed materials then move to the plant roots both by diffusion and convection. It is assumed in this concept that the plant root has sites which adsorb the cations strongly enough to take them away from the chelate, or that the chelated complex enters the plant where internal mechanisms separate the chelate from the cation. An equation describing the diffusion of the chelated complex is,

where the subscript c refers to the chelated phase. By adding Eq. (40) to Eqs. (23) and (24), and performing operations parallel to those used in developing Eq. (27), Eq. (41) is obtained which includes the effects of a chelated complex on the diffusion of an adsorbed cation. dqldt = [(C,9/C,)D,+ g (C,JC,) D,

+ (C,lCd D,]dColdx

(41)

where Co is the concentration of the cation in an equilibrium phase in which the chelate is not present. If the adsorbed or solid phase was sufficiently large that the removal of ions into the chelated phase did not affect the chemical potential of the adsorbed phase, Co would not change with the introduction of a chelating agent. If the adsorbed phase was limited and p was decreased by removal of part of the cation into the chelated phase, Cowould be decreased. As may be seen from Eq. (41), the role of the chelate phase in diffusion is similar to that of the adsorbed phase, except that for strongly adsorbed cations g < 1 (i.e., the adsorbed ion is highly immobile). Although the diffusion coefficient, D,,of the chelated phase is generally slightly less than that, D,, of the ion itself C,/Co may be much greater than C,/C,, and most of the diffusive movement may be attributed to the chelated phase. Values of (C,/Co)/(C,/Co)of the order of 100 appear likely for Z n + + citrate systems (Hodgson et af., 1967). Extension of Eq. (41) to the transient case can be made through a development similar to that for Eq. (33), and Eq. (42) is obtained,

120

S. R. OLSEN AND W. D. KEMPER

where the term in the square brackets must be reasonably constant if the equation is to be valid. If the term is a function of concentration and changes appreciably through the range of concentration being considered, a nonlinear differential equation similar to Eq. (32) must be used. In general chelating agents may provide a mobile phase which will increase the diffusion of cations which are adsorbed on clay minerals. If the adsorbed cations are tightly held and their mobility is low, a chelated phase may provide a large increase in diffusion. Ion pair formation (e.g., Ca2+ SO4*CaS04) may create another soluble, mobile phase of the ion considered, which will act in essentially the same way as a chelated phase to increase diffusion. Although ion pair formation may be more common than chelation, the portion of the ions existing as ion pairs is usually only a fraction of those existing as free ions. Consequently, although ion pairs may contribute to diffusion in a small way, they will never cause the large increases that can be obtained by introduction of chelating agents.

+

Ill. Diffusion of Salts

A.

GENERAL

THEORY AND

EQUATIONS

I . Electrical Coupling of Ionic Movement Electroneutrality requires that net movement of a particular species of ion be accompanied by either concurrent movement of other ions of like charge in the opposite direction (interdiffusion), or concurrent movement of ions of opposite charge in the same direction. Diffusion of a salt is an example of the latter requirement. In equation form, this electroneutrality requirement in soils containing n species of ions may be expressed as

where Zi is the valence of the ion species i (negative for anions) and Ji is the flux of that species in a specific direction in moles per square centimeter per second (may be negative for some species). In soils containing many species of ions, movement of a particular species is usually a combination of interdiffusion and “salt diffusion.”

MOVEMENT OF NUTRIENTS TO ROOTS

121

However, in cases where a single anion species and cation species make up most of the ion population, a reasonably good approximation is

where the subscript c refers to the cation and a to the anion. In such cases the fluxes of the anions and cations are in the same direction and approximately equal (in terms of equivalents). 2 . Steady State The general steady-state equations based on Fick's law for such salt movement are essentially the same as Eqs. ( 1 ) through (4). The diffusion coefficient associated with the movement of the salt may be calculated from the concentrations of the mobile ions and their self-diffusion coefficients using Eq. (22) where the subscripts 1 and 2 denote the anion and cation, respectively. Because most soils contain large amounts of mobile adsorbed cations, the concentration of mobile cations is normally much greater than the concentration of mobile anions. If the soil solution has a concentration near 0.01 N and the soil has a volumetric exchange capacity of 0.26 me./cm.", the soil contains about 130 times as much cation as anion. If about 10% of the adsorbed cations are mobile (approximately true of Ca according to Van Schaik et al., 1966) the amount of mobile cation is about 14 times the amount of mobile anion. Substitution of this concentration ratio in Eq. (22) shows that the salt diffusion coefficient applicable in this soil system will be practically equal to the selfdiffusion coefficient of the anion. At high soil solution concentrations (above 0.05 N ) the applicable salt diffusion coefficient will still be closer to the anion diffusion coefficient than the cation coefficient. Use of a diffusion coefficient which varies with concentration greatly complicates the mathematics of diffusion. The practical solution has generally been to assume a constant diffusion coefficient about midway in the range of the expected concentration. The error involved in this approximation is practically negligible (< 2 % ) when there is more than nine times as much mobile adsorbed ion as there is free electrolyte in the soil. Diffusion of salt requires a concentration gradient, and these gradients tend to induce movement of the soil solution toward the region of higher concentration (Kemper and Rollins, 1966). This movement of the soil solution retards the net movement of salt. The role of this osmotically induced convective flow in redistributing the salt will be discussed in Section 111,A,4.In unsaturated soils the retarding effect of osmotically

122

S. R. OLSEN A N D W. D. KEMPER

induced movement in the liquid phase is at least partially balanced (and in some cases overbalanced) by movement of water in the vapor phase to the region of higher salt concentration where the water condenses and viscous flow carries salt away from the zones of high salt concentration. 3. Transient State Salt Dzyusion and Adsorption-Desorption Reactions a . Relation to Previous Discussion and Equations. Adsorption or exchange reactions were not important in steady-state considerations because by definition if a component is being adsorbed, it must be desorbed at the same rate when a steady state is maintained. However, the more usual transient state involves net adsorptions or exchange, particularly of the diffusing cation, as a salt diffises into a soil. In this process an anion species such as NO:r will slowly change partners, as it moves with the invading cations so that it becomes more associated with the inhabitant cations. This change of partners will normally have little effect on the diffusion rate of the anions-particularly when the total salt concentration is less than 0.05 N [see previous discussion and Eq. (22)l. Consequently, if the “salt diffusion” is considered to be the diffusion of an anion species such as NOs- and the cation partner, or mixture of partners accompanying it, all boundary conditions and solutions expressed in Eqs. (1 5) to (2 1) are applicable to this “salt diffusion.” When there is no adsorption of the anion species, B = 8. If the anion species is adsorbed, the concentration of the anion per unit soil volume ( C ’ )in the concentration range of interest may be expressed approximately as C ’ = CB

+ 66 + k

(45)

where b and k are constants, then the proper value of B associated with Eqs. ( 1 2) through (2 1) is B

=

actlac = o + b

Olsen et al. (1962) found such a relationship to be applicable to phosphorus at low solution concentrations. b, Source Equation Solutions. Jost ( 1 952, pp. 16- 19) develops equations describing concentration of a component as a function of distance (from the source), time (since the source was applied), and the quantity of material in the source. Assumptions in Jost’s development included complete solubility of the applied material, no adsorption of the diffusing material, and no movement of the soil solution.

MOVEMENT OF NUTRIENTS TO ROOTS

123

( i ) Plane source equation. Consider that a material is applied uniformly on a surface at Q ’ me./cm.2. The concentration C - of the diffusing anion in me.1cn.1.~of solution (or Cvin m e . / ~ m of . ~ soil) is given as a function of time, t (seconds) and depth, x (cm.) by Eq. (47) C-

=

C,/B

=

( Q ’ I B W ) exp (-x2/4T’)

(47)

where T’ = D,t/B, D , (cm.Ysec.) is the porus system diffusion coefficient of the salt, and B is the capacity factor which is equal to the volumetric moisture content 8 if the component is not adsorbed. Jost’s development can be easily extended to the condition where adsorption occurs if the adsorption is proportional to the solution concentration C - , as indicated in Eq. ( 4 8 )

c, = 8 c - + b c - .

148)

+

When such proportional adsorption occurs, B = 0 b. (ii) Line source equation. Consider that a material is applied uniformly in a line within a soil mass at Q ” me./cm. Using the same terminology and symbols as above except r (cm.) is now distance from the line source,

C-

=

( C , / B ) = ( Q ” / B 4 7 r T ’ )exp (-r2/4T’)

(49)

Under some conditions this equation may be used to approximate the dispersion of a fertilizer after it has been applied in a narrow band within the soil. (iii) Point source equation. Consider that quantity Q ” ’ (me.) of material is applied as a granule within the soil mass and that r is the distance from the center of the original granule to any point in question. Using the terminology and symbols in (i)

Under some conditions this equation may be used to approximate dispersion of fertilizer from granules after these grandules have been buried in the soil. c. Mathematical Approximations for Adsorption-Desorption Reactions. In the case of adsorption of the diffusing component, the assumption of a linear or proportional relationship between solution concentration and adsorption [Eqs. (45) or (48)] may yield surprisingly

124

S. R. OLSEN AND W. D. KEMPER

close approximations of the ion distributions [using Eqs. (15) to ( 2 1) and (47) to ( S O ) ] even when the actual adsorption-solution concentration relationship deviates appreciably from being linear or proportional. In cases where the amount of nutrient adsorbed cannot be approximated by a straight-line function of the solution concentration but the adsorbed nutrient can be expressed as a more complicated function of concentration, differential equations can be developed, and programmed for computer solution. 4 . Transient State Salt Diffusion and Exchange Reactions

The principal difference between exchange and the adsorption discussed in the previous section is that adsorption was assumed to be a function of solution concentration alone. In exchange reactions, the amount of the invading cations adsorbed is a function of solution concentration of both the inhabitant and invading cations, as well as the difference in energy of adsorption. With phosphorus, adsorption appears to be a linear function of P solution concentration in the important concentration ranges in some soils, even though the actual adsorption may involve the exchange of H z P 0 4 - for OH-. Apparently in the concentration ranges considered, the amount of OH - liberated is sufficiently small that the buffering capacity of other soil constituents is able to hold OHconcentration of the soil solution essentially constant. In such cases the equations presented in Section III,A,3 apply. In some cases the concentration of ions which can compete for the exchange spot is appreciably changed by intrusion of the invading ion as it is adsorbed and releases other ions from the exchange complex. Differential equations for these systems may be developed from Fick’s first law as follows. The rate of accumulation dq’ldt of an invading cation species (one ion of the salt applied as a fertilizer) in a volume element 1 X 1 x dx (where x is the direction of flow) is

If the cations have similar adsorption characteristics, the quantity of invading cation species on the adsorbed phase in this volume will be (CJC,) E dx, where Ci is the solution concentration of the invading cation, C T is the total cation concentration in solution, and E is the volumetric exchange capacity of the soil. Thus the amount q ’ of the invading cation in the soil volume dx is

MOVEMENT OF NUTRIENTS TO ROOTS

125

and

Since aq’lat = (aq’/aCi)(aCi/at),from Eqs. (5 1) and (53)

Equation (54) has not been solved analytically for pertinent boundary conditions but it may be rearranged, and put in the finite difference form

If the adsorption energies of the invading and inhabitant cations are different, the quantity of species i on the exchange complex in the volume 1 x 1 x dx will not be (Ci/CT)Edx. For instance, if species i is adsorbed less strongly than the other cation, the amount of i adsorbed may be closer to (Ci/CT)’ E dx, in which case a development similar to the preceding equations yields 2 E C? A C T

ACi x ~

+

Ax

Many other functional relationships between the amount of cation species adsorbed and its equivalent fraction in solution can be similarly differentiated. If one assumes that the anions (i.e., NOa)are not adsorbed and the initial salt concentration is negligible then for one dimensional flow from a surface, Eq. (47) can give the total cation concentration C T needed in Eqs. (55) and ( 5 6 ) (i.e., C-= C T ) .as a function of Q ‘ , x , D,, and t where D , is practically the diffusion coefficient of the anions. Equations (55) and ( 5 6 ) and similarly derived equations are then amenable to computer solution for Ci as a function of Q ’ , E, t, x, and

126

S. R. OLSEN A N D W. D. KEMPER

D;. Note that D,’, the diffusion coefficient of the cation may be appreciably different from that of the anion, since there is some opportunity for interdiffusion of cations and therefore mobile adsorbed cations can contribute to diffusion in the manner illustrated by Eq. (26). In Fig. 10 typical results of a computer solution are shown where the following assumptions were made: Q =0.162 rne./cm2, E = 0.07 m e . / ~ m . ~ ,

INVADlNG CATIONS

INHABITANT CATIONS AFTER 1 DAY

.--. 1

2

3

4

5

6

DEPTH

7

(CM)

- -

-------.-__--_ ---_---_

L A

8

-7

L I

9

10

11

12

FIG. 10. Cation diffusion following application of a salt at a plane source as a function of depth in soil.

D,

=

(6Ci

1.6 x

cm.?/sec., D,’

=

1.6 x

cm.2/sec., 6

=

0.3, q’ =

+ E C i / C T )dx, and the initial salt concentration of the soil mass was

negligible. The initial conditions assumed were that all the salt was applied at the surface, but it was allowed to diffuse without adsorption for 6 hours [according to Eq. (49)] at the end of which time the invading cations came to local equilibrium with the inhabitant cations in the solution and adsorbed phases. The finite difference portion of the computer program begins from the concentrations at this time. (Allowing this short initial period of diffusion allows the computer solution to start from continuous concentration functions. This is necessary since finite difference solutions cannot begin from a plane source condition because infinitely

MOVEMENT OF NUTRIENTS TO ROOTS

127

large derivatives occur.) I t was also assumed that the adsorption characteristics of the two cations were the same. Note that the invading cations are preceded by a wave of released habitant cations which accompany the anions of the fertilizer salt in their diffusion into the soil mass.

5 . Role of Electrical Potentials and Osmotically Induced Viscous Flow The three main factors responsible for motion of ions in clay-water systems are movement of the solution, electrical potential gradients, and thermal motion of the ions (coupled with a concentration gradient). Concentration gradients of salts can activate all three of these factors, by osmotic inducement of solution movement (which causes streaming potentials), by diffusion itself, and by causing diffusion potentials. Diffusion potentials result when one of the ions of a salt has a higher diffusivity than the other. Because they are less hydrated the anions of most salts diffuse faster than the cations, resulting in a progressively more negative potential in the direction of lower salt concentration. This potential gradient causes a retardation of anion movement and acceleration of cation movement in the direction of lower salt concentration. Kemper and Van Schaik ( I 966) present equations and calculations of the effect of osmotically induced convective flow, diffusion and diffusion induced electrical potential gradients on movement of ions in clay-water systems. Some of their results are shown in Table I . In some cases the osmotic movement and electrical forces moved more cations than the thermal motion, or diffusion. However, since the osmotic and electrical factors move cations in opposite directions their combined effect was usually smaller. As a result of the osmotic flow, the measured diffusion was always slightly less than the diffusion calculated from Eq. (22). While the osmotic movement and electrical potential incurred by salt gradients are academically intriguing, they change the salt diffusion coefficients only slightly from those calculated using the diffusion coefficients of the respective ions in Eq. ( 2 2 ) [or from Eq. (9), Kemper and Van Schaik, 1966)l. In general, changes in the diffusion coefficient of a salt due to concentration differences will seldom be greater than 20% and is usually only a few percent in the concentration range of the study. Since the differential equations can be solved for many more boundary conditions when assuming a constant diffusion coefficient than when assuming a concentrationdependent coefficient, a constant coefficient is often assumed. The error incurred in this assumption is usually less than the errors associated with other assumptions, such as constant water content.

TABLE 1 Contribution of True Diffusion, Electrical Forces, and Osmotically Induced Mass Flow to Macroscopically Measured “Diffusion” of Salts across Clays according to Kemper and Van Schaik (1966) NaCl across Na-clay

CaCl across Ca-clay [Il

High-Low Side: Conc. ( N ) : 0.010-0.0005

High-Low 0.055-0.003

High-Low 1 .o-0.1

High-Low 0.002-0.000 1

High-Low 0.05-0.003

s

Transfer rates 108 x me./cm.’/sec.

Parameter

E >

Anions

Due to diffusion Due to electrical force Due to mass flow Total

.0

0.27 -0.01 -0.02

2.7 -0.2 -0.4 __

0.24

2. I

113 - 19 - 7

0.164 - 0.003 - 0.004

2.14 -0.37 - 0.07

z

U

3

~

~

87

0.157

1.70

u x

Cations Due to diffusion Due to electrical force Dut to mass flow

rn

0.17 1.21 - 1.14 ~

Total Calculated total using Eq. (22)

1.8 4.1 -3.8 __

74 20 - 7

0.064 0.129 -0.036

0.84 0.98 -0.12 __

~

0.24

2.1

87

0.157

I .70

0.27

2.6

94

0.161

1.70

5

M

b

MOVEMENT OF NUTRIENTS TO ROOTS

129

B. EFFECTSOF SOILPROPERTIES O N SALTDIFFUSION 1 . Diflusion Coeficients Kemper and Van Schaik (1966) show a relationship between volumetric water content, 0, and the diffusion coefficient, D,,, of NaCl in Na-bentonite and the diffusion coefficient in water, D o , of the type

D,

- -- KebO

DO

(57)

where K and b are constants. Data collected on soils by Olsen et al. (1965) and Porter et al. ( 1 960) fit this equation reasonably well with b = 10, and K being a function of surface area of the soil. As the surface area increases K decreases. This relationship was applicable in the range of moisture contents between 0.33 and 15 bars suction, but it cannot be extrapolated beyond these limits. For the soils studied (ranging from sandy loam to clay soils) K ranged from 0.005 to 0.001. At constant water content, D , decreases as clay content increases. However, if a sandy and a clay soil are compared at the same suctions, the clay soil generally holds so much more water that D , is larger in the clay soil. Negative adsorption of salt from mineral surfaces and increased tortuosity are probably major factors causing decreased values of D ,as clay contents increase at constant water content. When soils are at moisture contents greater than the water content at 15 bars suction, diffusion coefficients will seldom, if ever, be lower than cm.2/sec., and will normally be greater than lop7.Some authors have reported salt diffusion coefficients in moist soils as low as lo-” cm2/sec. These low values are almost invariably obtained using transient state systems, and the “diffusion coefficient” is really a diffusion coefficient divided by a capacity factor. 2. Capacity Factor Most of the ions needed by plants are adsorbed by mineral surfaces in the soil. In the case of all the cations and phosphorus, the amount of adsorption is generally proportional to the surface area (which is directly related to the exchange capacity). Consequently the capacity of a soil to adsorb various salt ions generally increases as the clay content rises. Clays with greater amounts of surface area usually have a greater exchange capacity. Generally, as soils become more acid, their capacity to adsorb cations decreases, but their capacity to adsorb anions may markedly increase.

130

S. R. OLSEN AND W. D. KEMPER

More detailed aspects of these specific forms of adsorption are discussed in detail in the sections on cation and anion diffusion. As fertilizer salts diffuse through soils, cations exchange with inhabitant cations, pH of the media changes as the salt becomes more dilute, and precipitation reactions may occur. For instance, Ca[H2PO412 changes to less soluble forms. As the ions in solution are used by plants, these slightly soluble phases may contribute ions to the solution phase. If the solid phase consisted of large, pure crystals, solution concentration at the surface of those crystals would be maintained essentially constant as the ions diffused away until the solid phase had all dissolved. From this point of view, the linear relationship found by Olsen et al. (1 962) between P in solution and labile P is surprising. Apparently there are a multitude of slightly soluble minerals with varying degrees of purity and many different adsorption energies at the various adsorption sites whose combined effect is the relatively constant capacity factor observed. IV. Combined Diffusive a n d Convective (Viscous) Flow

A. EQUATIONS FOR STEADY-STATE CONDITIONS

I . Equations f o r Radial Flow to Plant Roots A general differential equation for steady-state radial movement of a plant nutrient to a root is

where Q is the total uptake rate of the nutrient (i.e., me./cm. rootlsec.), W is the rate of convective movement of solution into the root ( ~ m . ~ / c m . root/sec.), r is distance (cm.) from the center of the root, C is the concentration of the nutrient (me./cm.3), 2 n-r D n(dC/dr)and W C represent the diffusive and convective components, respectively, of nutrient movement. The velocity, V , of solution to the root is related to W and r by V = Wl2n-r 9 . If one considers a root of radius a and assumes that the concentration is maintained at Cb at a distance b from the center of the root, Eq. ( 5 8 ) yields C

=

Q/w+ (cb- Q / W ) -

t)lYiZiDh

(59)

If C is known at the root surface (i.e., C = C , at r = a ) but is not known

MOVEMENT OF NUTRIENTS TO ROOTS

131

farther out in the soil, C, and a may be substituted for C band b in Eq. (59). If the uptake of ions, (3, is the unknown, and C = C , at r = u and C = Ch at r = b are known, rearranging Eq. (59) and remembering that ( b l a ) W l 2 nlih = ,Iw In ( b / a ) l / Z n D h ,gives

As Gardner ( 1 965) states, in the limit, a s W -

0, Eq. (60) reduces to

which is the equation for diffusion alone. In the concentration range of interest, the rate of uptake, Q , may be a function of the concentration C, a t the root surface and may be represented by

where C l is a limiting solution concentration below which the plant will not take up the nutrient but will lose nutrient to the soil solution. Under these conditions Eq. (59) yields

and

which is identical to Eq. (53) of Gardner ( 1 965) when Ct is equal to zero. 2 . Variations Expected in DjJ It has been assumed that D,, is a constant. If DlI is not a constant, but can be expressed as a function of distance (i.e., DI,= Djl0+ A T , Dtt = DtIO In ( r / a ) , etc.), Eq. ( 5 8 ) is still easily integrable and equations similar to (59) and (60) may be developed for these conditions. The uniformity of water content in soils up to suctions of 1 or 2 bars [as concluded by

132

S. R. OLSEN A N D W. D. KEMPER

Gardner and Ehlig (1962)l raises questions as to whether such refinements of D, are necessary under the conditions experienced by most growing plants. To simplify Eq. (58) we assumed that all solution is moving through any boundary r a t the velocity W / 2 m O . Because the velocity of solution in a pore is proportional to the diameter of the pore squared, solution moving toward the roots is traveling at a range of velocities. This variation in velocity causes “hydrodynamic dispersion” which is similar to thermal motion (diffusion) in that “mixing” occurs, with the nutrient tending to move more rapidly from regions of high to regions of low concentration. This dispersive movement is with respect to the solution. The similar responses of diffusion and hydrodynamic dispersion to concentration gradients has encouraged most investigators to lump the effects of diffusion and hydrodynamic dispersion into a single coefficient which we have designated by Dtr. This hydrodynamic dispersion plus diffusion coefficient could be defined as

Dh = D, + Dlld where D, is the coefficient for diffusion in a porous media in which no convection is taking place and D / , d , the hydrodynamic dispersion coefficient is a function of the solution velocity. Nielsen and Biggar (1962, 1963) have studied the combined coefficient D h as a function of flow velocity in soil and glass beads. Part of their data is plotted in Fig. I I . Although there is considerable scatter, for the various reasons which they discuss, the general indication is that for flow rates less than 0.2 cm./day the contribution of hydrodynamic dispersion to the combined coefficient Dh will be small. Since, as discussed later in this section, flow rates to roots are in this order of magnitude, we shall assume in the following discussion that Dh = D,, the thermal diffusion coefficient. 3 . Inferences from These Steady-State Equations

Equation (59) states that if the concentration, Q / W , of the nutrient in the solution entering the root is less than the solution concentration Cr, maintained in the soil at b, in the region between the root and b, the concentration will be greater than C b . This increase will be greatest at the root surface, will increase as the uptake of water increases and will decrease if the diffusion coefficient Dn is higher (which allows the nutrient to diffuse away from the root faster). When Q/W is greater than Cb, concentration near the root will be lower than Cb. The contribution of viscous flow is given as a fraction of the diffusive

MOVEMENT OF NUTRIENTS TO ROOTS

I

ao1

ID

03

10

100

133

1000

FLOW VELOCITY (CM/DAY)

FIG. I I . Variation in a dispersion-diffusion coefficient as a function of velocity of solution. (Data from Nielsen and Biggar, 1962, 1963.)

contribution, and as a function of [W In ( a / b ) ] / 2DI, ~ in Fig. 12, calculated according to Eq. (60). If we assume b/a = 7.4 (and therefore In(b/a) = 2 ) and that C,/C,)= 0.2, then when W = 257 Dkr, viscous flow would contribute eight times as much movement of nutrient to the plant as would diffusion. According to the data of Porter et al. (1960) and others, a reasonable range of diffusion coefficients in soils from about 3 to 0.3 bars of suction would be from to cm.2/sec. For W = 27r D,, , the uptake rates would need to be 0.054 and 0.543 ~ m . ~ / c m . root/day. Corn plants studied by Anderson and Kemper (1964) were taking up water at an average rate of 0.02 cm.Ycm. rootlday. However, if only a fraction (i.e., 0.2 to 0.5) of the roots are “active” in the uptake of ions and water, W may be near 257 D / , in some cases, and in such cases convection will move more nutrient to the plant than will diffusion. Somewhat similar conclusions could be drawn for the case of uptake being proportional to concentration at the root surface [Eq. (6411.

B.

SOLUTIONS FOR

TRANSIENT-STATE CONDITIONS

Steady-state equations are developed assuming either that there is no adsorption or, if the diffusing component is adsorbed on the solid, the

134

S. R. OLSEN AND W. D. KEMPER

I

[W ln(b/a)]

/ 277’711

FIG.12. Relative contribution of convection and diffusion as rate of convective movement ( W )of the soil solution to the plant root increases.

rate of adsorption a t any particular volume element is equal to the rate of release. Consequently, only in the transient state do we account for the ability of the solid to take away or supply nutrients to the solution phase. To make this accounting, the adsorption and desorption isotherms of the nutrient on the mineral must be known, as discussed in the previous sections on diffusion alone. In those sections we have assumed that diffusive movement was so slow that chemical equilibrium was practically the case at all times. It seems likely that convection (or viscous flow) could take place so rapidly in soils systems that chemical equilibrium between the solution and adsorbed phase would often not be the case. However, in the following discussion we shall assume that W is generally less than 0.1 ~ m . ~ / c root/day, m. that consequently the chemical equilibria are generally attained, and that we can use isotherms obtained under a series of equilibrium conditions.

MOVEMENT OF NUTRIENTS TO ROOTS

135

A development similar to that for Eqs. ( 5 ) to (12) leads to Eq. (65) as the differential equation for radial flow of a component to a cylinder (root) including both viscous and diffusive movement of the nutrient.

B ac a 2 c w I ac +I+--D,, at ar2 277Dt) r ar

(

Geering (1967) developed a solution for Eq. (65) for the case where uptake is proportional to concentration, C , , at the root surface, r=a. The solution has not been generally used because it is in the form of complex Bessel functions. However, Geering (1 967) noted that when W = 277 D,, Eq. (65) becomes identical to the equation for radial flow through an infinite region bounded internally by a sphere, and that Crank ( 1 956) has provided a solution for this differential equation when uptake by the sphere was proportional to concentration at the root surface. Thus, when W = 277 Dh and at r = a , t > 0

and

where h = k/(277a Dh) and k is a “plant characteristic” that must be obtained from a study where concentration at the root surface is known. Equation (67) was used to obtain the solid lines shown in Fig. 13, which represent Cr/Co (or uptake rate/initial uptake rate) when the viscous flux W = 277 D t , . The dashed lines in this figure are taken from Carslaw and Jaeger’s (1959) Fig. 43 for the case where W = 0. The lines representing a viscous flux of W = 277 Dl1 all extrapolate to a finite nutrient uptake rate of l/ah at infinite time. If no viscous flow of water toward the root occurs (W = 0), the uptake rate of nutrient approaches 0 at large times as the nutrient is exhausted from the vicinity of the root. OF DIFFUSION A N D CONVECTION AS MECHANISMS C. COMPARISON FOR BRINGING NUTRIENTS TO THE ROOT SURFACE

As a root grows into a region, it displaces soil. This displaced soil contains nutrients, which are also displaced. After a root extends through

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S. R. OLSEN A N D W. D. KEMPER

2.0 -

t

Id ah = 1.0,

W = 277 Dp

ah=Q5, W = O

1

2

3

4 Dpt/Ba2

5

6

7

FIG. 13. Relative concentration at the root surface (or uptakelinitial uptake) as a function of time.

a soil region, nutrients may move to the root surface by diffusion or convection. The relative importance of these processes will vary for different species, soils, and nutrients.

I . Theoretical Considerations Figure 14 was prepared to illustrate the relative effectiveness of viscous and diffusive flow as the capacity of the solid phase to adsorb the nutrient increases. In Fig. 14 the case of ah = 2.5 is considered for B = 0 = 0.25

137

MOVEMENT OF NUTRIENTS TO ROOTS

w ah= 2.5 FOR ALL LINES

3"

1

4

2

I

5

6

Dp:/12

FIG.14. Relative concentration at the root surface (or uptakelinitial uptake) as a function of time for variations in B and W.Dashed portion of bottom curve is an extrapolation.

(approximately the case for nitrate) and for B = 100 (a reasonable case for phosphorus). The curves in Fig. I3 were taken from Fig. 15, which is Dpt/Ba2 0.0001

0.001

0.01

03

1.o

10

0.9 . 0.8 .

0.7 . 0.6.

>0.5. 0.4 .

0.3 -

0.2

-

FIG. 15. An expanded range for similar curves in Figs. 13 and 14.

an expanded form of the ah = 2.5 curves in Fig. 13. Under the conditions assumed, when B = t9 = 0.25 and W = 2.rr D l l ,convective plus diffusive flow would move more than twice a s much nutrient to the plant root than woulddiffusiveflow alone ( W = 0). However, when the nutrient is strongly

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S. R. OLSEN AND W. D. KEMPER

adsorbed by the soil ( B = 100) uptake during the time from t = 0 to f = 6 a2/D, was only about 5% greater for the viscous plus diffusive flow (W = 2.rr D,,) than for the diffusive flow (W = 0) alone. As discussed in the section on steady state, W = 2m- DI, is probably near the upper limit of water fluxes to plant roots which occur in soils. When 0 < W < 27r Dh a linear interpolation is a good approximation of uptake rates, etc., for those who do not wish to use Geering’s (1 967) Bessel function solution. It appears that convective flow may often be the dominant means by which nitrate moves to plant roots and that diffusive flow accounts for the movement of most of the phosphate. However, an accurate estimate of the contribution of these two types of movement to supplying nutrients to the plant roots will be gained only when adsorption and desorption isotherms (nutrient on mineral) are known, and when k , C l , and other factors determined by the plant can be estimated with reasonable accuracy. From the complex nature of solutions to the transient case systems it is apparent that additional variable factors such as variable diffusion coefficients and capacity factors effectively preclude an analytical solution. Computer solutions of finite difference forms of the partial differential equations similar to Eq. (65) will probably provide the answers for these complex conditions. Dr. M. H. Frere (private communication) is working on such solutions. His results which we have seen to date indicate a great potential for handling complicated conditions and producing reasonable results.

2. Experimental Observations a. Nitrate and Chloride. The amount of N as NO3 moving to a root surface by convection can be indicated only in a general way because wide variations in concentration of NO; in solution have been observed (Barber, 1962). Mineralization of organic matter adds continually to the NO3- content of the solution. Barley and Naidu (1963) estimated that wheat obtained about half of its needed N by convection, and the rest by diffusion on a soil with low levels of N. Convection may transfer surplus quantities of NO3- to root surfaces when levels of NO3- and transpiration ratios are moderate or high; however, calculations about the supply of N by this mechanism may be misleading in terms of the actual contribution to the plant. Clarke (1 966) observed a linear relation between uptake of N by wheat and the D, for NO3- in Wanbi sand and Urrbrae loam. This result did not prove a direct causal link between uptake and D,, however, because 6 and C , (C, = CO) varied simultaneously with D,. Clarke ( 1 966) inter-

MOVEMENT OF NUTRIENTS TO ROOTS

139

preted his data to mean that diffusion exerted control on uptake through its effect on the rate of transfer of ions to the root. In nutrient solutions uptake of N usually reaches a maximum around I mM, but in Wanbi sand uptake of N was linear from 0 to 70 mM. These results indicate that diffusion can be important in uptake of N even though the usual contribution by convection, if the transpiration ratio is 300, would appear to be more than adequate to meet the needs of the plant. The experiments of Clarke (1966) do not show what might happen under natural conditions where concentration of NO3- varies inversely with 8. Paul (1965) measured the uptake of C1- by wheat under conditions where C,, was constant, but C varied inversely with 8. Paul ( 1965) observed that uptake of CI - increased with increased 8 or D, for a constant C , at low values of C , . A similar effect would be expected for NO3-. However, when C , was large an increase in 8 o r D, caused a decline in uptake, presumably because C falls as 8 increases and the effect of concentration on uptake became more important. The possible role of diffusion in uptake of NO3- implies that a more reliable soil test for N probably should include an allowance for differences in D ,among soils. Since D , for NH4+is less than for NO3-, equal concentrations of these two ions in soil solutions does not mean equal availability to roots (Clarke, 1966). The uptake of C1- by plants tenus to follow a pattern similar to that of NO:,- although some plants may absorb relatively smaller amounts of C1- than NO3-. Paul ( 1 965) observed a linear relation between D, for C1- and 8 on a Wanbi sand when C was 10 or 20 mM in solution. At a low constant C in solution (0.67 mM) the uptake, Q, for CI- was a linear function of 0 or D,,. As C increased to 28 mM a slight curvilinear trend between D,, and Q appeared. Uptake of C1 was independent of 0 except at low concentrations. The balance of these factors depends on the magnitude of the effect of 8 on D,,and C,and the plant response over the range of concentration involved. Paul ( 1 965) found that a given increase in 8 caused a fivefold increase in D p , a fourfold decrease in C, but only a twofold variation in Q. Therefore, Q increased with a rise in 8 even though C decreased fourfold over the range of 8. b. Phosphorus, Sulfur, Molybdenum. In contrast with NO3- and CIthese ions are taken up in much smaller amounts, especially Mo, by plants and their values for D p are less. Each of these ions will be adsorbed to varying degrees by the solid phase, so Eqs. (121, ( 1 3), or ( 14) must be used to calculate the amount diffusing to roots. Uptake of P by roots of corn was measured by Olsen et al. (1961) on three soils as a function of 8 and concentration of P. At the same value of

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S. R. OLSEN AND W . D. KEMPER

C the roots absorbed twice as much P from a Pierre clay as from Tucumcari fine sandy loam. Values of D , were calculated from the diffusion of CI- in these soils. Diffusion coefficients were measured also by transient- and steady-state methods (Olsen el al., 1965). The flux to the roots was calculated and compared with observed values of Q . These agreed reasonably well, which indicated that the main differences in Q among the soils was accounted for by variations in D, and ( b+ 13). Later experiments with improved methods for measuring Q by roots confirmed these results wherein a fivefold difference in Q at the same C could be explained by variations in D , and ( b 0). Thus, the differences between soils in their capacity to supply P to roots was elucidated. These results on alkaline, calcareous soils may have important implications in developing better soil tests for P. Similar experiments have not been tried as yet on acid soils with high capacities for fixing P. Lai and Mortland (1 96 1) have measured an apparent diffusion coefficient for SO$- in a clay paste. From the value of D in bulk solution, it is expected that D, for SO$- will be about twice as great as for P. To calculate the flux of SOg- to a root, it will be necessary to determine a capacity factor for SO12-, unless the soil does not adsorb SO,2-. If soils contain gypsum, the concentration of SOP- in a saturated solution will be so high that Sod2-can move by convection in amounts needed to supply the plant. Plants require Mo in amounts several thousand times less than P, and the concentration in solution of Mo in soils is usually smaller than P by this same order of magnitude. Lavy and Barber ( 1 964) estimated D,/B values for Mo in 11 soils by radioautographs. These values ranged from 0.5 to 8.4 X cm.2 sec.-'. Radioautographs of corn roots in soil treated with 99 Mo revealed that convection and diffusion functioned in uptake of Mo. Evidence of diffusion occurred mainly on soils which contained < 4 ppb. of M o in a saturation extract of the soil. Apparently, the same root in a soil obtained Mo by convection in an older section of the root and by diffusion in a younger section. c. Potassium, Calcium, Magnesium. Need for K by plants is usually two- to tenfold greater than for Ca and Mg, but the concentration of K in saturation extracts is generally five- to tenfold less. In most soils much of the K required by plants probably moves by diffusion to the roots (Barber, 1962). In contrast most of the Ca and Mg may be supplied by convection according to the initial work of Barber (1962) and Barber et a / . (1963). Later work indicated that convection was inadequate in supplying Ca and Mg on a soil leached with water (Oliver and Barber, 1966a). Place and Barber (1964) measured the D,/B values for Rb in a Raub

+

MOVEMENT OF NUTRIENTS TO ROOTS

141

silt loam to which they added three rates of Rb. Then they brought the soils to five different levels of water. Movement of Rb was followed by radioautographs. Values of D,/B were linearly related to the water content of the soil for each level of Rb. As the concentration increased cm.2 set.-'. from 0.5 to 900 ppm, D,AB rose from 1.2 to 13.0 X Uptake of Rb by corn roots was highly correlated with D J B that had been varied by varying the water content. The data support their conclusion that uptake of Rb was related to the rate of diffusion of Rb. Evans and Barber ( 1 964) found that uptake of Rb by corn roots was highly correlated with D,/B for eight soils. Their results were complicated, however, by differences in fixation of Rb, and these two effects on uptake could not be separated. A study of uptake of Rb by corn roots from kaolinite plus agar mixtures, where fixation of Rb was nil, revealed that uptake was highly correlated with (D,/B)”’. This latter relationship would be expected from Eq. (20). A definitive relationship between uptake and D J B , applicable to several soils, will require information about the values of B , Co,and C,. in Eq. (20) for each soil. The total uptake of Ca, Mg, K, and Na by soybeans has been separated into contributions from convection, diffusion, and “root interception” by Oliver and Barber ( I 966a). Their study revealed that convection was the most important mechanism for Ca on a Sidell silt loam. A subsoil of this same soil was leached with water to reduce soluble salts and then mixed with sand (0 to 80% sand). As the proportion of sand rose, diffusion increased in importance as a mechanism for supplying the roots with Ca and Mg. Other experiments indicated that K was supplied mainly by diffusion. The concepts and assumptions for calculating uptake of ions by “root interception” were outlined by Oliver and Barber ( 1 966a). In effect they assume that all the exchangeable and soluble ions will be absorbed from a volume of soil equal to the volume of the roots. Since these nutrients are initially displaced rather than ingested by the roots, “root interception” is an improbable phenomenon. Since Oliver and Barber (1 966a) calculate the contribution of diffusion in their studies by subtracting from total uptake the amounts from convection plus root interception, the real effect of diffusion as a transport mechanism is minimized in their calculations. d. Aluminum, Boron, Copper, Iron, Manganese, Strontium, Zinc. Diffusion coefficients for these ions in soil have not yet been published. Oliver and Barber (1966b) attempted to separate the total uptake into mechanisms of supply due to “root interception,” convection, and diffusion. The experiments were conducted on the subsoil of a Sidell silt loam. The calculations of uptake from “root interception” and diffusion

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S. R. OLSEN A N D W. D. KEMPER

are subject to the same limitations discussed previously. The total content of these ions, inorganic plus any combinations with organic matter, in the saturation extract were used in calculating uptake by convection. This study indicated that diffusion was the most important mechanism for Fe, Mn, and Zn. “Root interception” plus convection was interpreted as being more significant for Al, B, Cu, and Sr. However, the illogical nature of the “root interception” concept casts some doubt on the interpretation of their data. At present their results furnish interesting qualitative indications of the relative importance of diffusion and convective flow in causing movement of these nutrients to the root surface. V. Role of the Plant

A. ANATOMICAL FEATURES OF ROOTS The boundary conditions which the plant imposes on the diffusive and convective processes in the soil can be visualized better by considering briefly certain anatomical features of the root and a single cell. As dissolved material or water from the soil solution enters a root the ions or molecules pass through an epidermal layer of cells including those with root hairs, then a layer of cortical cells with the endodermis at the inner boundary. Beyond the endodermal cells the ions traverse the stele including the xylem vessels and phloem cells. An individual cell has a cell wall, cytoplasm, and vacuole. The cytoplasm is bounded by two membranes, the plasmalemma at the outer surface and the tonoplast at the inner surface, which also serves as a boundary for the vacuole. Cytoplasmic connections occur between cells by plasmadesma which are assumed to form a continuous pathway, the symplasm, from the epidermis to the xylem vessels (Arisz, 1956). The cells of the root cap readily absorb ions, but these cells lack connections to the xylem so they are ineffective in transmitting the ions to the shoot (Wiebe and Kramer, 1954) and do not absorb large quantities of water. The location of the rate-limiting barrier to the diffusive flow of ions is important in defining the possible contribution from root hairs, the mathematical boundary conditions, and the surface area of the root contacting the external solution. Either the epidermal or the endodermal cells have been considered to be this membrane. In a single cell the plasmalemma and the tonoplast have been suggested as the permeation barrier (Brouwer, 1965; Hendricks, 1966). If the ions or solutes in the external solution could diffuse freely into the endodermal cells or to the tonoplast of a single cell in the epidermal layer, then the root-hair cells would be bypassed since they would act like any other cell in the cortex or the

MOVEMENT OF NUTRIENTS TO ROOTS

143

remaining epidermal cells. This condition would mean that a concentration gradient would be established at the endodermis, or the tonoplast of a single cell, and as a result, the root-hair cell could not be any more important in diffusive flow than the other epidermal cells. The weight of evidence favors the epidermal cells and the plasmalemma of single cells in the epidermis and cortex as the rate-limiting barrier (Arisz, 1964; Brouwer, 1965; Hendricks, 1966; Laties, 1959; MacDonald and Laties, 1963; MacRobbie and Dainty, 1958; Spanswick and Williams, 1964). Therefore, concentration gradients can be established at the surface (plasmalemma) of root-hair cells. At present experiments have not been conducted to show clearly whether or not the root hairs increase significantly the rate of uptake of ions by diffusive processes. Their potential for increasing uptake is readily apparent from Eq. (20), and calculations indicate that for equal surface areas of root hair and main root the flux of P to the root hair can be four times greater than to the main root if the same concentration gradient is assumed. Thus, the ions in the soil solution diffuse or are carried by convection into the cell walls of the epidermal and cortical cells, or the water free space. The next step is an active transport of the ions across the plasmalemma into the cytoplasm. Probable mechanisms for transfer of cations, anions, and molecules across the plasmalemma have been reviewed elsewhere (Brouwer, 1965). The significance of this transfer, with regard to diffusion in soil, is that it lowers concentration in solution at the root surface which induces diffusion toward the root. Subsequent steps in transfer, such as movement across the tonoplast into the vacuole or a direct movement from the cytoplasm into the xylem, have less direct effects on diffusive processes. These later steps enter the picture mainly as they affect the rate of uptake into the cytoplasm from the external solution. These roles of these anatomical features raise questions about what boundary is significant in measuring the surface area of roots. Fried and Shapiro (1961) concluded that diffusion of ions through the free space would not likely limit the rates of active uptake. If the concentration within the free space were maintained equal to the adjacent external solution, possibly the area of the plasmalemma would be more important than the area of the external root. B. THEPLANT ROOTAS

A SINK FOR

NUTRIENTS

This aspect of the plant is clearly related to the concentration gradient that the root can establish between its surface and the external solution. The size of the sink as affected by length and radius of the root will be treated later. The actual gradient may change with time, but we will

144

S. R. OLSEN AND W. D. KEMPER

consider first the role of the plant in maintaining a low concentration of ions at the root surface. The maximum concentration gradient develops initially if the root can absorb the nutrient so rapidly that concentration of the nutrient in soil solution at the root surface is practically zero. A number of attempts have been made to determine the lowest value to which the plant can decrease the concentration. Unless adequate provision has been made to replenish the solution, such values will be too large (Asher et al., 1965). With 8 species Asher and Loneragan (1967) found that the roots absorbed P from solutions at 0.04 p M at pH 5.4. Russell et al. ( 1958) showed that rye and barley absorbed P from solutions at 0.032 p M (0.001 ppm. P). Olsen ( 1 950) observed that rye reduced the NOa- in solution to 0.3 p M . Barley decreased K in solution to values less than 0.025 pM (Williams, 1961). Although the plant has the ability to absorb ions at these low concentrations, this fact does not imply that roots in a soil reduce the concentration to these levels. The rate of uptake will depend on concentration, and it is also coupled with diffusive and convective processes in the soil. A better estimate of the C , at the root surface is likely to be made by determining the concentration distribution in soil near the root, using the diffusion equations, independently measured values of D , and B and boundary conditions (i.e., initial solution concentration and uptake rates). Calculations of concentrations of P in solution at the surface of corn roots revealed that it takes 6 to 8 days for C , to reach 0.1 Co. In two soils Cowas 5.2 p M and 1 1.0 p M (Olsen el al., 1962). After 6 to 8 days, these roots probably absorb P at a lower rate and new roots at different locations in the soil have become the major suppliers of P to the plant. In soil a corn root does not appear to lower the concentration of P to some minimum, fixed value. Instead the rate of uptake appears to be a linear function of the concentration of P in solution on a given soil. After a day or more of absorption the value for C , is roughly a fixed fraction of the initial Co [Olsen and Watanabe, (1966)l. Thus, it is likely that the root does not develop or maintain the maximum concentration gradient that might be expected from its ability to lower the concentration of P in water cultures to such low values. Experiments of this type have not yet been reported on other ions or on acid soils with very low concentrations of P in the soil solution. The root serves as a sink for ions or molecules until the absorption mechanisms become saturated. As this condition is approached the mass flow of water to the root surface may cause an increase in concentration relative to the soil and diffusion will occur away from the root. Several examples of this phenomenon have been illustrated (Barber, 1962;

MOVEMENT OF NUTRIENTS TO ROOTS

145

Barber et al., 1963; Lavy and Barber, 1964; Oliver and Barber, 1966a,b) for Al, Cu, Ca, Mg, Mo, S, and Sr. Legumes exhibit a mechanism for partly excluding silica from the soil solution whereas gramineous species absorb silica as a function of the concentration of Si(OHh in the solution and the amount of water transpired (Jones and Handreck, 1965). C. RELATION BETWEEN WATERTRANSPORT AND ION TRANSPORT IN PLANTS The relation between water transport and ion transport in plants has been difficult to define, and a clear understanding or agreement has not evolved. Viscous flow of the soil solution may increase the effective diffusion coefficient (by causing significant hydrodynamic dispersion) in addition to causing convective movement of the nutrient. The data of Nielsen and Biggar ( 1 963) (Fig. I 1 of this article) and the development in Section IV, have provided a means for evaluating the contributions to uptake of these mechanisms. The translocation of nutrients from the root to the shoot may affect the concentration of ions at the root surface and, therefore, the concentration gradient in Eq. ( 18). The symplasm connecting epidermal cells to the xylem vessels provides a passage where diffusion of ions may be important, especially in conjunction with the flow of water through the cells. This mechanism of transport is not very sensitive to inhibitors of active uptake (Brouwer, 1965). An important concept emerging from many studies (Russell and Barber, 1960; Brouwer, 1965) is that the effect of transpiration on salt uptake may vary greatly depending on the nutrient status of the plant. Generally, the results observed by Broyer and Hoagland ( 1943) have been confirmed, viz. that transpiration increased uptake of salts when high salt concentrations were used but not when low salt concentrations prevailed. Later studies have indicated that water transport through the root increases the active component of ion uptake and a small passive component as well (Brouwer, 1965). Thus, the transpiration stream can be important because of its relation to the saturation of ion absorption and accumulation in the roots of intact plants. The root may become saturated with ions, which in turn will decrease the flux of ions to the root more quickly at low rates of transpiration than at higher rates. Therefore, diffusive flux as well as convection will be related to the rate of transpiration in intact plants. D. THERELATION BETWEEN ION UPTAKE A N D CONCENTRATION

The rate of transport of ions to the root by diffusive processes in the soil depends upon the rate of removal of ions from the root-soil interface by ion absorption and transport processes in the plant (Bouldin, 1961).

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S. R. OLSEN A N D W. D. KEMPER

The mathematical boundary conditions that attempt to relate this mutual dependence are shown in Eqs. (15), (16), and (17). To apply these equations it is necessary to evaluate the constant, h or M, that relates uptake to concentration in solution. The value of M will be constant usually for only a limited range of concentration, and M decreases as concentration increases over a wide range. This limitation may not prove serious, however, if the concentration at the root surface varies less than a factor of 10 after the first few minutes of uptake. Bouldin ( 1 96 I ) suggested that values of M should be chosen corresponding to the concentration expected at the root surface after an absorption period of 4 to 8 hours. This boundary condition has many attractive features inviting it to be used and tested, so other limitations or precautions need to be considered. Essentially, M is the rate constant for the active transport of ions, salts, or molecules probably across the plasmalemma. The value of M should be measured in a well-stirred and aerated nutrient solution that approximates the nutrient concentrations likely to be encountered by roots in the soil, and adequate provision must be made for replenishing the solution (Asher er al., 1965). Other factors may influence M as well, such as, age of the root, its nutrient status at the beginning of the experiment, interaction with other ions, pH, kind and degree of microbial growth on the root surface (Bowen and Rovira, 1966), and possibly HC03- concentration (Olsen, 1953; Sutcliffe, 1962). When this boundary condition is applied, it will be appropriate to have some measure of the degree that the conditions of water culture approximate those found in the soil solution. Curves relating uptake to concentration often follow a Langmuir isotherm, but over a limited low range of concentrations the relationship may be approximately linear. These values of M from the lower range of concentration may represent more accurately the concentration at the root-soil interface. For example, nearly maximum rates of uptake of NO3- by wheat from water cultures have been observed at concentrations of I mM (Kihlman-Falk, 1961). In Wanbi sand Clarke (1966) found a linear relation between uptake rates of NO3- by wheat from nil to 70 mM. Bouldin (1961) showed that the concentration of P at the surface of barley roots will fall low enough under soil conditions so that the uptake mechanism is no longer saturated, although initially the concentration in solution was 5 times that required to saturate the absorption mechanism.

E. ROOTSYSTEMS AS RELATED TO DIFFUSION The relative importance of the length, diameter, surface area, age, rate of elongation, and root hairs of roots for nutrient absorption by plants has

MOVEMENT OF NUTRIENTS TO ROOTS

147

been difficult to measure. This problem stems from the complexity of actually making the physical measurements of the geometry of the root and an extreme lack of information about the ionic environment of roots. Microorganisms in the rhizosphere and mucigels around the roots add to this complexity (Jenny, 1966). The development of equations based on diffusion and convection in Sections I1 and IV has provided a more definitive means of studying these problems although the applications have just begun. Nye (1966b) has contributed useful equations and discussions of this problem. Root diameter is especially important for the conditions where potentially diffusible ions are adsorbed by the solid phase. For example, with P, calculations from Figs. 4 and 5 show that Q for 24 hours can be 4 times greater from root hairs than from the main root. The dimensions were chosen so that equal surface areas existed for the root hairs and the main root per centimeter length of the main root. Bouldin ( 1 961) has made similar comparisons. Lewis and Quirk (1 965a,b) presented data indicating that root hairs could account for 80 to 85% of the total uptake of P by wheat in soil. Place and Barber (1964) observed that corn roots with hairs absorbed 2.5 to 5 times more Rb from soil than roots without hairs, but the differences may have been due to poorer root-soil contact. The presence of root hairs allows the root system to absorb P at a faster rate from a given soil. This difference does not mean that C , at the root surface is smaller, but that the average concentration gradient is greater because the average distance is smaIIer from the root surface into the soil supplying P. Root hairs will have a greater flux of C1- and NOs- to their surface than the main root during initial periods of absorption. After a few hours, however, the flux over the root hair and the main root will be nearly the same so the root hairs would not add continually to the total rate of uptake (Passioura, 1963; Barley, 1964; Nye, 1966b). The effect of root length can be illustrated by calculations from Fig. 4 for roots with a = 0.05 cm. and a = 0.025 cm. In 24 hours the uptake g./cm., respectively, (Olsen et al., would be 9 X lo-* g./cm. and 5 x 1962), but the smaller root would have 4 times the length if an equal volume of root is assumed. Therefore, the uptake per plant could be at least twice as much for the plant having roots with the smaller radius. These differences in uptake of P due to length and radius of root would also be similar for CI- or NO,-. Since dqldt will be greater in the early stages of uptake, elongating roots would likely absorb more ions per centimeter length than the stationary root. The time sequence in the initiation and growth of root

148

S. R. OLSEN AND W. D. KEMPER

hairs could alter this picture. Barley ( 1 964) has estimated that the flux of CI- to roots of wheat in soil is twice as great for the elongating root as for the stationary root. Passioura (1 963), Miles ( 1 9 6 3 , and Anderssen and Radok ( 1 964) have suggested models to examine the importance of root elongation. These models have not been assessed as to their predictive value for actual systems as yet. The effect of aging, presence or absence of root hairs, and microbial effects will need to be evaluated as well in order to define the true effect of root elongation. The root cap absorbs ions readily, but it does not appear to transmit these ions to the older root or the shoot (Wiebe and Kramer, 1954). Thus, the absorption mechanism in a root cap may become saturated rather soon and as such it would not continue to serve as a sink for ion uptake. Considerable information about root systems in soil has been collected and large variations among species has been observed. In testing models of diffusive flow the actual roots in the experiment need to be measured and their absorptive properties determined.

F. DISTANCES FROM THE ROOTTO WHICH NUTRIENTS AREEXTRACTED A major interest in diffusion of ions to roots stems from its potential application in determining the availability of nutrients and in gaining a clearer understanding of the processes involved in availability of nutrients. Gardner (1965) has reviewed data on the movement of N in soil and to roots in relation to diffusion coefficients, water content of soil, and convection of ions. In general, the total NOa- content in the top 15 em. of soil may be considered to be nearly all available and some movement of N to the roots may occur from outside the volume of soil occupied by the roots. The volume of soil contributing ions to the root can be much less than the volume occupied by the roots in the case of P and other ions with much smaller values of D,/Bthan for NOs-. Olsen et af. (1962) calculated the concentration distribution of P in the soil solution from the root surface into the soil with time. After 1 and 3 days P had moved a distance of 0.5 and 0.9 mm., respectively, from the root surface and a distance of 1.3 mm. after 10 days. If root hairs had been absorbing P as well, the distance of movement would be less but the volume of soil contributing to total uptake would be larger. After 1 and 3 days the roots had removed 9 and 12%, respectively, of the labile P in these volumes of soil. A more intensive removal of P occurred from the volume of soil 0.1 mm. from the root surface and the roots absorbed 33% of the labile P in this volume in 1 day. These corn roots were absorbing P at a rate comparable to rates observed from field experiments. Since the roots absorb P more slowly

MOVEMENT OF NUTRIENTS TO ROOTS

149

as they age, these calculations indicate that only a fraction of the labile P will be absorbed by a crop and that this fraction will vary with distance from the root surface. If this fraction varies considerably among soils, such information could be useful in interpreting soil tests which measure the contribution of the total soil volume. The size of root systems for various species has been measured by Dittmer (1938), viz. 5.5, 11.8, and 26.2 crn.2/cm.3of soil for oats,Avena sativa; winter rye, Secale cereale; and bluegrass, Poa pratensis, respectively, in the top 15 cm. of soil. These measurements included root hairs which contributed about 90% of the total surface area. If the roots for oats are assumed to be 0.05 cm. radius, all the soil volume is within 1 mm. g./cm.2 sec., of the roots. If these roots absorbed P at a rate of 4 X as observed by Olsen et al. ( 1 962) for corn, they would take up 1.17 X 1 O3 g. of P per day. If we assume a growth period of 12 weeks, the amount of P absorbed would be at least 10 times greater than expected. Therefore, zones of removal must be overlapping (particularly in the case of root hairs), and only a fraction of the root surface is in the stage where uptake is as rapid as was the case for the fresh young roots used in the studies of Olsen el al. (1962).

G. EFFECTOF WATER CONTENT OF SOIL ON T H E ABSORPTION MECHANISM OF THE ROOT When metabolic processes control ion uptake in the root cells, water stress in the soil may control water relations in the plant and increased water stress may reduce ion uptake. A check in growth induced by lack of water in young tomato plants depressed the net accumulation of N and P (Gates and Bonner, 1959). Gates and Bonner (1959) also observed a lower rate of synthesis of RNA in the leaves of these plants. Dean and Gledhill (1956) found that roots preconditioned at a high water stress absorbed less P than control roots. Paul ( 1 965) observed a lower rate of C1 absorption by roots pretreated in soil at a high water stress. Olsen et al. ( I 962) plotted the concentration of P in solution at the root surface with time for roots in soil at 0.33 and 9 bars water suction. At a given time the concentration was greater around the roots at 9 bars but the rate of uptake was less. This result indicated that a high water stress inhibited physiological processes in the root. Possible differences in root-soil contact were not studied. REFERENCES Anderson, W. B., and Kemper, W. D. 1964. Agron. J . 56, 453-456. Anderssen, R. S.. and Radok. J. R. M. 1964. Australian Math. SOC.,Symp. Water, Soil, Plants, Adelaide, South Australia, 1964.

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Arisz, W. H. 1956. Protoplasma 46, 5-62. Arisz, W. H. 1964. Acta Botan. Neerl. 13, 1-58. Asher, C. J., and Loneragan, J. F. 1967. Soil Sci. 103,225-233. Asher, C . J . , Ozanne, P. G., and Loneragan, J. F. 1965. Soil Sci. 100, 149-156. Barber, S. A. 1962. Soil Sci. 93,39-49. Barber, S . A., Walker, J. M., and Vasey, E. H. 1963. Agr. Food Chem. 11,204-207. Barley, K. P. J. 1964. Australian Math. Soc., Symp. Water, Soil, Plants, Adelaide, South Australia, 1964. Barley, K. P. J., and Naidu, N. A. 1963. Australian J . Exptl. Agr. Animal Husbandry 4, 39-48. Bolt, G. H., Shainberg, I., and Kemper, W. D. 1967. SoilSci. 104,444-453. Bouldin, D. R. 1961. Soil Sci. SOC.A m . Proc. 25,476-480. Bowen, G. D., and Rovira, A. D. 1966. Nature 211,665-666. Bray, R. H. 1954. Soil Sci. 78,9-22. Brouwer, R. 1965. Ann. Rev. Plant Physiol. 16,241-266. Broyer, T. C., and Hoagland, D. R. 1943. Am. J . Botany 30,261-273. Carslaw, H. S., and Jaeger, J. C. 1959. “Conduction of Heat in Solids,” 2nd ed. pp. 335341. Oxford Univ. Press, Oxford. Clarke, A. L. 1966. Ph.D. Thesis, Univ. of Adelaide, Adelaide, South Australia. Crank, J. 1956. “The Mathematics of Diffusion.” Oxford Univ. Press, Oxford. Dean, L. A., and Gledhill, V. H. 1956. Soil Sci. 82,71-79. Dittmer, H. J. 1938. A m . J . Botany 25,654-657. Evans, S . D., and Barber, S. A. 1964. Soil Sci. SOC.Am. Proc. 28, 56-57. Fried, M., and Shapiro, R. E. 1961. Ann. Rev. Plant Physiol. 12,91-112. Gardner, W. R. 1965. Agron. Monograph 10,550-572. Gardner, W. R., and Ehlig, C. F. 1962. Agron. J. 54,453-456. Gardner, W. R., and Mayhugh, M. S. 1958. Soil Sci. SOC.Am. Proc. 22, 197-201. Gates, C. T., and Bonner, J. 1959. Plant Physiol. 34,49-55. Geering, H. R. 1967. M.Sc. Thesis, Cornell University, Ithaca, New York. Graham-Bryce, I. J. 1963. J . Soil Sci. 14, 188-200. Hendricks, S. B. 1966. Soil Sci. SOC.Am. Proc. 30, 1-7. Hodgson, J. F., Lindsay, W. L., and Kernper, W. D. 1967. Soil Sci. SOC. A m . Proc. 31, 4 10-4 13. Jenny, H. 1966. Plant Soil 25,265-289. Jenny, H., and Overstreet, R. 1939. J . Phys. Chem. 43, 1185-1 196. Jones, L. H. P., and Handreck, K. A. 1965. Plant Soil 23,79-96. Jost, W. 1952. “Diffusion.” Academic Press, New York. Kemper, W. D., and Rollins, J. B. 1966. Soil Sci. SOC.A m . Proc. 30, 529-534. Kemper, W. D., and Van Schaik, J. C. 1966. Soil Sci. SOC.A m . Proc. 30,534-540. Kemper, W. D., Maasland, D. E. L., and Porter, L. K. 1964. Soil Sci. SOC.A m . Proc. 28, 164- 167. Kihlman-Falk, E. 196 I . Physiol. Plant 14,4 17-438. Lai, T. M., and Mortland, M. M. 1961. SoilSci. Sac. A m . Proc. 25, 353-356. Laties, G. G. 1959. Ann. Rev. Plant Physiol. 10,87-112. Lavy, T. L., and Barber, S. A. 1964. Soil Sci. SOC.A m . Proc. 28,93-97. Lewis, D. G. 1963. Ph.D. Thesis, Univ. of Adelaide, Adelaid, South Australia. Lewis, D. G., and Quirk, J. P. 1962. Intern. SOC.SoilSic. Trans. Comm. IV, V , Palmerston N , NewZealand,pp. 132-138. Lewis, D..G., and Quirk, J. P. 1965a. Intern. Atomic Anergy Agency Tech. Rep?. Ser. 48.

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Lewis, D. G., and Quirk, J. P. 1965b. Nature 205,765-766. I OW, P. F. 1962. Soil Sci. 93,6-15. MacDonald, J. R., and Laties. G. G. 1963. Plant Physiol. 38,38-44. MacRobbie, E. A. C . , and Dainty, J . 1958.3. Gen. Physiol. 42,335-353. Melsted, S. W., and Bray, R. H. 1967. Soil Sci. 63,209-225. Miles, J. W. 1965. Proc. Roy. SOC. A284, 137-145. Moreno, E. 1957. Ph.D. Thesis, Univ. of California, Berkeley, California. Nielsen, D. R., and Biggar, J. W. 1962. Soil Sci. SOC.A m . Proc. 26, 216-221. Nielsen, D. R.,and Biggar, J. W. 1963. Soil Sci. SOC.A m . Proc. 27,lO- 13. Nye, P. H. I966a. J . Soil Sci. 17,I6-23. Nye, P. H. 1966b. PlantSoil25,81-105. Oliver, S., and Barber, S. A. I966a. Soil Sci. Soc. Am. Proc. 30,82-86. Oliver, S., and Barber, S. A. 1966b. Soil Sci. SOC.Am. Proc. 30,468-470. Olsen, C . 1950. Physiol. Plant 3, 152-164. Olsen, C . 1953. Physiol. Plant 6,848-858. Olsen, S. R., and Watanabe, F. S. 1963. Soil Sci. SOC.Am. Proc. 27,648-653. Olsen, S. R., and Watanabe, F. S. 1966. Soil Sci. SOC.A m . Proc. 30,598-602. Olsen, S. R., Watanabe, F. S., and Danielson, R. E. 1961. Soil Sci. SOC. A m . Proc. 25, 289-294. Olsen, S. R., Kemper, W. D., and Jackson, R. D. 1962. Soil Sci. Soc. Am. Proc. 26,222227. Olsen, S. R., Kemper, W. D., and Van Schaik, J. C. 1965. Soil Sci. SOC.Am. Proc. 29, 154- 158. Parsons, R. 1959. “Handbook of Electro-chemical Constants.” Academic Press, New York. Passioura, J. B. 1963. Plant Soil 18, 225-238. Paul, J. L. 1965. Agrochimica 9,370-379. Place, G . A., and Barber, S. A. 1964. Soil Sci. SOC. Am. Proc. 28,239-243. Porter, L. K., Kemper, W. D., Jackson, R. D., and Stewart, B. C. 1960. Soil Sci. SOC.A m . Proc. 24,460-463. Russell, R. S., and Barber, D. A . 1960. Ann. Rev. Plant Physiol. 11, 127-140. Russell, R. S., Russell, E. W., and Marais, P. G. 1958.3. Soil Sci. 9, 101-108. Salvinien, J . , Marignan, R., and Cordier, S. 1954. Compt. Rend. Acad. Sci. 238,888-890. Schofield, R. K., and Graham-Bryce, I. J . 1960. Nature 188, 1048-1049. Shainberg, I., and Kemper, W. D. 1966a. Soil Sci. SOC.Am. Proc. 30,700-706. Shainberg, I., and Kemper, W. D. 1966bSoil Sci. SOC.A m . Proc. 30,707-713. Spanswick, R. M., and Williams, E. J. 1964. J . Exptl. Botany 15, 193-198. Sutcliffe, J . F. 1962. “Mineral Salts Absorption in Plants.” Macmillan (Pergamon), New York. Van Schaik, J. C . , and Kemper, W. D. 1966. Soil Sci. SOC.A m . Proc. 30,22-25. Van Schaik, J. C . , Kemper, W. D., and Olsen, S. R. 1966. Soil Sci. SOC. A m . Proc. 30, 17-22. Wiebe, H . H . , and Kramer, P. J. 1954. Plant Physiol. 24, 342-348. Williams, D. E. 1961. Plant Soil 15, 387-399.

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THE ELECTRON MICROPROBE X-RAY ANALYZER AND ITS USE IN SOIL INVESTIGATIONS Michel

P. Cescas,

Edward H. Tyner, a n d Lawrence J. G r a y ' University of Illinois, Urbana, Illinois

I. 11. 111.

IV.

V.

V1.

VII.

Introduction ........ ...................................................................... Description of the on Microprobe Analyzer ...... .._. . .................. A. Basic Components ....................................................................... Methods for Analysis .......................................................................... A. Modes of Operation for Signal Generation a play .............. .... .... B. Resolution of Characteristic X-Rays .......... ............................ Fundamentals of Quantitative X-Ray Analysis ........................................ A. Wavelength Shift ..... ............... B. Initial Corrections .... ... C. Corrections for Interactions of Incident Electrons and Generated X-Rays with Atoms: Single Standard Analysis ... ........ ., .... . D. Analysis by t h e Multi The Quality of X-Ray Mic A. Precision and Accuracy B. Spatial Resolution ............ ......... .. . .. . . . .... .. . . . ................ ..... . ..... ... .... C. Detection Limits .......................................................................... Applications to Soil Investigations .... ................. A. Preparation of Soil Specimens.. .. B. Electron Microprobe Analysis of C. Analysis of Unpolished Micron to Submicron Specimens ................... Summary and Conclusions ............... .................. .................. References

Page 153 i55 155

157 157 158 162 163 163

164 173 176 176

177 178 179 179 183 186 191 192

I. Introduction

The microchemical procedures commonly employed in soil laboratories include scaled-down modifications of macromethods, methods using color reactions, and methods based on various physical phenomena exhibited by the deexcitation of energized atoms. The composition of sam'Supported in part by AEC Contract AT( I 1 - 1)- 1 198. Mr. Gray acknowledges support in part from the U. S. Naval Undersea Warfare Center, Pasadena, California.

153

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

ples obtained by these methods is reported on a bulk basis. With the electron microprobe analyzer, the chemical composition of microscopic regions of samples as minute as 1 p3 can be determined nondestructively and in situ. Thus, for the first time it is possible to analyze the composition of small precipitates that might form within the diffusion zones of fertilizer particles; study diffusion, weathering, and small particle homogeneity; supplement the petrographic examination of soil thin sections with chemical analysis; and study directly many other pedological problems the understanding of which has been impeded by the lack of chemical methods applicable to the analysis of microscopic to submicroscopic soil volumes. Whenever samples are bombarded with energetic electrons, the sarnples emit the characteristic X-rays of the elements present. The employment of this phenomenon for qualitative and quantitative chemical analyses with a finely focused electron probe was first conceived by Hillier (1947). Castaing and Guinier (1950) and Castaing (1 95 l), however, were the first to construct an operational electron microprobe. Subsequently, Castaing and Descamps ( 1 953, 1955) contributed substantially to the basic theory of quantitative electron microprobe analysis. The historical development of the electron microprobe analyzer is reviewed by Birks (1963) and Elion ( 1966). The first commercial instruments based on Castaing’s design were delivered by CAMECA, a French Company, in 1956. Today some fifteen manufacturers compete in a rapidly expanding market. Approximately 300 instruments are currently in use, about 30 of which are located at American universities. The electron microprobe analyzer was initially used in metallurgical investigations. In recent years, its use has been rapidly extended to other disciplines. Some applications in mineralogy and geology are rereported by Guillemin and Capitant ( 1 960), Agrell and Long (1 960), Philibert ( 1962), Adler ( 1963a,b, I966), Smith ( 1 965), and Keil ( 1967); in biochemistry by Andersen and Hasler ( 1966),and in biology and medicine by Engstrom (1963), Tousimis (1964), and Galle (1967). Three extensive bibliographies which include most recent applications of the electron microprobe analyzer are those by Heinrich ( 1966a), Campbell et al. ( 1 966), and Campbell and Brown ( I 968). Applications in soil science are few. These have been made by Rausell-Colom et al. (19651, Cescas and Tyner (1967a,b), Tyner et al. (19671, and Gillespie and Elrick (1968). The objective of this review is to acquaint the reader with the general characteristics of electron microprobe analysis, as approached from both the instrumental and theoretical aspects, and to discuss the use, poten-

THE ELECTRON MICROPROBE X-RAY ANALYZER

155

tialities, and limits of electron microprobe analysis as an analytical tool for soil studies. II. Description of the Electron Microprobe Analyzer

When a small highly energetic electron beam bombards a solid target a number of phenomena occur. These are outlined in Fig. 1 . Only several, however, are of sufficient immediate interest to warrant discussion in this review. Quantitative chemical analysis with an electron microprobe analyzer is performed by employing the characteristic X-radiation from the sample. The backscattering of incident electrons and the cathodoluminescence phenomena which are suitable for qualitative and semiquantitative analysis will also be of interest to soil scientists.

,

BREMSSTRAHLUNG

I

-

RADIATION/

~

~

1 -

CHARKTERISTIC RADIATION

~

EMISSION SPECTRA

ABSORPTION SPECTRA

BACKSCATTER ELECTRONS

I. X-RADIATION

SECONDARY ELECTRONS ~ ~ ~ AUGER ELECTRONS

2. U.V. RADIATION s 3. OPTICAL RADIATION

I. X-RAYS 2. ELECTRONS

LI ELECTRON

BEAM INTERACTIONS WITH SPECIMEN-

CHEMICAL REACTIONS PHYsicAL REACTIONS

FIG. 1. Interactions of an electron microprobe beam with a specimen. (After Elion, 1966.)

A. BASICCOMPONENTS Although electron microprobe analyzer designs vary in detail and degree of sophistication, all have the common basic components shown in Fig. 2 . 1 . The Electron Beam System

This system consists of an electron gun for the generation of an electron beam and two electromagnetic lenses. The lens system provides for the control of probe intensities varying from 0.001 to 10 microamperes (PA.) and probe diameters varying from 0.1 to 100 microns (p).

2 . The X-Ray Detector System Two systems are employed for X-ray detection and energy and/or

156

M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

wavelength characterization. The wavelength dispersion system employs one or more fully focusing X-ray spectrometers with associated propor-

ELECTRON GUN

ELECTRON DETECT OBJECTIVE LENS

FIG.2. Schematic diagram of the arrangement of the basic components of an electron microprobe analyzer.

tional counter detector and electronic counting circuitry. The less common energy dispersion system utilizes a proportional counter in line with a multichannel analyzer. 3 . Backscattered Electron Detector

Some electrons from the incident beam are backscattered by the target. The backscatter properties shown by a sample are primarily a function of the average atomic number (2)of that sample. Heterogeneity in sample composition, phase changes, and inclusions within samples are details which can be readily detected from backscattered electron measurements. 4 . The Optical Viewing System

A metallographic microscope provides for the location and observation of the sample in reflected light. Generally the sample can be viewed even when under electron beam bombardment. Some instruments are also designed to accept transmitted or polarized sample illumination systems. The optical system allows the position, sphericity, and size of the electron probe to be adjusted while observing the visible fluorescence from a cathodoluminescent material such as alumina or yttria.

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157

5 . The Sample Holding System This system serves to bring the samples and standards under the electron probe for successive analysis. The holder designs vary, but usually the specimens are placed on a sample stage which is equipped with X, Y , and rotational drives. Further details relative to the components discussed under headings 1 to 5 and their variations are given by Castaing (1 95 I), Wittry ( 1 957), Birks ( 1 963), Fisher (1 963), Ogilvie (1 964), Adler (1966), Elion ( 1966), and Keil ( 1 967).

6 . The Vacuum System During analysis, the electron beam optics column and X-ray specmm. Hg o r better. The trometers are maintained in a vacuum of probe column is usually compartmentalized to permit the change of the electron gun filament and/or the specimens without breaking the vacuum in other parts of the instrument. I l l . Methods for Analysis

A. MODESOF OPERATION FOR SIGNAL GENERATION AND DISPLAY The radiation and charged particle signals generated by the interaction of the electron probe with the target (Fig. 1 ) can be employed for different types of analysis as is illustrated in Fig. 3. Depending upon the CATHODOLUMINESCENCE

SECONDARY ELECTRON

D ELECTRON METHOD

TRANSMITTED ELECTRON METHO

FIG.3. Electron microprobe analytical techniques. (Modified from Elion, 1966.)

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M . P. CESCAS, E. H. TYNER, A N D L. J. GRAY

objectives of the analysis, the electron probe is used in either a static or in a scanning mode.

I . The Static Mode The electron probe is operated in a fixed position on a sample area preselected by manipulation of the sample stage controls. Qualitative and quantitative spot analyses are then performed by using either the wavelength or energy dispersion methods for isolating the X-rays characteristic of the various elements present in the sample. Also backscattered electron intensity and sample current can be used to closely approximate the average atomic number (2)of the volume under probe bombardment. 2 . The Scanning Mode The electron probe in response to the action of two sets of deflection plates or coils automatically sweeps over the area being analyzed. The signals commonly used are backscattered electron intensity, sample current, and characteristic X-ray intensity. The intensity of the chosen signal modulates an oscilloscope beam. The spot on the oscillosope screen has a synchronous and simultaneous position relative to that of the electron probe on the sample. The intensities of the signals can also be monitored along a chosen traverse of the sample to give for example, X-ray intensity line profiles. The composition profile thus obtained can be compared with intensity line profiles from standards for the rapid semiquantitative estimation of an element. Examples of the various data obtainable by the sweeping mode are illustrated in Fig. 4. B. RESOLUTIONOF

CHARACTERISTIC

X-RAYS

Two methods for the resolution of the characteristic X-rays emitted by a sample are used in electron microprobe analysis. 1. Wavelength Dispersion

The spectrum of the X-rays emitted by the sample is analyzed by one or more X-ray spectrometers equipped with fully focusing curved and

ground analyzing crystals of particular d spacings. With a static electron probe, the X-ray angle of incidence with the diffracting crystal is continuously changed although the X-ray take-off angle from the sample remains constant. For a crystal of given d spacing, wavelengths within a certain range will successively fulfill the B r a g condition (nh = 2 d sin 8 1. The limits of 8 are defined by the spectrometer configuration. The wavelengths of the characteristic lines observed permit the qualitative identification of the elements present in the sample (see Fig. 5).

THE ELECTRON MICROPROBE X-RAY ANALYZER

FIG.4. Examples of backscattered electron and X-ray sweeps and line profiles.

159

160

M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

Because of the limited 0 working range of the spectrometer, crystals with different d spacings ;ire required to cover the full X-ray spectrum

I

I

2.3200

I

I

2.3300

I

I

I

2.3400

1

2.3500

Spectrometer odometer reading (“.=reading x

2.01 36

FIG. 5 . Intensity of phosphorus (Ka)line from a wavelength scan obtained with an ADP crystal.

A.)

(1.0 to 90 in which element identification is made. The most commonly used crystals and their useful range in angstrom units are listed in Table I. TABLE I Effective Wavelength Ranges, d Spacing, and Diffracting Planes for Various Crystals Used in the ARL EMX Electron Microprobe Analyzer d spacing

Diffraction

Wavelength

Crystal LiF NaCl S O z(quartz) EDdT (ethylenediamine &tartrate) ADP (ammonium dihydrogen phosphate) K A P (potassium acid phthalate) Mica PbSD (lead stearate decanoate)

2.0136 2.8203 3.3436 4.4040 5.3200 13.3160 9.9750 50.10

200 200 1011

020 101 1010 002

-

1 .OO-3.77 1.41-5.29 1.67-6.26 2.20-8.27 2.66- 10.00 6.57-24.16 10.00- 18.70 25.0-93.0

THE ELECTRON MICROPROBE X-RAY ANALYZER

161

Currently, attempts are being made to increase the ease of detecting very light elements (Be to F) with blaze angle gratings (Nicholson and Wittry, 1963). Commercial spectrometers using this device, however, are only currently becoming available. Characteristic X-ray lines can be readily resolved with only four crystals (LiF, ADP, KAP, and PbSD) for elements from 2 = 5(B) to Z = 94(Pu). A wavelength scan, using an ADP crystal, which revealed the presence of P in a minute mineral inclusion present in hypersthene is shown in Fig. 5 . Analysis indicated that the inclusion was apatite (see Fig. 14). For quantitative analysis, the intensities of selected characteristic lines are almost always measured with sealed proportional counters and/or flow proportional counters fitted with very thin windows. The amplified detector signals are displayed on scalers, chart recorders, and/or cathode-ray oscilloscopes. Step-by-step quantitative analysis can be determined along a sample traverse across phase boundaries, surface deposition layers, diffusion zones, etc., by moving the sample under the beam in successive micron steps (see Fig. 11). Qualitative information can be obtained more quickly by moving the sample continuously under the beam. 2. Energy Dispersion The principles and applications of this method are discussed by Dolby (1959), Dolby and Cosslett (1960), Liebhafsky et al. ( I 960a), Birks and Batt ( 1963), Fergason ( 1 9651, and Schippert et al. (1967). In energy dispersion analysis no crystals are employed. Within the linear range of a proportional counter, the height (or amplitude) of the output pulse is proportional to the energy of the X-ray quantum being detected. Since the energy of the X-ray quantum is inversely proportional to wavelength, E = hv = (hclh), an energy spectrum analysis can be performed for all the X-rays emitted by a specimen. A multichannel analyzer in line with the proportional counter (sealed or flow) samples the whole energy spectrum. The energy spectrum can be displayed on a cathode ray tube for visual observation and photographic recording. The spectrum can also be printed-out numerically, punched out directly on computer tape or cards, or plotted directly using an X-Y recorder. The resolving power by the energy dispersion method is limited to elements which are three atomic numbers apart (Castaing, 1960). This is much less than the resolution attainable with wavelength dispersion systems. Before suitable diffracting materials (blaze angle grating and

162

M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

PbSD) were available, this procedure was the only method for the analysis of light elements where the wavelength differences resolved are relatively large. An important aspect of the energy dispersion method is that rapid surveys and semiquantitative analysis can be made in a fraction of the time required for similar analysis by the wavelength dispersion method. For quantitative analysis, areas under the peak of the pulse height distribution of the particular unknown and standards are compared. Overlapping of pulse height spectra may occur. When this happens, the simultaneous equation method of Dolby ( 1 959) is used to unfold the overlapping spectra into individual pulse height spectra. The energy dispersion method is most useful when employed in conjunction with the wavelength dispersion system. IV. Fundamentals of Quantitative X-Ray Analysis

Quantitative analysis is performed with a static probe. Castaing ( 195 1) initially analyzed metallurgical binary systems. The approach

consisted of comparing the directly generated intensity ZA,u of a strong characteristic line, e.g., AK,, emitted by the element A in the sample (unknown) with the directly generated intensity I A , s of the same line from a pure element A (standard) under identical analytical conditions. In the first approximation, Castaing (195 1) showed:

where CAis the concentration of element A in the sample, expressed as the weight fraction. Taking into account the variation in the electron scattering and deceleration properties for elements of different atomic number, Castaing developed his second approximation:

where aiis the experimentally determined specific electron deceleration and scattering power for the ith element in the sample. From the beginning, Castaing recognized that the conversion of X-ray intensity to mass concentration was complicated by the fact that the

THE ELECTRON MICROPROBE X-RAY ANALYZER

163

X-ray intensities measured by the detector system were not the true intensities generated within the sample or the standard by the electron probe and that corrections had to be applied to obtain the true intensities. A. WAVELENGTH SHIFT

Before any counting is done, one must be aware of the possibility of shift of analytical line wavelength between the standard and unknown. White et af. (1965) stated that “nearly all K lines (including satellites) of wavelength greater than about 2.0 A can be expected to show some variation in wavelength, depending on how the element is combined chemically.” As examples, White et al. (1 965) indicated that the wavelength of the line for aluminum metal is about 7.959 A.whereas for aluminum in a-AlaOs it is about 7.982 A. Other examples can be found in the works of Koffman and Moll (1966), Fischer and Baun (1 965a), and Colby ( 1967). Keil ( 1 967) says that wavelength shifts can be considered negligible for lines with a wavelength less than 4 A.,that is, with elements 2 2 18 for K lines and 2 3 48 for L lines. Fischer and Baun (1964, 1965a,b, 1966a,b) and Hart and Pilney (1 967) show the important deviations which can be obtained when wavelength shifts occur from a pure metal to one of its oxides. Lee and Campbell (1965) have shown that besides chemical bonding, variation in room temperature may alter the analyzing crystal lattice through thermal effects. This is usually no problem since most of the instruments are kept in air-conditioned rooms. The wavelength of the peak intensity of the analytical line must be determined separately on both the standard and unknown. When using standards close in composition to the sample and having the same crystal structure as the sample the shift may be negligible. B. INITIALCORRECTIONS Corrections to be applied to raw data can be divided into two categories. First, the corrections inherent to the interactions of the incident electrons and the generated X-rays with the atoms of the sample, and second, corrections of an instrumental nature. All data from the electron microprobe must always be corrected first for deadtime, drift, contamination, and background. 1 . Deadtime

For a finite time following each pulse, the detector and associated electronics are insensitive to further pulses. This lag in detection system response is called deadtime. Wittry ( 1 964b) strongly advocated a cor-

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

rection for deadtime. Heinrich et al. (1965) have discussed several forms for the relation between the true counting rate and the measured counting rate. If N is the true counting rate and N ’ is the measured counting rate, then:

N=

” (1 - rN‘)

(3)

where T is the deadtime. Several methods of measuring 7 have also been presented by Heinrich et al. ( 196% and Sawatzky and Jones ( 1 967). 2 . Drift and Contamination During analysis a decrease in the counting rate with time may occur. This is usually attributed to short-term instrumental drift and/or carbon contamination of the sample surface. The latter results from the cracking by the electron probe of organic vapors (oil from vacuum pumps, etc.) present in the microprobe enclosure and their uneven deposition on the regions being analyzed. Usually the errors resulting from these causes are negligible and are ignored. In other cases, when the decrease in counting rates with time is appreciable, the errors are assumed to be linear with time and are corrected by extrapolation procedures (Brown, 1966). Carbon contamination can be minimized by cooling the specimen surroundings (Ong, 1966), by heating the sample, or by directing a small gas jet over the probe impact area (Castaing, 1960; Moll and Bruno, 1967). 3 . Background

In addition to the characteristic X-rays emitted by the element being analyzed, the measured intensity also includes continuous radiation from the sample itself, cosmic rays, electronic noise, and fluorescence produced by the spectrometer components. The extraneous signals from these sources are called background. The most generally applicable method for background correction is that initially used by Castaing (1951). In this, the X-ray intensities above and below the peak wavelength are measured and averaged. The wavelength for these rneasurements is generally 5 / 2 times the width at half maximum of the X-ray line intensity distribution measured from the peak wavelength. The calculated background contribution is then subtracted from the measured peak intensity. C. CORRECTIONS FOR INTERACTIONSOF INCIDENTELECTRONS A N D GENERATED X-RAYSWITH ATOMS: SINGLESTANDARD ANALYSIS Two methods of reducing electron microprobe X-ray intensities to concentrations are used, namely, the single standard or “absolute” and

THE ELECTRON MICROPROBE X-RAY ANALYZER

165

the multiple standard methods. Deadtime, drift, and background corrections are made for both. For the “absolute” method, additional mass absorption, fluorescence, and atomic number corrections are made.

I . Mass Absorption Some of the X-rays generated within the sample are absorbed by the sample itself. To obtain the true characteristic X-ray intensities generated, absorption corrections must be made. Correction procedures based on either experimental measurements or theoretical treatments have been advanced by Castaing (1 95 l ) , Birks ( 1960), Theisen ( I965), Green ( 1963, 1964), Philibert ( 1 963), Duncumb and Shields ( 1966), Barman ( 1967), and Heinrich ( 1967). The proposed model of Philibert (1 963) as modified by Duncumb and Shields ( I 966) and Heinrich ( 1 967) gives the closest agreement over a wide range of conditions when compared to well defined standards (Colby 1965, 1966). If fA,u(x) is the absorption correction for the intensity of A radiation in the unknown (u) and fA&) is that for A radiation in the pure standard A (s) then:

where CA is the concentration of element A in the unknown and K is the ratio of measured intensities from the unknown and the standard corrected for deadtime, background, and drift. The factorf(X) is defined as:

The term

x is given by:

where ( p / ~is )the ~ mass ~ absorption coefficient of the ith element for A radiation; Ci is the concentration in weight fraction of the ith element evaluated in the first approximation as the ratio of the measured intensities, Ki;and e is the X-ray take-off angle, the angle between the sample surface and the X-ray path to the diffracting crystal.

166

M. P. CESCAS, E. H. TYNER, AND L.

. I .GRAY

The modified Lenard coefficient, CT,is obtained from:

E, being the accelerating voltage in kilovolts, and E, the excitation potential of the analytical line also expressed in kilovolts and tabulated by Bearden (1 964, 1967). Finally:

where A and Z are the average atomic weight and average atomic number. Tables of mass absorption coefficients have been published by Adler and Goldstein ( 1 965), Heinrich ( 1966b), Frazer ( 1967), and Henke et al. (1967), and Gray and Wert (1968). When tables are not available, expressions given by Leroux ( 1 96 l ) , Heinrich ( I 966b), Frazer ( 1 967), and Taylor (1967) can be used to calculate the mass absorption coefficients. 2 . Fluorescence Secondary fluorescence by a characteristic line occurs whenever the wavelength of the fluorescing radiation falls below the absorption edge corresponding to the analytical line. The X-ray continuum will also fluoresce the characteristic line, because part of the continuum radiation falls below the absorption edge of the analytical line. Both of these possibilities are illustrated in Fig. 6. The measured line intensity, e.g., K,(A), would be enhanced by these secondary fluorescence effects and must be corrected to obtain true generated intensities. a. Correction for Secondary Fluorescence by a Characteristic Line. Castaing (1951) derived an expression for K line fluorescence by K lines. This relation was modified by Reed (1965) to allow for K-K, K-L, L-L, and L-K fluorescence. Other expressions attempting to obtain the fluoresced intensity have been derived by Wittry ( 1 964a) and Birks (1961). The method of Reed ( 1 965) for binary compounds appears to be the most generally applicable, from comparative studies made by Colby ( 1 965). Reed’s relation considers the accelerating voltage used and the type of fluorescence, e.g., L-K and K-K types.

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167

Ka(A)

t

Electron beam wavelength

K(A) Absorption edge wavelength

FIG. 6. Typical emission spectrum from compound target. Shaded region represents part of spectrum capable of fluorescing K(A) lines.

If [A,,, is the directly excited intensity of the analytical line and If is the intensity contributed to the analytical line due to fluorescence by a shorter wavelength characteristic line from element B, then:

.. -

- J j \u,

- I/

where:

CB= weight fraction of element B r,

ratio of mass absorption coefficients on either side of the absorption edge for element A (absorption jump ratio) wB = fluorescence yield for element B A = atomic weight of element A B = atomic weight of element B LIB = E,,/E, for element B ( ~ / p )= : mass absorption coefficient of element A for B radiation =

(p/pIB=

7 (")P '

the specimen

C,= mass absorption coefficient for B radiation by

168

M . P. CESCAS, E. H. TYNER, A N D L. J. GRAY

CT

v=-

where u is defined in Eq. (7)

Pij= a constant which takes different values for different fluorescence l;PLL=1; PKL=2.4andPLK=0.42. types2:PKK= If

ImA

is the measured intensity of A radiation it follows that:

Since I

f =

KfA

IA,u

Therefore:

and

Numerical values of the absorption jump ratios are either obtained from Lindstrom (1955) or Henke et al. (1967), or calculated from Heinrich's (1966b) or Frazer's (1967) data. The fluorescence yields are given by Fink et al. ( I 966) or Hagedoorn and Wapstra (1 960) or calculated from the semiempirical relation due to Burhop ( 1955):

(e) 1/4

=A+BZ+CZ3

with the coefficients A , B , and C given by Colby (1 967) listed in Table 11. b. Correction for Secondary Fluorescence by the Continuum. Castaing and Descamps (1955) and Kirianenko et al. (1963) showed that the contribution, by continuum fluorescence, to the total X-ray line intensity 'Private communication from John W. Colby, Bell Telephone Laboratories, Allentown, Pennsylvania.

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THE ELECTRON MICROPROBE X-RAY ANALYZER

TABLE I1 Constants for Calculating Fluorescence Yields ( w ) ~ Coefficients

K

A

-0.03795 0.03426 -0.11634 X lo-'

€3

C 'I

L - 0 . 1 I107 0.01368 0.21772 x

M -0.00036 0.00386 0.20101 x 10-6

After Col by ( 1967).

was generally small. This fluorescence radiation is generated deeper in the specimen than the primary radiation, resulting in greater absorption of the former. Theoretical considerations are difficult since fluorescence by the continuum is produced by a whole spectrum of wavelengths. Castaing and Descamps (1955), Green and Cosslett (1961), and Henoc (1962) give correction procedures for pure elements and binary compounds. The expression derived by Henoc is very complex, but is also considered to be the most accurate (Henoc et al., 1964). Henoc followed the same approach as Castaing ( 1 95 1) for the fluorescence by characteristic lines, but he integrated over all the continuum from the short wavelength limit to the absorption edge of the particular analytical line. Corrections for secondary fluorescence by the continuum are seldom made. Henoc's relation is very lengthy and its current limited usage does not warrant inclusion in this review. The interested reader is referred to Henoc's doctoral dissertation. c . Errors Resulting from Fluorescence Effects. It is possible in soil studies, as in mineralogical investigations, for secondary fluorescence to give rise to bothersome errors. These are illustrated in Fig. 7. Figure 7a illustrates how iron may be reported erroneously in an analysis of a small iron-free inclusion present in a iron-host mineral. Smith (1965) stated that the iron analysis of a sillimanite needle in contact with a biotite crystal containing 10 percent iron gave an apparent iron content varying from 0.1 to 0.5 percent iron for sillimanite, even though the latter contained no iron. The continuum emitted by the sillimanite excited iron in the biotite. These effects can be large when the inclusion diameter approaches, within a factor of 3, the probe diameter. However, the errors can be minimized by reducing the probe size and by using an excitation potential as low a s possible for exciting the inclusion. These operations will reduce the electron penetration depth and increase the minimum wavelength of the continuum. Figure 7b illustrates analysis made near the boundary of two phases varying in iron content. Fluorescence of the iron-rich region by charac-

170

M . P. CESCAS, E. H. TYNER, A N D L. J. GRAY

teristic lines and/or the continuum from the iron-poor region will cause an overestimation of iron in the iron-poor region. This is a most difficult error to correct. Electron

Fe radiation

Zone of Fe excitation by continuum

Phase boundory

FIG.7. Examples of fluorescence effects: (a) fluorescence of the matrix by the continuum generated in an inclusion containing no elements with characteristic lines capable of exciting iron; (b) fluorescence due to the continuum and characteristic lines from a phase containing elements with lines capable of exciting iron.

Although, in many cases, fluorescence corrections can be ignored, it is important that the reader recognize that there are instances where fluorescence may lead to serious analytical errors. For example, the characteristic lines of iron generated in small iron oxide inclusions 0.5 to 1.0 p below the surface of a silicate particle would fluoresce the silicon analytical K line resulting in an artificially high silicon X-ray intensity. 3 . Atomic Number

Castaing’s second approximation, Eq. (2), introduced the factor a which represented the overall effect of the atomic number upon X-ray generation within a sample. The factor a includes a specific deceleration power and an electron backscatter coefficient and must be determined experimentally. Investigations dealing with the effect of atomic number on X-ray intensity have been reported by Poole and Thomas (1962, 1963, 1966), Archard and Mulvey (1963), Birks (1963), Thomas (1963, 1964), Duncumb and da Casa ( 1 967), and Philibert ( 1965, 1967).

T H E ELECTRON MICROPROBE X-RAY ANALYZER

171

In general, the atomic number effect reduces the apparent concentration of heavier elements and increases the apparent concentration of lighter elements. A relation taking into account the effect of 2 on X-ray intensity is given by Duncumb and Shields ( 1 963):

where: Q is the ionization cross-section and is a function of EIE,; S is the stopping power and is a function of Z , A , and E; R is the backscatter loss factor and is a function of 2, A , E, and E,. The values of R and S may be taken from graphs prepared by Thomas (1964). The stopping power can also be obtained from the Bethe law (1930) as given by Nelms (1956,1958):

J where e is the base of natural logarithms, E is the energy of electron along its path within the sample, and J , the mean ionization potential, may be obtained from an expression given by Duncumb and da Casa ( 1967): 75.5 z!= 14.0 ( 1 .(t-e-n.l ?) +---

-

22'7.5

-

2

ioo+2

The backscatter loss factor can also be found from an integral expression by Duncumb and Shields (1963) or tables prepared by Colby.2 The ionization cross section can be determined from the expression of Webster ef al. (193 I): 1 QE = constant In U Urn

(19)

where m = 0.837 and E, is expressed in electron volts and here U = (EIE,). Substitution of Q (Eq. 19), S (Eq. 17), and R values into Eq. (16) will permit calculating CA by performing the integration. However, Colby (1 967) has simplified Eq. ( 16) to the form:

172

M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

R, K=CAR,

(m),(7.061 + In E In J , ) (m),(7.061 + In E - In J,) -

+

with E = (E, E J 2 . The atomic number effect assumes importance when the analyzed sample contains elements separated by three atomic numbers or more. The relationship between the above method for evaluating the effect of atomic number on X-ray intensity and the Castaing a-coefficient approach [ Eq. ( 2 ) ] has been discussed by Duncumb and Shields ( 1 963). The difficulty which arises with an experimentally determined coefficient is simply that in some cases the a is constant throughout the composition range of, for example, some binary system, whereas in other cases, a varies with Ci (Poole and Thomas, 1962). In particular, one is not able to predict beforehand, for a given system, the dependence of such a coefficient on composition.

4 . Sequence of Applying Corrections f o r the “Absolute” Method To obtain concentration as the ratio of the true intensities from the measured intensities the sequence of corrections listed below is followed: a. Correct standard and unknown intensities for deadtime, background, and drift. b. Calculate the ratio of the intensities of the unknown to the standard, and approximate, with Eq. ( I ) this ratio to concentration of the element being analyzed. c. Correct for mass absorption, secondary fluorescence, and atomic number effects. These correction formulas are applied in a reiterative manner to the set of elements, in the unknown, sequenced in an order determined by the wavelength of the analytical line. The iterations are continued until the resultant concentration for each element differs from the Ci input to that iteration by less than a specified amount, usually less than 0.001 weight percent. Reed and Mason ( 1 967) have shown that the Wegstein method of iteration is the most practical. The complete correction procedure for one element, A, in a compound and for one iteration only may be written as:

(m),(7.061 + In E - In J,) (m),(7.06 1 + In E - In J , ) The first term in brackets is the mass absorption effect correction factor, the second term the characteristic line fluorescence correction factor for one exciting line (when necessary), the third term is the atomic number

THE ELECTRON MICROPROBE X-RAY ANALYZER

173

effect correction factor, and K is the ratio of the fixed-time measured peak intensities for the standard and for the specimen corrected for deadtime, background, and drift.

D. ANALYSIS BY

T H E MULTIPLE-STANDARD

METHOD

In Section I I C , we have discussed the “absolute” or single standard method. Applying the formulas to a complex matrix of more than three elements, such as a soil, by hand calculation would indeed be formidable. With present-day computers, however, this is not impossible. Various computer programs have been developed by Hobby and Wood (1 967), Frazer el al. ( I 9661, Criss and Birks ( 1 966), Brown ( I 966), Lifshin and Hanneman ( 1966), Beaman ( 1967), Colby ( 1 967), and Gray ( 1 968). Some of these programs can handle data for up to eight elements. The mathematical correction procedures inherent to the emission of X-rays can be byTpassed by the use of calibration curves established empirically with numerous natural or artificial mineral standards, providing the analyses are carried out under identical analytical conditions. The multiple-standard or empirical method has been successfully used in mineralogical investigations by Adler (1963a,b, 1966), Keil and Fredriksson ( 1 963, 1964), Andersen and Hasler ( 1966), Smith and Stenstrom ( 1 9 6 9 , Smith ( 1 965, 1966a,b), Howie and Smith ( 1 966), Smith and Ribbe ( 1 966), and Ribbe and Smith ( 1966). The advantages of this method are its relative simplicity and, in some cases, its time-saving features. The primary disadvantage is the frequent unavailability of mineral standards of known composition having sufficient homogeneity on a micron scale. Although some mineral standards of known composition and homogeneity may be secured from different sources, one needs to assemble a personal collection. Keil (1967) discusses four criteria for assembling minerals of known composition to serve as microprobe standards. “First, the homogeneity of many grains of the mineral has to be checked by several hundred point-to-point electron microprobe analyses. Second, the mineral sample in question has to be checked optically as well as by means of electron microprobe techniques for eventual contamination due to inclusions of foreign minerals, exsolution lamellae of composition different from the host, reaction rims, etc. Third, the composition of the minerals as given by the chemical analysis has to be checked by analyzing the compound with the electron microprobe using well-established standards such as pure elements, minerals, synthetic compounds, as references. Particular emphasis should be given to elements which are known to be difficult to determine by conventional chemical methods. Fourth, the

174

M. P. CESCAS, E. H. TYNER, AND L. J. GRAY

chemical analysis of the mineral should be checked for gross inconsistencies, such as appreciable deviations of the total percentage from 100 percent, or appreciable deviations from the theoretical structural formula, if the latter is well-established.” For the analysis of many silicates, Smith (1965, 1966a,b) has developed simple empirical formulas using measured intensities corrected only for deadtime, background and drift. A calibration curve obtained for Fe by Smith and Stenstrom (1965) is shown in Fig. 8. Ziebold and Ogilvie ( I 963) working with binary alloys concluded that accurate measurements require the use of carefully prepared calibration standards if one is to convert X-ray data to mass concentrations. Ziebold and Ogilvie (1964, 1966) have observed that for a binary system the plot of CIK vs. C is approximately linear. For the alloy AB where A radiation intensity is measured and corrected for background, deadtime and drift:

where a A B is a coefficient valid only for the particular A-line measured and for a given set of experimental conditions, i.e., excitation potential, probe size and intensity, etc. Rearranging Eq. (22) gives:

I-K -K

‘AB

1 - C* 7

If atom fractions are of interest then Eq. (23) is written as:

where X , is the atom fraction of A. For multicomponent systems Eq. (23) is written:

where iilNis an averaged coefficient:

175

THE ELECTRON MICROPROBE X-RAY ANALYZER

It must be noted that for a complex sample containing N elements, several binary standards are required for each of the ZfJ= (N-i) combiI

lOOr 90 Microprobe pru,'nput for

80 -

70 60 50

-

40

-

0

5

10

15

20

25

30

35

40

45

50

55

Iron ( w t %)

FIG.8. Microprobe output for iron versus weight-percent iron for a series of olivine minerals. (After Smith and Stenstrom, 1965.)

nations of elements, and twice this number of equations of the form of Eq. (23) must be solved to obtain the (aij)coefficients. These values are then substituted into N equations of the form of Eq. (25)which are solved simultaneously to yield the Ci. Equation (25) has been shown to be very satisfactory for binary systems. More data, however, are still necessary to prove that the relation is valid for multielement samples. Its use would appear, for the time being, to be limited to binary systems. Few such cases will be encountered in soil studies. However, at least two examples of the application of the Ziebold and Ogilvie correction procedure have been made to ternary systems: Ingersoll e? at. ( 1967) and Ziebold and Ogilvie (1964). Ziebold and Ogilvie ( 1 964) have also developed an empirical correla-

176

M. P. CESCAS, E. H. TYNER, AND L. J. GRAY

tion of the conversion factor (aij)with the important factors involved in electron microprobe analysis. Bence and Albee (1 967) give an empirical value for the factor (aij) entering Eq. (23) for calibration purposes in the microanalysis of silicates and oxides. Mineralogists use and prefer Smith’s multiple standard calibration curve method. It can be expected that soil scientists will tend to follow this procedure because of its relative apparent simplicity. However, the Smith procedure has limitations, the chief of which is the availability of many well-defined series of minerals homogeneous on a micron scale and the necessity of also occasionally applying theoretical corrections (e.g.., fluorescence corrections). The Ziebold-Ogilvie semiempirical approach also requires a series of binary standards homogeneous on a micron scale, a limitation similar to the previous method. It is further limited by the difficulty of extending the mathematics to systems having more than three elements. The method of analysis which will eventually predominate will be the pure elemental standard (99.99+) or a stoichiometric binary compound when the use of the pure element is not practical, e.g., Fe304or Si02 for oxygen. Limitations of this method are the uncertainties in fundamental physical constants, e.g., atomic fluorescence yield, mass absorption coefficients particularly for long wavelengths, and the approximations involved in deriving the working equations. V. The Q u a l i t y of X-Ray Microanalysis

Andersen (1 967) defines the quality of an electron microprobe X-ray analysis in terms of the degree of spatial resolution attained, the sensitivity of detection and the precision of measurement. A. PRECISION AND ACCURACY

The definitions and factors affecting precision and accuracy in electron probe microanalysis have been tabulated by Ziebold (1 967). These are given in Table 111. Yakowitz and Heinrich (1967) and Heinrich and Yakowitz (1967) have discussed semiquantitatively the effect of various errors on absorption and fluorescence corrections, respectively. They considered the effect on the analytical results of both errors of an instrumental nature and errors in fundamental constants. However, assuming as valid the definitions given in Table 111, care must be taken to ensure that the precision of the data collection is greater than the errors in the correction procedures. Only in this case will the limitation of the accuracy of an

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177

TABLE Ill Factors Affecting Precision and Accuracy in Electron Probe Microanalysis ‘ Precision: Measurements that relate to scatter of dispersion among test results without assumption of any prior information 1. Statistics of X-ray counting (Poisson statistics) a. Signal from unknown b. Signal from reference standard c. Background measurements d. Calibration measurements 2. Other sources of scatter a. Stability of electronics b. Sample positioning (reproducibility of “focus”) c. Sample preparation (surface roughness or irregularities) Accuracy: Measurements that relate to difference between average test results and true result when the latter is known or assumed 1. Error in relative intensity a. Sample preparation b. Background measurements c. Counting system errors (dead time error, peak shift) 2. Error in calibration a. Empirical method: accuracy of equations; uncertainty in true composition b. Computational methods: accuracy of equations; uncertainty in physical properties (absorption coefficients; X-ray yields; etc.)

After Ziebold ( I 967).

analysis be the precision of the physical constants, the precision of the analysis to which the microprobe results are compared, or the accuracy of derived equations. B. SPATIAL RESOLUTION

The spatial resolution is determined by the volume of the sample which, through excitation by the primary electron beam, contributes to the quantitative analysis. The spatial resolution depends on the accelerating potential, the excitation potential of the characteristic line being used for the analysis, and the range of the primary electrons in the specimen. The depth of penetration of the electrons, x,, in microns, can be found from the relation given by Castaing ( 1960): A

x, = 0.033 (E;.‘ - EA.7)-= PZ

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

where p is the density in grams per cubic centimeter and E, and E, are expressed in kilovolts. The total diameter, 6, of the analyzed volume is approximately equal to:

where d is the electron probe diameter. Wittry (1958) recommends using E, = 1.5 E, in order to obtain an optimum resolution compatible with the other aims of the analysis.

C. DETECTION LIMITS Two detection limits are considered.

1 . The Weight Fraction Detection Limit Andersen (1 967) defines this limit as “the concentration of the element which is required to produce an intensity larger than the intensity of the background at the position of the analytical line by an amount equal to three times the standard deviation of the background intensity.” This criterion of three times the standard deviation of the background had been statistically developed by Liebhafsky et al. (1960b) and is commonly used in X-ray emission spectrographic work. It is written as: N,-N,>3.\lN, where NTis the average total number of counts recorded at the peak and NBis the average of the counts obtained for background alone. When this condition is fulfilled, the element in the unknown from which NT was obtained is guaranteed present in a concentration corresponding to NT. When this condition is not fulfilled, the presence of that element is not proved. Colby (1 967) presents some data for thin-film analysis using a minimum detectability based on a signal 20% above background. Ziebold ( 1 967) presents a convenient method for estimating precision before actually performing an analysis. Any of these methods described may be used. 2 . The Absolute Detection Limit This limit is defined by Andersen and Hasler ( 1 966) as the number of atoms or grams of the element detectable and is equal to the product of

THE ELECTRON MICROPROBE X-RAY ANALYZER

179

the weight fraction detection limit and the analyzed mass. Their results, obtained from a tooth analysis for calcium, phosphorus, sodium, and carbon, are shown in Table IV. VI. Applications to Soil Investigations

A. PREPARATION OF SOILSPECIMENS 1. Surface Relief of Specimens and Emergence Angle Relationships

Depending upon the design of the objective lens of the instrument the X-ray emergence (take-off) angle varies from 6 to 52.5 degrees. X-ray intensity measurements from rough poorly polished surfaces can lead to false concentrations (Picklesimer and Hallerman, 1966). This effect is attributable to the variation in emitted intensity induced by relief differences. Such differences result in a variance in X-ray path length in the specimen. The K-ratio obtained from poorly polished surfaces would be different at different beam position, contrasted to K ratios obtained from flat highly polished surfaces. Flat highly polished surfaces are, therefore, necessary for precise analysis. The measured intensities are made-up of two components, the primary (characteristic) and secondary (secondary fluorescence) X-ray radiation. The primary X-rays, as illustrated in Fig. 9, are on the average generated closer to the surface than secondary X-rays. The emergence angle affects the absorption of both intensity components. However, absorption is proportionately greater at low take-off angles for secondary X-rays, most or all of which may be absorbed as is illustrated in Fig. 9 A'B'. Most soil minerals are made-up of light elements (2 13) for which absorption effects are usually much greater than fluorescence effects. In general, fluorescence radiation, when it exists, represents only a small fraction of the total radiation emitted and in many cases can be neglected. Considering the relative magnitudes of the two effects, it is evident that mass absorption effects should be minimized by using high take-off angle instruments. In the case of very small particles, the control of relief through polishing is virtually impossible. However, analysis of small unpolished particles can be performed. The methods used and associated problems will be discussed in Section VI, C .

-

2 . Polished Thick Specimens

a. Preparation of Sample Holders and Sample Embedding. Samples

1

TABLE IV Detection Limits of Ca, P, Na, and C in Tooth" Ca Accel. Potential

(kV) 3 5 10 15 20 25 30 35

(I

Weight fraction (%)

0.578 0.037 0.020 0.0 15 0.0 I3 0.0 12 0.0 10

P

Absolute limit (g. x 10-14) I

0.130 0.076 0.200 0.554 1.448 3.458 7.162

After Andersen and Hasler ( 1966).

Weight fraction (%) 0.627 0.124 0.034 0.023 0.02 1 0.0 I9 0.018 0.0 17

c

Na

Absolute limit (g. x 10-14) 0.086 0.07 I 0.086

0.260 0.850 2.250 5.512 12.406

Weight fraction (%)

Absolute limit (g. x 10-14)

0.196 0.045 0.024 0.02 I 0.022 0.024 0.027 0.032

0.032 0.066 0.250 0.922 2.91 I 8.567 23. I79

0.05 1

Weight fraction (%) 0.04 I 0.032 0.038 0.043 0.047 0.057 0.078 0.099

Absolute limit (g. x 10-13 0.0 I3 0.024

0.107 0.520 1.989 7.101 24.864 71.963

T H E ELECTRON MICROPROBE X-RAY ANALYZER

181

to be polished are mounted in specimen holders consisting of brass disks (2.5 cm. diameter X 1 cm. thickness) cut from 1-inch brass rod. Holes HIGH TAKE-OFF ANGLE=52,5O

LOW TAKE-OFF A N G L E Q I ~ '

e-

e-

POORLY POLISHED

FLAT POLISHED SURFACE

FIG.9. Effect of surface relief and X-ray take-off angle on X-ray intensities.

are drilled through the disks, the soil specimens are inserted and embedded in an epoxy resin of the same type used in electron microscopy. Examples of brass specimen holders are illustrated in Fig. 10. Cold-setting acrylic resin, thermal-setting diallyl phthalate with glassfiber fillings, bakelite, and copper-impregnated diallyl phthalate materials were initially used either for the preparation of sample holders or for embedding purposes. Cold-setting acrylic resins were found to be unsatisfactory because of their tendency to soften and vaporize under the electron probe. The use of the thermal-setting compounds was discontinued because of possible change in phase and composition of the specimens. With brass disks, probe current standardization and carbon coating control are less difficult. The use of brass disks and epoxy for embedding is recommended for soil specimens (Cescas, 1968). b. Polishing Operations. Polishing of embedded specimens to secure relief-free surfaces can be done mechanically or manually. The techniques and equipment employed are similar to those used for polishing metallurgical and mineralogical specimens. The following polishing method was used and found to be satisfactory for soft and hard soil concretions and soil minerals. Silicon carbide paper and paper disks

182

M. P. CESCAS, E. H. TYNER, AND L. J. GRAY

FIG. 10. Typical brass specimen holders with embedded soil samples, prior to final polishing.

were used for rough grinding and polishing in steps down to 600 grit. The final polishing was carried out in stages with diamond paste and oil on nylon cloth laps with 8, 3, 1, and 1/4 p grades of paste. Between each grade size, during either the rough or final polishing steps, the specimens were ultrasonically cleaned in an organic liquid neutral to the embedding material (alcohol for epoxy) and finally the samples were washed with alcohol, dried in a warm air stream, and stored in desiccators. c. Coating of Specimen Mounts. The samples must be coated with a few hundred angstroms of a conductor to prevent charge accumulation and excessive heating by the electron probe. For soils, carbon coating is preferable because it permits good optical observation of the sample and introduces no interference in the normal analysis of other elements. The carbon was sputtered onto the specimen surfaces (200 to 400 A. thick) in an evaporator of the type used in electron microscopy. 3. Soil Thin Sections

We have not attempted to prepare thin sections (20 to 30 p) of soils in their natural structured states for microprobe analysis. It is doubtful whether the impregnating media (Cady, 1,965) commonly used in the preparation of soil thin sections possess sufficient thermal stability for use in microprobe a n a l y ~ i s . ~ 3Gillespie and Elrick (1968) analyzed soil thin sections but gave no details regarding impregnating media.

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183

Geologists have prepared thin rock sections for microprobe analysis (Cadwell and Weiblen, 1965; Smith, 1965). From their experiences, the preparation of soil thin sections suitable for both petrographic examination and electron microprobe analysis appears to be feasible. For friable rocks, Smith (1 965) used an epoxy solution as the impregnating medium. Impregnation with methacrylate would warrant investigation. Magdalena ( 1960) describes the preparation of a monomeric methacrylate solution of low viscosity capable of penetrating pores of a few hundred angstroms for the preparation of ultrathin microporous metal specimens for examination under an electron microscope. An ingenious method of sampling the paint films of oil paintings to establish their authenticity has been developed by Peterson and Ogilvie as cited by Birks ( 1963). A hypodermic needle is inserted into the picture surface and the withdrawn core containing several layers of paint pigments is analyzed with an electron microprobe analyzer. This sampling method, although not a substitute for soil thin sections, might be useful for electron microprobe analysis of clay skins on ped surfaces and other applications.

B. ELECTRON MICROPROBE ANALYSIS OF SOILS

I . Weathering of Micas and Errors in Silicate Analysis Rausell-Colom et al. (1 965) studied the artificial weathering of micas. Their publication appears to be the first reported use of the electron microprobe in a soil investigation. Mica flakes (biotite, phlogopite, lepidolite, and muscovite) were sealed in ampules with lithium, sodium, magnesium, calcium, strontium, and barium chloride solutions and equilibrated for various periods of time at 20" and 120°C.The weathering of the mica flakes was followed by optical microscopy, X-ray spectrography, X-ray diffraction, flame emission spectrography, and electron microprobe X-ray analysis. Point-by-point electron microprobe X-ray analysis for calcium and potassium made along a traverse crossing the optical boundary of weathered biotite is shown in Fig. 11. The results demonstrate the replacement of potassium by calcium at the edge of the flake. In soil studies involving silicate analysis, it is necessary to be aware of certain analytical problems associated with the thermal effects arising from electron bombardment of specimens. These include cratering, surface diffusion and volatilization. White et al. (1966) measured the effects of probe size and specimen current on the potassium analysis of muscovite. The rate of potassium volatilization losses is illustrated in Fig. 12.

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

Large volatile losses of potassium occurred with decreasing electron probe size and increasing specimen current. Potassium loss from feld-

DISTANCE FROM EDGE OF FLAKE, p

FIG. 1 1. Mica-weathering demonstrated by point-by-point analyses for potassium and calcium. (After Rausell-Colum et a/., 1965.)

z 5:

2

m

0.7-

0.06118.

0.6-

3 0.560. 0.4W

2

0.3-

td 0 . 2 [L

0.1

l

l

l

l

’

r

l

’

l

’

I

I

I

I

I

1

r

l

I

I

FIG. 12. Potassium volatilization losses during the analysis of muscovite. (Modified from White el a/., 1966.)

spars by volatilization was also stated to be serious. The effects of beam “damage” on feldspar analysis is discussed by Baird and Zenger (1 966). Cratering and volatilization of sodium and potassium have been reported

THE ELECTRON MICROPROBE X-RAY ANALYZER

185

by Borom and Hanneman ( 1967) during electron microprobe analysis of alkali silicate glasses. Adler ( 1 963a) reported serious cratering with quartz and opal. It is evident when analyzing silicates that the conditions of analysis must be carefully chosen to minimize or avoid the errors which might arise from local probe heating effects. In general, the errors decrease with lower accelerating voltages, lower specimen currents, and larger probe diameters. 2 . Ferromanganiferous Concretion Analysis The zoned and nonzoned concretions illustrated in Fig. 13 were isolated from the sand fractions of the soils of the Morrow plots by

FIG. 13. Iron and manganese distribution in zoned and nonzoned concretions (accelerating voltage 15 kV.,probe diameter about 1 p).

Cescas and Tyner ( 1 967a,b) and Tyner et a f . (1 967). The iron content of the core of zoned concretions was found to be as high as 36% iron. The manganese content for the core areas varied from 3 to 8% manganese. The outer shell, on the other hand, was dominated by manganese. Contrasting the composition of zoned with nonzoned concretions, the

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

iron and manganese contents of nonzoned concretions tended to be much lower and the iron and manganese uniformly distributed throughout the concretions. The iron and manganese solubilities required for the local concentration of these elements at the site of concretion formation was attributed to reducing environments characterized by low redox potentials. The redox potential of the Fe2+-Fe3+couple is considerably lower than that for Mn2+-Mn4+.Conceivably, the rapidity with which oxidative conditions are restored to the soil system could affect the order in which nucleation and precipitation of iron and manganese occur. Zoned concretions were postulated to have formed in slowly oxidizing environments where iron was precipitated prior to manganese. Nonzoned concretions are postulated to have formed in very rapidly oxidizing environments where simultaneous iron and manganese precipitation occurred. 3 . Mineral Inclusion Analysis

The mineralogical homogeneity of the silt and sand fractions of soils can be verified or established by electron microprobe analysis. Syers et al. (1967) studied the distribution of the inorganic phosphorus in a chronosequence of New Zealand soils developed on volcanic ash. Apatite inclusions present in weathering soil particles represented the dominant mode of occurrence for the phosphorus in the parent material and many of the youthful soils. Electron microprobe analysis on samples supplied by Syers confirmed the presence of apatite in hypersthene sand grains, an example of which is illustrated in Fig. 14. Microprobe analysis also revealed the presence of magnetite, ilmenite, and a spinel in these same hypersthene particles (Cescas, 1968).

C. ANALYSIS OF UNPOLISHED MICRONTO SUBMICRON SPECIMENS The emphasis in the previous sections was on the analysis of polished specimens. Physically and chemically, however, the clays and the claysize particles represent the most active soil fractions. Dissolution techniques, X-ray diffraction, eIectron microscopy, infrared analysis and differential thermal analysis have contributed much to our knowledge of these components. With the aid of the new electron microprobe analyzers, it may be possible to determine further the homogeneity of clay minerals and the nature of the morphologically similar amorphous substances associated with the crystalline phases. Examples of microprobe small particle analysis of radioactive fallout contaminants from Russian and Chinese nuclear tests are reported by Mamuro and Fujita (1963) and Mamuro et al. (1965); of dust particles

FIG.14. Elemental distribution of silicon, calcium, and phosphorus from an apatite inclusion in a hypersthene host mineral (accelerating voltage 15 kV., probe diameter about I P).

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

in air-pollution studies by Bayard (1967); of synthetic and natural clay minerals by White (1964) and White et al. (1966); of submicroscopic precipitates in stainless steel by Banerjee and Bingle (1966); and of micrometeorites, extracted precipitates, and fine mineral particles ranging in size from 0.3 to 5 p by Schippert el al. (1 967). 1 . General Considerations The preparation and the analysis of particles in the 0.25 to 20 p range presents special difficulties. First, good polished sections of particles less than 20 p, irrespective of their hardness, are difficult to prepare. Second, some electrons will be transmitted through clay-sized (<2p) and other fine particles. Bayard (1967) has shown that X-ray intensity from small aluminum spheres decreases with sphere size, as is illustrated in Fig. 15. If one compares the aluminum X-ray intensity generated by

I

loo

0

I-

I

6

7

/I-

I

2

3

4 Diameter. p

5

8

9

FIG. 15. Size versus intensity for aluminum particles based on 600 particles. (After Bayard, 1967.)

an unknown aluminum compound particle of 3 p diameter with the intensity generated by a “thick” aluminum standard, a smaller K would result. A low aluminum concentration would then be obtained for the unknown. This difficulty can be avoided by using elemental ratios from the unknown compared to elemental ratios from compound standards (White et al., 1966). Bayard, however, pointed out that it was necessary

189

THE ELECTRON MICROPROBE X-RAY ANALYZER

to choose analytical lines having critical excitation potentials of about the same magnitude. T h e importance of this is shown in Fig. 16. Element ratios diverged very rapidly with decreasing particle size (< 5 p ) when large differences in the excitation potentials of the analytical lines of lead and sulfur were employed. It is not always possible, however, to have analytical lines with such close excitation potentials. It is then necessary as shown by Bayard to compile ratio vs. size graphs.

r)J \

L a , Vc = 10.55KV.

Pb

S K a , VC = 2.31 KV.

Pb Ma. V c = 2.35KV. S Ka, Vc= 2.31 KV.

I

2

3

4 Size, p

5

6

7

8

FIG. 16. Ratio of lead to sulfur in lead sulfide. Actual ratio is 6:46: I . (After Bayard, 1967.)

Additional problems associated with the analysis of submicron particles exist.4 First, it may be necessary to measure background as well as peak intensities; second, it may be difficult to establish that a given claysized particle represents a monomineralic species rather than an aggregate of several clay minerals; third, the time required to achieve significant counting statistics may be excessive; and fourth, long counting times tend to aggravate drift probtems arising from surface contamination and chemical changes due to thermal effects, e.g., volatilization of alkali elements. etc. Private communication from E. W. White, Materials Research Laboratory, Pennsylvania State University, University Park, Pennsylvania.

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M. P. CESCAS, E. H. TYNER, A N D L. J. GRAY

2 . Sample Preparation and Analysis Two methods of sample preparation similar to those used in electron microscopy may be employed. In the first method, a small droplet of a sample suspension ultrasonically dispersed in a neutral volatile liquid (alcohol, water, etc.) is placed on a polished metal rod or flake substrate fitting the instrument sample holders. After evaporation of the liquid, the particles are distributed at random over the metal surface. White et al. (1 966) recommended analysis of particles separated by no less than 5 to 10 particle diameters to avoid interference from neighboring particles (secondary fluorescence). The substrate may be beryllium, aluminum, boron carbide, carbon-coated bakelite, or graphite. White et al. (1 966) did not coat mineral samples with a conducting layer prior to analysis. However, as is illustrated in Fig. 17, defocusing of the beam by charged particles may occur (Bayard, 1967). Coating is therefore desirable.

Cu substrata

FIG. 17. Defocusing effect of charged particle. (After Bayard, 1967.)

In the second method, a small droplet df the dispersed suspension is deposited on an electron microscope grid previously covered with a thin film of collodion or Formvar. After drying, carbon (100 to 300 or aluminum (50 to 100 A) depending upon the objectives of the analysis

A)

THE ELECTRON MICROPROBE X-RAY ANALYZER

191

is evaporated on the grids. This method is particularly useful when electron microscopy, electron diffraction, and electron microprobe analysis information are desired for the same specimens. However, collection of such data requires the use of separate electron microscope and electron microprobe instruments. For the analysis of aluminosilicates, aluminum is not a suitable coating. Separate grids coated with aluminum or gold should be prepared for electron diffraction calibration purposes. Single instruments combining the features of the electron microscope and the electron microprobe are now commercially available. The methods of sample preparation are similar to those used where separate instrumentation is employed. Analysis performed with these instruments have been reported by Cooke and Duncumb (1967), Conty and Finley (1967), and Schippert et u1. ( 1967). VII. Summary and Conclusions

The electron microprobe analyzer is an exciting new analytical tool. Soil scientists will now be able to analyze nondestructively and in situ the chemical composition of microscopic to submicroscopic soil volumes. It will be possible to study directly many problems the understanding of which has been impeded by lack of chemical methods applicable to particles as small as 0.25 p. I n electron microprobe analysis, the sample is bombarded with a small, finely focused highly energetic electron beam or probe. The characteristic X-radiation emitted by the elements present is analyzed by one or more X-ray spectrometers equipped with fully focusing curved and ground analyzing crystals or by energy dispersion analysis in which the energy of the X-ray quanta for all X-rays emitted are detected and resolved by means of a multichannel analyzer in line with a proportional counter. Electron microprobe analysis although simple in concept is complicated in practice by the fact that the X-ray intensities measured by the detector systems are not the true intensities generated within the sample or standard. Thus, when the single standard method of analysis is employed, corrections are necessary for mass absorption, secondary fluorescence and atomic number effects arising from the interaction of incident electrons and generated X-rays with atoms in the sample. Multiple standard methods which obviate the need for corrections represent an alternative to the single standard method. These employ calibration curves generated from the X-ray intensities measured from a series of well-defined, homogeneous multielement compounds. At the

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present time, the method of Smith and co-workers based on the use of curves obtained from a series of well-defined mineral standards will in general be the most practical for soil analysis. Precise quantitative results in many cases will be the objective of the analysis. Not to be overlooked, however, are the scanning procedures (X-ray intensity and line profiles). There will be many instances where considerable information relative to the distribution and semiquantitative contents of various elements present in soils can be secured by these techniques. The microprobe analyzer is capable of detecting, for most elements., concentrations as low as 100 ppm. The detection limits depend on operating conditions. It must be emphasized, however, that an absolute detection limit of 0.0 1% ( 100 ppm.) permits the nondestructive measurement of elemental components as low as to 10-l6 gram within a p3 analyzed volume. From this viewpoint, no other instrument currently available surpasses the electron microprobe. The nature and size of the soil material to be analyzed dictates the method of sample preparation. For coarse silt-sized and larger particles, polishing prior to analysis is necessary. Details for the preparation of polished and unpolished specimens are described. Examples of electron microprobe analysis of soil concretions, mineral inclusions, and micas are given. REFERENCES Adler, 1. 1963a. Electron probe analysis of minerals. Symp. X-ray EIectron Probe Anal., ASTM Spec. Tech. Publ. No. 349,183- 192. Adler, I. 1963b. In “Advances in X-ray Analysis” (S. M. Mueller, G. Mallett, and M. Fay, eds.), Vol. 7,426-432. Plenum, New York. Adler, 1. 1966. “X-Ray Emission Spectrography in Geology.” Elsevier, Amsterdam. Adler, I., and Goldstein, J. 1965. Absorption Tables for Electron Probe Microanalysis NASA Tech. Note D-2984. Agrell, S. O., and Long, J . V. P. 1960. In “X-ray Microscopy and Microanalysis” (A. Engstrom, V. Cosslett, and M. Pattee, eds.), pp. 391-400. Elsevier, Amsterdam. Andersen, C. A. 1967. The Quality of ><-RayMicroanalysis in the Ultrasoft X-Ray Region, Brif.J.App1.Phys. IS, 1033-1043. Andersen, C. A., and Hasler, M. F. 1966. Extension of Electron Microprobe Techniques to Biochemistry by the Use of Long Wavelength X-Rays, In “X-Ray Optics and Microanalysis” (R. Castaing, P. Deschamps, and J. Philibert, eds.), pp. 3 10-327. Hermann, Paris. Archard, G. D., and Mulvey, J. 1963. In “X-ray Optics and X-ray Microanalysib“ (H. H . Pattee, V. E. Cosslett, and A. Engstrom, eds.), pp. 393-410. Academic Press, New York. Baird, A. K., and Zenger, D. H. 1966. In “Advances in X-ray Analysis” (G. R. Mallett, M. Fay, and W. M. Mueller, eds.), Vol. 9,487-503. Plenum, New York.

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Banerjee, B. R., and Bingle, W. D. 1966. In “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, and D. B. Wittry, eds.), pp. 653-664. Wiley, New York. Barman, M. L. 1967. A proposed extension to Philibert’s absorption correction. Trans. 2nd Natl. Conf. Electron Microprobe Anal. N o . 6. Bayard, M. 1967. Microprobe analysis of small particles. Trans. 2nd Natl. Conf. Electron Microprobe Anal. N o . 36. Beaman, D. R. 1967. A Computer program for quantitative electron probe microanalysis. Trans. 2nd Natl. Con$ Electron Microprobe Anal. N o . 11. Bearden, J. A. 1964. X-ray wavelengths. US.A t . Energy Comm. Rept. NYO 10586. Bearden, J. A. 1967. X-ray wavelengths. Rev. Modern Phys. 38,78-124. Bence, A. E., and Albee, A. L. 1967. An empirical method for the electron microanalysis of silicates and oxides. Trans. 2nd Natl. Conf. Electron Microprobe Anal. N o . 32. Bethe, H. (Von). 1930. Zur theorie des Durchgangs Schneller Korpuskularstrahlen durch Materie. Ann. Phys. 5,325-400. Birks, L. S. 1960. Technique for calculating X-ray intensities in the electron probe microana1yzer.J. Appl. Phys. 31,1297- 1298. Birks, L. S. 1961. Calculation of X-ray intensities from electron probe specimens. J . Appl. Phys. 32,389-390. Birks, L. S . 1963. “Electron Probe Microanalysis.” Wiley (Interscience), New York. Birks, L. S., and Batt, A. P. 1963. Use of a multi-channel analyzer for electron probe microanalysis. Anal. Chem. 35,778-782. Borom, M . P., and Hanneman, R. E. 1967. Surface damage effects in alkali silicate glasses during electron microprobe analysis. Trans. 2nd Natl. Conf. Electron Microprobe Anal. N o . 35. Brown, J. D. 1966. Comprehensive computer program for electron probe microanalysis. Anal. Chem. 38,890-894. Burhop, E. M. S. 19.55. Le rendement defluorescence.J. Phys. Radium 16,625-629. Cadwell, D. E., and Weiblen, P. W. 1965. Diamond disc preparation of polished thin sections for electron microprobe analysis. Econ. Geol. 60,1320- 1325. Cady, J. G . 1965. In “Methods of Soil Analysis 1” (C. A. Black, ed.), pp. 604-631. Am. SOC.Agron., Madison, Wisconsin. Campbell, W. J., and Brown, J. D. 1968. X-Ray Absorption and emission. Anal. Chem. 40,346R-375R. Campbell, W. J., Brown, J . D., and Thatcher, J. W. 1966. X-ray absorption and emission. Anal. Chem. Ann. Rev. 38,41613-439R. Castaing, R. 195 1. Application des sondes electroniques a une mkthode d’analyse ponctuelk chimique et cristallographique. Thesis, University of Pans. Castaing, R. 1960. Electron probe microanalysis. Advan. Electron. Electron Phys. 13, 3 17-386. Castaing, R., and Descamps, J. 1953. Sur la repartition en profondeur d e I’emission X d’une anticathode. Compt. Rend. Acad. Sci. 237,1220- 1222. Castaing, R., and Descamps, J. 1955. Sur les bases physiques de I’analyse ponctuelle par spectrographie X. J. Phys. Radiation 16,304-3 17. Castaing, R., and Guinier, A. 1950. Application des sondes electroniques a I’analyse metallographique. Proc. Congr. Electron Microscopy, Dev? 1949, pp. 60-63. Martinus Nijhoff, The Hague. Cescas, M. P. 1963. Ph.D. Thesis, University of Illinois, Urbana, Illinois. Cescas, M. P., and Tyner, E. H. 1967a. Chemical analysis of soil particles. A new approach to the study of climatic cycles, weathering and soil formation. Illinois Res. 9,8-9.

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Cescas, M. P., and Tyner, E. H. 1967b. Electron probe microanalysis of soil concretions. Soil Sci. SOC.A m . Proc., to be published. Colby, J. W. 1965. Comparison of analytical procedures for quantitative microprobe analysis. Natl. Lead Co. Ohio, Cincinnati, Ohio. Rept. 969. Colby, J. W. 1966. In “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, and D. B. Wittry, eds.), pp. 95-188. Wiley, New York. Colby, J. W. 1967. Quantitative microprobe analysis of thin insulating films. Advan. X-Ray Analysis, 11, to be published. Conty, C., and Finley, P. 1967. Design and application of a combined instrument electron microprobe microscope. Trans. 2nd Nail. Conf. Eleciron Microprobe Anal. N o . 47. Cooke, C. J., and Duncumb, P. 1967. Applications of a combined electron microscope and electron probe microanalyzer, EMMA. Trans. 2nd Nail. Con$ Electron Microprobe Anal. N o . 49. Criss, J. W., and Birks, J. S. 1966. In “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, D. B. Wittry,eds.),pp. 217-235. Wiley, New York. Dolby, R. M. 1959. Some methods for analysing unresolved proportional counter curves of X-ray line spectra. Proc. Phys. SOC.(London) 73,8 1-94. Dolby, R. M., and Cosslett, V. E. 1960. In “X-ray Microscopy and Microanalysis” (A. Engstrom, V. Cosslett, and H. Pattee, eds.), pp. 35 1-357. Elsevier, Amsterdam. Duncumb, P., and Shields, P. K. 1963. The present state of quantitative X-ray microanalysis: Part I, physical basis. Brit. J. Appl. Phys. 14,617-625. Duncumb, P., and Shields, P. K. 1966. I n “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, and D. W. Wittry, eds.), pp. 284-295. Wiley, New York. Duncumb, P., and da Casa, C. 1967. Atomic number and absorption correction accuracy obtained in practice. Trans. 2nd Nail. Conf. Electron Microprobe Anal. N o . 4. Elion, H. A. 1966. “Instrument and Chemical Analysis Aspects of Electron Microanalysis and Macroanalysis.” Macmillan (Pergamon), New York. Engstrom, A. 1963. In “X-ray Optics and X-ray Microanalysis” (H. H. Pattee, V. E. Cosslett, and A. Engstrom, eds.), pp. 23-3 I . Academic Press, New York. Fergason, L. A. 1965. In “Advances in X-ray Analysis’’ (G. R. Mallett, M. Fay, and W. M. Mueller, eds.), Vol. 9,265-272. Plenum, New York. Fink, R. W., Jopson, R. C., Mark, H., and Surft, C. D. 1966. Atomic fluorescence yields. Rev. Modern Phys. 38,5 13-540. Fischer, D. W., and Baun, W. L. 1964. The effect of chemical combination on K X-ray emission spectra from magnesium, aluminum and silicon. Tech. Rept. AFML-TR64-350. Fischer, D. W., and Baun, W. L. 1965a. I n “Advances in X-ray Analysis” ( G . R. Mallett, M. Fay, and W. M. Mueller, eds.), Vol. 9,329-343. Plenum, New York. Fischer, D. W., and Baun, W. L. 1965b. Effect of chemical combination on the soft X-ray L emission of K, CI and S using a stearate soap film crystal. Tech. Repi. AFML-TR65- 139. Fischer, D. W., and Baun, W. L. 1966a. In “Advances in X-ray Analysis” (J. B. Newkirk, and G. R. Mallett, eds.), Vol. 10,374-388. Plenum, New York. Fischer, D. W., and Baun, W. L. 1966b. The effects of electronic structure and interatomic bonding on the soft X-ray emission spectra from aluminum binary systems. Tech. Rept. AFML-TR-66- 19 1. Fisher, R. M. 1963. Electron optical design of electron probes. A m . SOC.Tesiing Mater. Spec. Tech. Publ. 349,88-127. Frdzer, J. 2. 1967. A computer fit to mass absorptions coefficient data. Trans. 2nd Nail. Conf. Electron Microprobe Anal. N o . 13.

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Frazer, J. Z., Fitzgerald, R. W., and A. M. Reid. 1966. Computer program EMX and EMXZ for electron microprobe data processing. Unpubl. Rept. Scripps Inst. Oceanography Ref. 66, June 20, 1966. Galle, P. 1967. Electron probe microanalysis of biological ultrathin sections. Trans. 2nd Natl. Conf. Electron Microprobe Anal. N o . 41. Gillespie, J . E., and Elrick, D. E. 1968. Micromorphological characteristics of an Oneida soil profile. Can. J . Soil Sci. 48,133-142. Gray, L. J. 1968. Ph.D. Thesis, Univ. of Illinois, Urbana, Illinois. Gray, L. J., and Wert, C. A. 1968. Quantitative microanalysis of refractory metal carbides. Advan. X-Ray Analysis, 12, to be published. Green, M. 1963. I n “X-ray Optics and X-ray Microanalysis” (H. H. Pattee, V. E. Cosslett, and A. Engstrom, eds.), pp. 361-377. Academic Press, New York. Green, M. 1964. The angular distribution characteristic X-ray radiation and its origin withina solid target. Proc. Phys. SOC. (London) 83,435-45 I. Green, M., and Cosslett, V. E. 1961. The efficiency of production of characteristic Xradiation in thick targets of pure elements. Proc. Phys. SOC.(London) 78, 1206- 12 14. Guillemin, C., and Capitant, M. 1960. Utilisation de la microsonde electronique de Cashing pour les etudes mineralogiques. 21st Intern. Geol. Congr. Copenhagen 21,201 -204. Hagedoorn, H. L., and Wapstra, A. H. 1960. Measurements of the fluorescent yield of the K-shell with a proportional counter. Nuclear Phys. 15,146- 15 I . Hart, R. K., and Pilney, D. G. 1967. Effect of spectral line shift on microprobe data. Trans. 2nd Natl. Conf. Electron MicroprobeAnal. N o . 31. Heinrich, K. F. J. 1966a. I n “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich and D. 9. Wittry, eds.), pp. 841-1030. Wiley, New York. Heinrich, K. F. J. 1966b. I n “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich and D. B. Wittry, eds.), pp. 296-377. Wiley, New York. Heinrich, K. F. J. 1967. The absorption correction model for microprobe analysis. Trans. 2nd Natl. Con$ Electron Microprobe Anal. N o . 7. Heinrich, K. F. J., and Yakowitz, H. 1967. Quantitative electron probe microanalysis: fluorescence correction uncertainty. Trans. 2nd Natl. Con$ Electron Microprobe Anal. N o . 10. Heinrich, K. F. J., Vieth, D., and Yakowitz, H. 1965. In “Advances in X-ray Analysis” (G. R. Mallett, M. Fay and W. M. Mueller, eds.), Vol. 9,208-220. Plenum, New York. Henke, B. L., Elgin, R. L., Lent, R. L., and Ledinghan, R. B. 1967. X-ray absorption in the 2-to-200 A region. Air Force Office Sci. Res. 67-1 254. Henoc, J. 1962. Contribution a la microanalyse par sonde electronique. Thesis, University of Paris. Publication C N E T Etude No. 655 PCM Henoc, J., F. Maurice, and Kirianenko, A. 1964. Microanalyseur a sonde electronique: etude de la correction d e fluorescence due au spectre continu. Rapport CEA-R 242 1. Hillier, J. 1947. Electron probe analysis employing X-ray spectrography. U. S. Patent 2,4 18,029. Hobby, M. G . , and Wood, G. C. 1967. Computer correction of raw electron probe microanalyses of alloys and oxides.Acta Met. 75,143- 146. .Howie, R . A., and Smith, J . V. 1966. X-ray emission microanalysis of rock forming minerals. V. Orthopyroxenes. J . Geol. 74,443-462. lngersoll, R. M., Taylor, J. E., and Derouin, D. H. 1967. The Application of the Ziebold Correction Procedure for Electron Microbeam Probe Data to Three Ternary Copper Base Alloys. Advan. X-rayAnal. 11, to be published. Keil, K. 1967. The electron microprobe X-ray analyzer and its application in mineraiugy. Fortschr. Mineral. 44,4-66.

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Keil, K., and Fredriksson, K. 1963. Electron microprobe analysis of some rare minerals in the Norton County Achondrite. Geochim. Cosmochim.Acta 27,939-947. Keil, K., and Fredriksson, K. 1964. The iron, magnesium, and calcium distribution in coexisting olivines and rhombic pyroxenes of chondrites. J . Geophys. Res. 69, 34873515. Kirianenko, A., Maurice, F., Calais, D., and Adda, Y. 1963. In “X-ray Optics and X-ray Microanalysis” (H. H. Pattee, V. E. Cosslett, and A. Engstrom, eds.), pp. 559-576. Academic Press, New York. Koffman, D. M., and Moll, S. H. 1966. In “Advances in X-ray Analysis” ( G . R. Mallett, M. Fay, and W. M. Mueller, eds.), Vol. 9,323-328. Plenum, New York. Lee, F. S., and Campbell, W. J. 1965. In “Advances in X-ray Analysis” (W. M. Mueller, G . R. Mallett, and M. Fay, eds.), Vol. 8 , 4 3 1-442. Plenum, New York. Leroux, J. 1961. In “Advances in X-ray Analysis’’ (W. M. Mueller, ed.), Vol. 5, 153-160. Plenum, New York. Liebhafsky, H. A., Pfeiffer, H. G . , Winslow, E. H., Zemany, P. D. 1960a. “X-ray Absorption and Emission in Analytical Chemistry.” Wiley, New York. Liebhafsky, H. A., Pfeiffer, H. G., and Zemany, P. D. 1960b. In “X-ray Microscopy and X-ray Microanalysis” (A. Engstrom, V. Cosslett, and H. Pattee, eds.), pp. 321-330. Elsevier, Amsterdam. Lifshin, E., and Hanneman, R. E. 1966. Electron microbeam probe analysis. General Electric Rept. N o . 66-C-250, Part 11. Lindstrom, B. 1955. Roentgen absorption spectrophotometry in quantitative cytochemistry. Acta Radiol. Suppl. 125. Magdalena, T. 1960. Etude des materiaux microporeux durs par la technique des coupes ultra-minces. Proc. European Reg. Congr. Electron Microscopy, Delft 1960, Vol. 1, pp. 568-570. D e Nederlandse Vereniging Voor Electronen Microscopie, Delft. Mamuro, T., and Fujita, A. 1963. X-ray microanalysis of highly radioactive fallout particles. Ann. Rept. Radiation Center Osaka Prefect. 4,4-8. Mamuro, T., Fujita, A., Matsunami, T., Shirai, S., and Murakami, M. 1965. X-ray microanalysis of highly radioactive fallout particles originating from the first Chinese nuclear explosion. A n n . Repr. Radiation Center Osuku Prefect. 6, 14-22. Moll, S. H., and Bruno, G . W. 1967. Gas jet sample decontamination in the electron microprobe. Trans. 2nd Natl. Conf.Electron Microprobe Anal. No. 57. Nelms, A. T. 1956. Energy loss and range of electrons and positrons. US.Natl. Bur. Std. Circ. 577. Nelms, A. T . 1958. US.Natl. Bur. Std. Suppl. Circ. 577. Nicholson, J. B., and Wittry, D. B. 1963. I n “Advances in X-ray Analysis” (W. M. Mueller, G . Mallett, and M. Fay, eds.), Vol. 7,497-51 1. Plenum, New York. Ogilvie, R. E. 1964. Electron optical design of X-ray microanalyzers. Norelco Reptr. 11, 75-77. Ong, P. S. 1966. Reducing carbon contamination in an electron microprobe measuring low energy back scattered electrons. In “X-Ray Optics and Microanalysis” (R. Castaing, P. Deschamps, and J. Philibert, eds.), pp. 18 1- 192. Hermann, Paris. Philibert, J. 1962. The Castaing “microsonde” in metallurgical and mineralogical research. J . Inst. Metals 90,241-252. Philibert, J. 1963. In “X-ray Optics and X-ray Microanalysis” (H. H. Pattee, V. E. Cosslett, and A. Engstrom, eds.), pp. 379-392. Academic Press, New York.

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Philibert, J. 1965. L’Analyse Quantitative en Microanalyse par Sonde Electronique. Inst. Rech. Siderurgie, Ser. B, No. 5 I . Philibert, J. 1967. Aspects quantitatifs de la microanalyse a sonde electronique. Inst. Rech. Siderurgie. R e v . Phys. Appliqee, 229, to be published. Picklesimer, M. L., and Hallerman, G. 1966. The influence of the preparation of metal specimens on the precision of electron probe microanalysis. Oak Ridge Natl. Lab. TM- 159 1. Poole, D. M., and Thomas, P. M. 1962. Quantitative electron-probe microanalysis. J. Inst. Metals 90,228-233. Poole, D. M., and Thomas, P. M. 1963. In “X-ray Optics and X-ray Microanalysis” (H. H. Pattee, V. E. Cosslett, and A. Engstrom, eds.), pp. 41 1-417. Academic Press, New York. Poole, D. M., and Thomas, P. M. 1966. In “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, and D. B. Wittry, eds.), pp. 269-283. Wiley, New York. Rausell-Colom, J. A., Sweatman, T . R., Wells, C. B., and Norrish, K. 1965. In “Experimental Pedology” (E. H. Hallsworth and D. V. Crawford, eds.), pp. 40-72. Butterworths, London. Reed, S. J. B. 1965. Characteristic fluoresence corrections in electron probe microanalysis. Brit. J . Appl. Phys. 16,913-926. Reed, S.J.B., and Mason, P. K. 1967. Iterative methods in microprobe correction programs. Trans. 2nd Natl. Conf: Electron Microprobe Anal. N o . 12. Ribbe, P. H.. and Smith, J. V. 1966. X-ray emission microanalysisof rock forming minerals IV Plagioclase feldspars. J. Geol. 74,2 17-233. Sawatzky, A., and Jones, S. 1967. Correction for nonlinearity in X-ray counting systems of electron-probe microanalyzers. 1.Appl. Phys. 38,4758-4760. Schippert, M. A., Moll, S. H., and Ogilvie, R. E. 1967. Analytical applications of a combined electron microscope electron microanalyzer. Trans. 2nd Natl. Conf. Electron Microprobe Anal. N o . 48. Smith, J. V. 1965. X-ray emission microanalysis of rock-forming minerals I Experimental techniques. J. Geol. 73,830-864. Smith, J. V. I966a. X-ray emission microanalysis of rock-forming minerals I1 Olivines. J. Geol. 74,l- 16. Smith, J. V. I966b. X-ray emission microanalysis of rock-forming minerals VI Clinopyroxenes near the diopside-hedenbergite join. J . Geol. 74,463-477. Smith, J . V., and Ribbe, P. H . 1966. X-ray emission microanalysis of rock-forming minerals 111 Alkalife1dspars.J. Geol. 74, 197-216. Smith, J. V., and Stenqtrom. R . C . 1965. Chemical analysis of olivines by the electron microprobe. Mineral. Mag. 34,436-459. Syers, J. K., Williams, J. D. H., Campbell, A. S., and Walker, T. W. 1967. The significance of apatite inclusions in soil phosphorus studies. Soil Sci. SOC. A m . Proc. 31,752-756. Taylor, T. A. 1967. Additional relationship for use in improving the reliability of X-ray mass absorption coefficients. Anal. Chem. 39,405-406. Theisen, R. 1965. “Quantitative Electron Microprobe Analysis.” Springer, Berlin. Thomas, P. M . 1963. Outline of a method for correcting for atomic number effects in electron probe microanalysis. Brit. J . Appl. Phys. 14,397-398. Thomas, P. M. 1964. A method for correcting atomic number effects in electron probe microanalysis. A t . Energy Res. Est., H a w e l l , Publ. N o . 4593.

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Tousimis, A. J. 1964. Electron probe X-ray microanalysis of medical and biological specimens.ASTM Spec. I’ech. Publ. No. 349,193-206. Tyner, E. H., Cescas, M. P., Gray, L. J., and Luehrs, F. U. 1967. Iron and manganese distribution in soil concretions. Trans. 2nd Natl. Con$ Electron Microprobe Anal. N o . 37. Webster, D. L., Clark, H., and Hansen, W. W. 1931. Effects of cathode-ray diffusion on intensities in X-ray spectra. Phys. Rev. 37,115-1 35. White, E. W. 1964. Microprobe technique for analysis of multiphase microcrystalline powders. Am. Mineral 49,196- 197. White, E. W., Gibbs, G. V., Johnson, Jr., 0.G., and Zechman, G. R., Jr. 1965. X-ray wavelengths and crystal interchange settings for wavelength geared curved crystal spectrometers. Mineral Ind. Expf.Sta. Spec. Publ. No.-3-64. Penn. State Univ. White, E. W., Denny, P. J., and Irving, S. M. 1966. In “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, and D. B. Wittry, eds.), pp. 791-804. Wiley, New York. Wittry. D. B. 1957. An electron probe for local analysis by meanb of X-rays. CdU. f n s t . Tech. Thesis 188, AD-13409. Wittry, D. B. 1958. Resolution of electron probe microanalyzers. J . Appl. Phys. 29, 15431548. Wittry, D. B. 1964a. Fluorescence by K lines in electron probe microanalysis. 14 pp. (Correcied kJniv. Southern Calif. Eng. Center Repf. 84-204. 1962 ( A R L reprint). Wittry, D. B. I964b. In “Advances in X-ray Analysis. (W. M. Mueller, G. Mallett, and M. Fay, eds.), Vol. 7,395-418. Plenum, New York. Yakowitz, H., and Heinrich, K. F. J. 1967. Quantitative electron probe microanalysis: absorption correction uncertainty: 1. Advan. X-Ray Anal. 11, to be published. Ziebold, T. 0. 1967. Precision and sensitivity in electron microprobe analysis. Anal. Chem. 39,858-861. Ziebold, T. 0.. and Ogilvie, R. E. 1963. Quantitative analysis with the electron microanalyzer.Ana1. Chern. 35,62 1-627. Ziebold, T . O., and Ogilvie, R. E. 1964. An empirical method for electron microanalysis. Anal. Chem. 36,322-327. Zieboid, T. O., and Ogilvie, R. E. 1966. In “The Electron Microprobe” (T. D. McKinley, K. F. J. Heinrich, and D. B. Wittry, eds.), pp. 378-389. Wiley, New York.

HYBRID WHEAT V. A. Johnson and J. W. Schmidt United States Department of Agriculture, Lincoln, Nebraska, and University of Nebraska, Lincoln, Nebraska

1. lntroduction ........................... ....................., .. ...... ...................... 11. Cytoplasmic Sterility .... ............, ................._................. . . ........ . ....... ... A. Early Japanese Research ........................................... B. The Use of Triticum timopheevi . ......................... C. Additional Sources of Male Steril D. The Nature of Cytoplasmic Male Sterility ......................................

Ill. A.

Fundamental Discoveries ........

..............

D. Sources of Restorer Genes ..................................

.................. ...................................................

1V.

v.

.........................

A. B.

Milling Quality Baking Quality ....

A.

Plant Height ..............................................................................

VI.

VII.

Seed Production ... A. Morphological B. Seed Set on M

............................. ..................................... s ....,., ................. ......

D. Male-Sterile Maintenance ................................................... E. Hybrid Seed Production .............................................................. VIII. IX.

Page i99 200 200 20 1 20 1 202 204 204 205 207 209 210 210 213 2 14 215 216 218 2 I9 219 220 22 I 222 222 224 226 228 228 229 230 232

I. lntroduction

Wheat is a major food cereal of the world. It is a dietary mainstay for millions of people. The importance of wheat as a human food provides in199

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V. A. JOHNSON A N D J. W. SCHMIDT

centive, then, for continuing worldwide effort to improve its productivity. Wheat is a self-pollinating crop species. Individual wheat flowers possess both male and female parts enclosed within the flower glumes. The anthers within a flower normally provide pollen to fertilize the ovary within the same flower. There is occasional outcrossing in wheat, but this is believed to occur with low frequency. Wheat, therefore, is highly inbred, and its improvement during the modern era has been as pure lines or varieties selected from segregating populations fojlowing handmade crosses. The discovery of an effective biological system in wheat involving cytoplasmic male sterility and pollen fertility restoration has opened a new approach to the further improvement of wheat. It provides the possibility of commercial utilization of F1 hybrids to achieve new levels of productivity. Developments leading to hybrid wheat are comparatively recent, many occurring since 1962. Published information, therefore, is 1imited. 11. Cytoplasmic Sterility

A. EARLYJAPANESE RESEARCH Numerous cases of observed male sterility resulting from intergeneric and interspecific crosses involving Triticum are on record. They were not suspect as being cytoplasmically induced since the sterility could be readily explained on the basis of chromosomal imbalance and incompatibility. The first published evidence of recognized cytoplasmically influenced male sterility in wheat was reported by Kihara (195 1). It resulted from his genome analyses of Aegilops caudata-Triticum vulgare combinations. Cytoplasmically induced male sterility from Aegilops ovata X Triticum durum and Ae. ovata x T . dicoccoides also was reported by Fukasawa in 1953. Kihara (1958) selected male sterile vulgare types from hybrid material in which the T . vulgare nucleus was substituted for the A e . caudata nucleus in Ae. caudata cytoplasm that remained sterile through successive backcrosses. Cytoplasmic male sterility on an intergeneric basis was firmly established by the Japanese research (Fukasawa, 1957, 1958, 1959; Kihara and Tsunewaki, 196 1). Undesirable side effects were found to be associated with the Aegilops cytoplasms. Delayed flowering and maturity are associated with A e . ovata cytoplasm. Ae. cauduta cytoplasm is linked with pistillody.

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20 1

B. THEUSE OF Triticum timopheevi Interspecific crosses within the genus Triticum were not studied for nuclear-cytoplasm interactions leading to male sterility until 1958. Kihara (1958) reported that the T . dicoccum nucleus substituted into T . timopheevi cytoplasm resulted in normal female and male fertility. Kihara and Tsunewaki (196 1) observed that normal fertility resulted from the substitution of the T . durum nucleus into T . timopheevi cytoplasm. It remained for Wilson and Ross (1962b) to clearly establish the existence of usable male sterility from the interaction of the common wheat nucleus with T. tirnopheevi cytoplasm. They identified pollen sterile plants from backcrosses of T . timopheevi with the BISON variety of T . aestivum as the recurrent parent. The male-sterile condition was maintained through many successive backcrosses. Among the cytoplasms that interact with the Triticum nucleus to produce a male-sterile reaction, T . timopheevi appears to offer the best possibilities for commercial use. Undesirable differences between T . timoplzeevi cytosteriles and their normal fertile counterparts have not been detected (Wilson, 1967). The few occasions in which the sterile was noted to be slightly later in heading than its B-line are explainable on the basis of delayed pollination and germination of the sterile. The degree of sterility is adequate in the tetraploids as well as the hexaploid wheats. Some genotypes in all classes of wheat into which sterile cytoplasm has been introduced have been observed to become partially fertile in some environments. Wilson (1 967) suggests that this is likely to occur in environments that are conducive for “large, well-extruded anther development” in normal varieties.

c. ADDITIONAL SOURCES OF

MALESTERILITY

Additional sources of cytoplasmic sterility have been identified. Crosses of T . zhukovskyi X T . aestivum (Maan and Lucken, 1967a; Nettevich and Fodorova, I966), T . boeoticum X T . aestivum (Maan and Lucken, 1967a), T . timonovum X T . aestivum (Nettevich and Fodorova, 1966), and A e . ventricosa X T . timopheevi (Oehler and Ingold, 1966) produce cytoplasmic sterility. The cytoplasm of T . zhukovskyi may not differ from that of T . timopheevi since zhukovskyi may be a hexaploid form of T . timopheevi (Wilson, 1967). Backcross progenies possessing alien cytoplasm with a wheat nucleus have been utilized for the detection of and transfer of cytoplasmic male sterility to wheat. Other breeding approaches have been suggested. A procedure for developing cytoplasmic male sterility utilizing wheat

202

V. A. JOHNSON A N D J. W. SCHMIDT

cytoplasm and sterility genes from an alien species has been suggested by Wilson (1967). A scheme proposed by Hermsen (1965) for development of male sterile and fertility restorer lines requires fertile cytoplasmgenetic sterile interaction.

D. THENATURE OF CYTOPLASMIC MALE STERILITY The production of normal anthers and fertile pollen in wheat requires balance and a harmonious relationship between specific chromosomal genes and so-called extranuclear genes or factors located in the cytoplasm (Rodriguez et al., 1967). Some cytoplasms are defective in that they possess mutant nonchromosomal genes that can inhibit normal anther and pollen development. If there exists in the nucleus corrective dominant chromosomal genes for male fertility restoration, the extranuclear cytoplasmic male sterility will not be expressed and normal fertile anthers and pollen will be produced. The effect of the cytoplasm from T . timopheevi seems to be specific for male sterility and little or no effects on the female structures and other traits of the wheat plant have been detected (Fig. 1). Cytoplasmic effects are transmitted only through the female, whereas chromosomal genes may be transmitted to progeny from either parent. The use of cytoplasmic male sterility in wheat for practical purposes depends upon this. Female transmission of cytoplasmic effects is employed to develop male sterile lines through a series of backcrosses of a cytosterile line to a variety lacking the fertility restoring chromosomal genes (or possessing chromosomal sterility genes). The latter variety serves as a maintainer line (B-line) for the self-sterile A-line. The cross of the self-sterile A-line with a line or variety possessing dominant chromosomal genes for male fertility restoration (R-line) results in an F, hybrid which is a normal self-fertile. The metabolic processes associated with cytoplasmic male sterility are not understood. Some observations provide limited information about the sterility phenomenon. Meiosis is normal and the first mitotic division occurs in the microspore. Pollen grains are produced in small thin anthers, but they are either empty or only partially filled with starch (Schmidt and Johnson, 1966; Johnson et al., 1967). Barnard ( 1 9 5 3 , in a histogenic study of the inflorescence and flower of T . aestivum, found that anthers are terminal microsporangia that arise from lateral branches of the flower primordium, whereas the megasporangium is terminal on the main axis of the flower primordium. Joppa et al. ( 1 966) studied microgametogenesis in the anthers of fertile and cytoplasmic male sterile winter wheat. Tapetal cells in the anthers

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of male-sterile wheat formed less starch and persisted longer than those in fertile anthers. It was suggested that vascular deficiencies in the stamen may cause sterility in male sterile wheat.

FIG. I . Left: A normal self-fertile wheat spike at anthesis. An open floret reveals a feathery stigma subtended by large anthers on greatly elongated filaments. Right: A malesterile wheat spike at anthesis. The stigma is normal but anthers are small and poorly extended from the flower. (Courtesy DeKalb Agricultural Association.)

Where sterility is less than complete in a head, the fertile florets are, without exception, found a t or near the base of the spike. The seed produced on a male-sterile wheat plant from cross-pollination tends to be

204

V. A. JOHNSON A N D J. W. SCHMIDT

wrinkled or somewhat shriveled as though it had failed to finish development normally (Johnson er al., 1967). These observations strongly point to a nutritional or metabolic block that occurs below the spike and which may begin even before meiosis (Fig. 2).

FIG. 2. Grain from a normal self-fertile wheat plant (& andI a) male-sterile plant (right). The kernels from the male-sterile plant are large but visibly wrinkled.

111. Fertility Restoration

A. FUNDAMENTAL DISCOVERIES The early work of Kihara and Fukasawa clearly established the existence of cytoplasmic male sterility in wheat and pointed to the possibility of a usable system for practical breeding. However, the information remained of academic interest only for nearly ten years. Little effort was made to exploit the phenomenon for practical wheat improvement until the efforts of Wilson and Ross in the United States. Cytoplasmic male sterility in itself offered little as a wheat improvement tool. It would permit large-scale production of F1 seed, but the resulting hybrid would be self sterile and could not be used for production of a seed crop. The second component of the system, fertility restoration, was needed to reestablish male fertility in the F1generation. Fukasawa (1958, 1959) demonstrated male fertility restoration when the nucleus from a 28-chrgmosome wheat, T . dicoccoides var. Kotschyanum, was substituted for the nucleus from A e . ovara in ovafa cytoplasm.

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A cytoplasmic male sterile hexaploid wheat derived from Fukasawa’s research was crossed to 124 hexaploid wheat varieties by Wilson and Ross (1961). Although slight fertility was observed in some combinations, adequate restoration was not found. Kihara ( 1958) reported that he could extract both male-fertile and female-fertile lines from T. timopheevi crossed with T. dicoccum (emmer). The first published report of T. timopheevi-derived fertility restoration in T. aestivum appeared in 1962 (Schmidt et al., 1962). The finding of cytoplasmic male sterility in selections from T. timopheevi X T. aestivum by Wilson and Ross pointed to the possibility of fertility restoration in T. timopheevi-derived hexaploid wheats. Genes for fertility restoration were found in the timopheevi-derived Nebraska experimental line 1279A9-111-4 X Nebred. Soon thereafter Wilson reported the successful transfer of male fertility restoration genes from T. timopheevi to hexaploid T. aesrivum (Roberts and Holland, 1962). The establishment of effective fertility restoration for T. timopheeviderived male sterility triggered a widespread research effort on hybrid wheat in the United States and other wheat-producing countries. Geneticists in more than 15 states in the United States are actively engaged in the research. Comprehensive hybrid research programs are under way in Mexico and Canada. Several United States-based commercial seed companies are engaged in hybrid wheat development programs.

B. INHERITANCE OF RESTORATION There are few precise genetic data to define clearly the inheritance of male fertility restoration. That more than a single dominant gene is involved (such as is available in corn and sorghum) seems certain. Identification of true restoration genes is complicated by environmental effects and the probable existence of sterility and fertility chromosomal genes that may function independently from the restoration genes in wheat but modify their expression (Wilson, 1967). The complexity of restoration inheritance is indicated by analysis of segregation in the Fz of two crosses made in Nebraska involving different restorer parent plants but a common male sterile parent (Table I). The Fz from cross 627 produced 44% fully fertile, 53% partially fertile, and only 3% sterile plants. In contrast, cross 628 produced 20% fully fertile, 69% partially fertile, and 1 1 % sterile plants in the Fz. Clearly, the number of restorer genes functioning in the two populations is different. The Nebraska data show similarity to data reported by Fukasawa (1958) for restoration by T. dicoccoides var. Kot. in male sterile dicoccum (Ae. ovata cytoplasm).

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V. A. JOHNSON AND J. W. SCHMIDT

TABLE 1 Seed Setting Fertility Observed in the F2 from Two Crosses of Male Sterile x Fertility Restorer Common Wheat Types in the Greenhouse at Lincoln, Nebraska"

Population

F2 from Cross 627 F2 from Cross 628 a

Total number of plants 277 207

Seed setting fertility classesh Total

0%

1-5%

2.9 11.1

9.0 14.5

25% 50%

75%

90%

100%

5.4 10.1

18.1 14.0

9.4 12.1

44.0 19.8

11.2 18.4

100 100

From Johnson et al. (1967). Values express percentage of plants in each class.

Lacadena et al. (1966) obtained evidence of heterogeneity for restoration in the Nebraska material and suggested that selection would be necessary in the restorer to obtain hybrids with homogeneous fertility. McCuistion (1968) studied the inheritance of two lots of the Nebraska restorer tentatively identified as possessing 1 gene and 2 genes for fertility restoration. Some selections from each lot fit 2-gene genetic ratios and others failed to fit either a 1-gene or 2-gene ratio. It was concluded that fertility restoration in the Nebraska material is more complex than a simple 1 -gene or 2-gene control. Livers ( 1 964a), working with a T . timopheevi X Marquis3 restorer line, obtained data indicating the operation of two major dominant genes with possible minor genes. Monosomic analysis of the restorer line by Robertson and Curtis (1 967) indicated that one of the genes is located on chromosome 1A and that chromosomes IB, 2A, 3D, 6A, and 6B of CHINESE SPRING may carry modifying genes for fertility restoration. Maan and Lucken (1967b) determined that the dosage of certain chromosomes and chromosome arms influences expression of partial fertility restoration in crosses of ms BISON with CHINESE SPRING aneuploids. Most observations to date indicate two or more genes operating in fertility restoration. Gene action for restoration has been variously suggested as cumulative, dominant, incompletely dominant, and complementary. Nebraska data involving 1279A-9-111-4 X Nebred indicate the restorer genes to function as incomplete dominants (Johnson et al., 1967). The failure of many fully fertile restorer lines to transmit full fertility to the F1 hybrid in contrast to other restorer lines that transmit fertility more fully to the F1 strongly suggests a gene dosage effect. Johnson et a!. (1967) suggest

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207

that test crossing of restorer progeny after each cross may be necessary. Rodriguez et al. (1967) support this. They write: “Test crosses must be made to a cytoplasmic male sterile line, using those plants which are thought to have complete pollen fertility restoration before proceeding to the next backcross. Only those plants that possess the capacity to completely restore fertility to the cytoplasmic male sterile parent in a test cross should be backcrossed again.” Wilson (1 967) suggests the action of T . timopheevi restorer genes to be additive on an individual gene basis but taken collectively on a character basis as dominant to nearly dominant. He suggests “cumulative dominance” as adequate to describe the general nature of restoration. A T . timopheevi-derived restorer when crossed with a series of sterile lines does not uniformly restore all of them. Wilson (1967) interprets this as due to variability among the sterile lines in their sterility gene number and/or effectiveness or to the presence of fertility genes that act in a complementary or additive fashion with the restorer genes. Excess sterility genes could act as inhibitors of pollen restoration. Deletion of sterility genes, duplication of fertility genes, or isolation of favorable mutations through radiation are suggested as possible methods to increase restoration expression.

C. ENVIRONMENTAL INFLUENCE Fertility restoration genes are sensitive to environment (Schmidt and Johnson, 1966; Johnson et al., 1967; Wilson, 1967). Degree of expression of fertility restoration has been found to be markedly different in artificially illuminated growth rooms, greenhouse, and field at Lincoln, Nebraska (Johnson ed al., 1967). Restoration has been consistently better in the field and growth rooms than in the greenhouse. Rajki and Rajki (1966) also obtained data indicating the greenhouse environment to be more severe for restoration than the field environment. McCuistion (1 968) found a plastic-covered greenhouse to be the most critical for measuring degree of fertility restoration. A glass-covered greenhouse and the field at Stillwater, Oklahoma, in that order were less critical. The greenhouse has been suggested as the most critical, and hence the most favorable, environment for the conversion and testing of restorer lines (Johnson et al., 1967; McCuistion, 1968). Wilson ( 1967) has classified environments as shallow-sterile, sterile, and deep-sterile, basing this on field observations of restoration from Mexico to the northern United States. He characterizes the northern latitudes of the United States as being “deep-sterile,” the climates of the more southern areas of the United States as “sterile,” and that of

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V. A. JOHNSON A N D J. W. SCHMIDT

Ciudad Obregon in Mexico as “shallow-sterile.” Restorers that are fully adequate in the shallow-sterile environment may be inadequate in the deep-sterile environment. According to Wilson, the intensity of sterility of an environment seems to be associated with short growing seasons, very cool temperatures, or limited plant development. He suggests cold temperatures as having the strongest effect on sterility. Wilson has schematically illustrated the fertility behavior of hypothetical restorer genotypes in variable environments (Fig. 3). According to this scheme,

Surplus fertility

Normal fertility

2

.-.--

Seed fertility

+ L

a3

Lc

0

$

.-L

Tip sterile

n

A

I

Basal fertility

Male sterile Shollow sterile

Sterile

Deeply sterile

Environment

FIG. 3. Schematic representation of the fertility behavior of hypothetical restorer genotypes in variable environments. (From Wilson, 1967.)

a restored hybrid having normal fertility in a shallow-sterile environment might become tip-sterile when grown in a deep-sterile northern environment.

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209

Intercrossing the fertile counterparts (B-lines) of easy-to-restore sterile lines has been suggested as a possible way to develop even more easily restored sterile lines (Wilson, 1967). The introduction into the sterile of a single gene for restoration also could give rise to female lines particularly suited for use in hybrid combinations in the deep-sterile environments of the more northern latitudes. In these environments, they would require pollen from their normal counterparts for maintenance and increase but would give rise to a more completely fertile hybrid when crossed with a restorer line.

D. SOURCES OF RESTORERGENES The presence of major genes for fertility restoration of T . timopheeviderived cytoplasmic male sterility in T . timopheevi itself was demonstrated by their successful transfer to common wheat by Wilson and R o s s . Although it has been assumed that the restoration discovered in 1279A9-111-4 X Nebred was derived from T . timopheevi, this has not been proved (Schmidt et al., 1962). Subsequent discovery that genes for fertility restoration of T . timopheevi-derived male sterility are present in numerous hexaploid and tetraploid varieties suggests the possibility that at least one of the genes in the Nebraska restorer may have come from a common wheat instead of T . timopheevi. Livers obtained evidence that a gene in CHEYENNE winter wheat is the same as one gene from T . timopheevi. Similar evidence was subsequently obtained in Nebraska. Fertility-restoring genes for T . timopheevi cytoplasm have been isolated in various common wheats, tetraploid wheats, spelt, and T . timopheevi derivatives. Preliminary test cross data from Oklahoma State University indicate that T . vavilovi and T . macha possess genes for partial fertility restoration of T . timopheevi-derived male sterility (E. L. Smith, personal communication). Triticum dicoccoides var. Kotschyanum appears to nearly completely restore male sterile durum in T . timopheevi cytoplasm, according to Wilson (1967). Kotschyanum also is reported to restore A e . ovum cytoplasmic sterility (Fukasawa, 1958), and may be a weak restorer for T . timopheevi cytoplasm with the hexaploid wheat nucleus. Porter et al. ( 1967b) report finding restoration in a composite of common wheats from the world collection maintained by the U. S. Department of Agriculture. Varieties of T . aestivum and T . durum reported to possess genes for male fertility restoration of T . timopheevi-derived cytoplasmic male sterility are listed. Several of the varieties are heterogeneous for restorer genes; this makes it possible to select from them genotypes that can be converted to male sterile status.

210

V. A. JOHNSON AND J . W. SCHMIDT Variety T . aestivum 1279A9-111-4 x Nebred T . rimopheevi X Marquis' Cheyenne Lancer 1tana Aztec Relief Selkirk Guide Selkirk X Cheyenne' Blackhawk Crim Minn. 11-59-9 Lathrop Chinese Spring Penjamo 62 Y-Kt (Af-My)* Kirik Pissi Khebra

Primepi Palmaress Professeur Marchal Florence-Aurore 8 193 T . durum Cappelli ND61-48

Number

Country

N B542437 (Kansas) C. 1.8885 C. 1. 13547 C. I. 12933 C. 1. 13016 C. 1.10082 C. I. 13100 C. I. 13856 C. I. 13884 C. I. 12218 C. I. 13465 C. 1.13826 C. I. 13457 C. I. 6223 C. I . 13924 P. I. 172534 P. 1. 167841 P. 1. 176277 P. 1. 277013 P. I . 277016 P. 1. 316002 P. 1. 316001 P. I. 264645 P. 1. 318511

U.S.A. U.S.A. USA. USA. U.S.A. U.S.A. USA. U.S.A. USA. U.S.A. U.S.A. U.S.A. U.S.A. U.S.A. England Mexico Colombia Turkey Turkey India Spain Spain France France Belgium France

P. I . 271896 C. 1. 13942

Italy U.S.A.

It is likely that many additional common wheat varieties will be found to possess genes for male fertility restoration. It has been suggested that the European land races Crimean, Mediterranean, and Noe probably possessed restoration genes for T. tirnopheevi cytoplasm because many varieties descended from them are reported to have varying degrees of fertility restoration (B. E. Mounter, personal communication). IV. Heterosis

A. YIELD It is assumed that the productivity of wheat hybrids wili be significantly superior to that of varieties. The pursuit of hybrid wheat research would

HYBRID WHEAT

21 1

be pointless unless this were so. Evidence can be found in the literature to support the belief that wheat hybrids will be more productive than varieties. A comprehensive survey of heterosis in wheat was made by Briggle ( 1 963). Among the 23 publications surveyed by Briggle, the magnitude of heterosis reported in wheat ranged from 0 to more than 100 percent. The studies for the most part were limited in scope and application. Many were based on a few plants grown in greenhouses. Field experiments were based on single F, rows with few or no replications. Heterotic effects on height, maturity, and yield components were more often measured than heterosis in grain yields. The early studies surveyed by Briggle may have only limited usefulness for predicting the performance of commercial hybrids because measurements of heterosis were frequently based on noncommercial unproductive varieties. Heterosis so measured is inadequate for commercial hybrid projections. Economic levels of heterosis in wheat must be based upon comparisons of hybrid performance with that of the most productive commercial variety available (Rodriguez et al., 1967). There have been recent published reports of heterosis studies in wheat. Most of the studies were based on limited space-planted seedings from handmade crosses. Hybrids between varieties in the Mexican wheat breeding program were evaluated at Ciudad Obregon, Sonora, in 1964. The experiments were space-seeded in replicated rows at the rate of 18 kg./ha. Twentythree of 25 hybrids significantly outyielded the highest-yielding check variety Huamantla Rojo; some by more than 30%. Hybrids from crosses of Mexican varieties with United States, Canadian, and Argentine varieties were evaluated at Ciudad Obregon in 1965. Thirteen of 20 outyielded Huamantla Rojo, with the highest yielders over 20% more productive (Rodriguez et al., 1967). Wilson (1967) reported hybrid yield increases of more than 30% from handmade crosses of North American and Mexican varieties. A hard red winter wheat hybrid exceeded its commercial parent by nearly 19% over a four-year period. Fourteen Canadian spring hybrids ranged in yield from 28% less than to 26% more than their most productive parent (Shebeski, 1966). Similar results with winter wheat hybrid combinations have been reported by Briggle et"al. ( I 964), Fonseca Martinez (1 965), Patterson and Bitzer ( 1 966), Livers and Heyne (1 966), Rajki and Rajki (1 966, 1967), Johnson et al. ( 1 967), and Merkle et al. ( 1 967). Durum spring wheat hybrids seeded at the rate of 28 kg./ha. were evaluated in North Dakota (Widner and Lebsock, 1967). Forty of 44

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V. A. JOHNSON A N D J. W. SCHMIDT

hybrids outyielded their more productive parent. The range in hybrid performance was from 81 to 184% of the best parent. The most productive hybrid yielded 15% more than the standard variety WELLS seeded at 28 kg./ha. but only 9% more than WELLS seeded at 56 kg./ha. Larrea (1 966) demonstrated heterosis for yield in 26 of 28 F1hybrids from spring bread wheat crosses in North Dakota. McNeal et al. (1965) were unable to demonstrate heterosis in three spring wheat crosses involving related parental varieties. In studies of hybrid performance in winter wheat by Fonseca Martinez and Patterson (1965), all F1 hybrids were higher yielding than their most productive parent. It has been relatively easy to demonstrate heterosis for yield in F1 hybrids from handmade crosses where low rate spaced seedings were used for yield determinations. In these low rate seedings, F, superiority in yield has been demonstrated even between closely related parental varieties. Such experiments have predictive value for commercial hybrid wheat production only if the yield superiority of spaceplanted hybrids persists under the higher normal commercial seeding rates, or if it can be demonstrated that high yield levels can be achieved from low as well as high seeding rates of the hybrids. To date, there is relatively little information to support this. An early study by Rosenquist (1 93 1) indicated that heterosis was expressed in some hybrids when seeded at the low rate of 4 inches between plants. Similarly, Pal and Nek Alam (1938) concluded that spacing between plants within rows influences the expression of heterosis in wheat. However, Bitzer ( 1 965) found a high correlation between close planting (15 seeds per foot) and hill planting (9 seeds per hill; hills spaced 1 foot apart) of soft winter wheat varieties in Indiana indicating that varieties gave similar response at each seeding rate with respect to other varieties. Pfeifer (1 966) measured significant heterosis for yield in several handmade soft winter hybrids seeded at the rate of 2 bushels per acre in Pennsylvania. A recent comprehensive 3-year study of hybrid performance under irrigation at five different rates of seeding was made by Briggle et al. (1967a,b). Both spring and winter hybrids were higher yielding on the average than their more productive parent at all seeding rates which ranged from less than 1 peck to more than 9 pecks per acre. In two of three years, the winter hybrid produced the highest yield at the 3% pecks-per-acre seeding rate, whereas the spring hybrid was the most productive at the 6 W pecks-per-acre rate. Wilson (1967) reported that restored hybrids having T . timopheevi cytoplasm and pollen restoring genes tested at normal seeding rates have

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not yielded much more than the best varieties. He suggests that qualitative and quantitatively controlled traits in the R-line need to be improved in order to obtain maximum hybrid yields. Similarly, the performance of restored hard winter wheat hybrids seeded at normal rates in Nebraska have been intermediate to the parent varieties in productivity. On the other hand, Rodriguez et al. (1967) suggest that higher hybrid yields relative to the parents might have been obtained with Mexican hybrids had heavier seeding rates been used. It would seem that the firm establishment of the yield superiority of wheat hybrids in commercial plantings must await more extensive testing of restored hybrids at normal rates of seeding. The relationship of hybrid performance at different seeding rates may be different under irrigated high-fertility situations than it is on dryland. Projected high cost of hybrid wheat seed has raised the question of possible use by growers of FB seed. Pfeifer (1966) and others have suggested the use of 3-way hybrids as a means of reducing seed production costs. The obvious advantage of FBseed is that it could be produced and marketed at much lower cost than F1 seed. The disadvantage would be decreased productivity and genetic segregation for male sterility, agronomic, and quality traits. Tests in Mexico show a sharp drop in productivity ( I8-22%) of the Fz below the F1 level, and further but smaller decrease in the Fa generation (Rodriguez et al., 1967). The yield depression was attributable to a reduction in tillering capacity and reduction of fertility in the third and fourth florets in many spikelets. It was concluded that the commercial use of advanced generations in wheat does not show much promise. Results of other studies of F n performance in relation to F1 and parent varieties in nearly all cases have shown sharp reductions from the FI yields with little remaining yield advantage over the parent varieties (Johnson et af., 1966; Larrea, 1966; McIlrath, 1967).

B. COMBINING ABILITY Limited studies of combining ability and gene action associated with heterosis in wheat have been conducted. Purdue research (Bitzer, 1965; Patterson and Bitzer, 1966) involving a seven-parent intercross showed partial dominance for the yield components, spike number, and kernels per spike. Slight overdominance was measured for the third component kernel weight as well as for yield itself. There was little indication of epistasis. Significant general combining ability for each yield component and for yield was demonstrated. Specific combining ability was shown for kernel weight only. The research strongly pointed to the importance of additive gene action.

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V. A. JOHNSON A N D J. W. SCHMIDT

Shebeski ( 1 966) found that the high quality spring wheat variety was an excellent combiner for yield and that it was a parent in each of the most productive hybrid combinations. Another variety, CANTHATCH, proved to be a poor combiner for yield. Results for F1 tester stock crosses indicated that a suitable F1 could be useful for measuring general combining ability. Individual components of yield were found by Shebeski to be significantly correlated with yield, but the inconsistency of their transmittal to the F1 hybrid from different parents suggested that they would not be useful for prediction of hybrid performance. Kronstad and Foote (1965) studied the Fl’s from eight varieties and two experimental selections of winter wheat crossed in all combinations. Diallel analyses indicated that a large part of the total genetic variation for grain yield and components of yield was associated with a significant general combining ability effect. Significant Fl variances for general combining ability were obtained by Larrea (1 966) for all three yield components and for grain yield. Specific combining ability variances were significant or highly significant for kernel weight, number of kernels per spike, and yield. Significant R correlations between yield and number of kernels per spike were found. General combining ability variances well in excess of specific combining ability variances for yield and each yield component pointed to the predominance of additive gene action in crosses of broadly divergent wheats studied by McIlrath (1967). Kernel weight and number of kernels per spike were of greatest importance in crosses in which heterosis for yield was observed. The prevalence of additive genetic variability for yield and yield components pointed to effective selection of productive homozygous lines. On the other hand, the occurrence of significant specific effects for yield and other traits suggested the likelihood of obtaining superior hybrids from crosses of diverse genotypes. Wells and Lay (personal communication) found 22 different spring wheat varieties to be dissimilar in general combining ability for yield and kernel weight. They also detected differences in specific combining ability for yield. It seems likely that the most productive wheat hybrids will come from high-yielding parents with the greatest genetic diversity (Patterson and Bitzer, 1966; Wilson, 1967). McNeal et al. (1965) also emphasized the importance of a wide genetic base for productive hybrids. PEMBINA

V. Quality of Hybrids

The extensive use of wheat grain as human food makes quality an important consideration in hybrid wheat development. It adds an ele-

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ment that was of little importance in hybrid corn and sorghum development. The term “wheat quality” requires specific definition to be meaningful. Until recently it related primarily to the processing characteristics of the wheat grain. A good milling quality wheat was one that possessed overall desirable or acceptable milling characteristics. Good baking quality related to the acceptability of a wheat in the bakeshop in terms of its processing characteristics as well as the characteristics of the resulting bakery product. Wheat in many countries provides the basic raw material for an array of food products, each with its peculiar requirements for the wheat grain. Thus, there have emerged broad classifications of wheat that reflect the end use to which the wheat will be put. In North America, South America, and Europe the major use of wheat is for the production of leavened bread. Lesser amounts are utilized for cakes, pastries, crackers, and pasta products. In many Asiatic countries the basic wheat food is an unleavened baked product. Each of these products imposes its own particular requirements upon the wheat grain. A more recent concept of wheat quality is concerned with the nutritional value of wheat as a human food. The amount of protein in the wheat grain as well as the amino acid composition of the protein are the important factors in this concept of quality (Johnson et al., 1968b). It seems reasonable that each concept of quality will be important in hybrid wheat development. The most productive wheat hybrids may come from parental combinations involving varieties that are diverse in origin and widely different in important agronomic and quality characteristics. Many such hybrids could possess general and specific quality characteristics that would be unacceptable to the milling and baking industries. It would be unfortunate if such hybrids became available for commercial production. Quality, then, must assume equal status with agronomic, disease, insect, and seed production considerations. Although research data on quality of wheat hybrids are meager, some relationships are emerging that provide significant guide1ines. A. MILLINGQUALITY The maintenance of acceptable hybrid milling quality should not present a serious problem in intraclass hybrids from parents of acceptable quality. If hard-textured wheat varieties are used as parents, the hybrid can be expected to mill as a hard wheat. Similarly, hybrids from soft-textured parents should exhibit soft wheat milling properties.

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V. A. JOHNSON AND J. W. SCHMIDT

Hybrids from parental lines having contrasting kernel textures could present milling problems due to endosperm segregation in the seed crop from the hybrid. The grain will be intermediate in average texture between that of the two parents but the individual kernels will exhibit a wide range in texture. Thus, the hybrid seed crop would actually be a mixture of kernel textures. As such it may be difficult to temper properly for optimum milling (Johnson et al., 1967; Rodriguez et al., 1967; Wilson, 1967). Improper tempering could reflect in low flour yields and generally poor milling performance. Rodriguez et al. (1967) report that several of the most promising Mexican experimental wheat hybrids exhibited milling deficiencies. These were attributed to improper tempering resulting from an endosperm population of widely differing textures. Johnson et al. (1967) reported milling yields of hybrids from hard wheat X soft wheat crosses to be intermediate to the parents. Hybrid milling yields, ranging from intermediate to the parent varieties to superior to that of the best parent, have been reported by Shebeski (1966) and Larrea (1966). Wilson (1967) has suggested that hybrids from hard wheats crossed with soft wheats might be successfully utilized in some production areas. This is based on his observation that some hybrids from soft wheat X mellow-gluten hard wheat combinations appear to have acceptable cookie-baking quality. Market classification of the hybrid from crosses of soft with hardtextured parents is also a potential problem. The hybrid could not be properly classified as either hard wheat or soft wheat. It may be significant from a milling standpoint that productive hybrids appear to be possible from intraclass parents of acceptable milling quality (Johnson et al., 1967). It may not be necessary to resort to hard wheat X soft wheat hybrid combinations. If such combinations are utilized, extreme care and judgment will need to be exercised by hybrid seed marketing agencies to avoid serious commercial milling problems.

B. BAKINGQUALITY Overall baking quality evaluation encompasses many specific characteristics. Protein level and the nature of the protein are both involved. The latter is reflected by such properties as gluten strength, doughhandling properties, and baking properties. Sophisticated tests to measure different specific baking characteristics are employed by quality control laboratories of the milling and baking industries and by federal and state wheat quality laboratories. Hybrids tend to be intermediate to the parents in most baking charac-

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teristics. These include dough handling properties and baking characteristics (Johnson et al., 1967; Wilson, 1967; Rodriguez et al., 1967; Larrea, 1966).

1. Protein Content Grain yield and the protein content of the grain tend to be inversely related in wheat varieties. As grain yields increase, the protein content of the grain can be expected to decrease. A serious anticipated quality implication of hybrid wheat, then, is a possible reduction in the protein content of productive hybrids. Contrary to this commonly observed relationship, Shebeski ( 1966) found a highly significant positive correlation (v = 0.684) between yield and protein in handmade hybrids from five different spring wheat varieties crossed in all possible combinations. F1 hybrid protein levels generally intermediate between the parent varieties have been reported from handmade crosses by Rodriguez et al. (1967), Larrea (1966), and McNeal et af. ( 1 965). Johnson et al. ( I 967) reported a hybrid protein level lower than that of either parent variety in one winter wheat cross. In another winter cross involving ATLAS 66, which is known to possess genes for high protein, the F1 hybrid produced grain that was intermediate to the parents in protein content. The use of varieties possessing genes for high protein was suggested as a possible means of avoiding serious protein depression in high-yielding wheat hybrids. High protein from ATLAS 66 is transmitted as an incompletely dominant character. Amino acid analyses of Nebraska high protein ATLAS 66-derived lines indicate that the high protein trait need not be associated with a less favorable balance of essential amino acids (Johnson et al., 1968b). Protein depression in T . timopheevi-derived commercial wheat hybrids may not be as much a problem as initially anticipated. Wilson (1967) reports that T . timopheevi genetic material may be higher in protein than normal wheat lines. Protein determinations of T . timopheevi-derived winter restorer lines and their hybrids at stations in Nebraska in 1966 and 1967 have led to a similar conclusion (unpublished data). This could be of much value in bread wheat hybrids but could result in a difficult problem in soft pastry wheats, in which low protein is desirable. 2. Gluten Strength Rodriguez et al. (1967) found hybrids from crosses involving weak gluten and strong gluten parents to be intermediate to the parents in gluten strength as measured by the sedimentation test, mixograph, and

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V. A. JOHNSON A N D J. W. SCHMIDT

alveograph. Pelshenke values for the hybrids were nearer to that of the weak gluten parent rather than the strong parent. Intermediate gluten strength of hybrids also has been reported by Wilson and Villegas (1966), Johnson et al. (1967), Wilson ( 1 967), McNeal et al. (1 96% and Larrea (1966). Shebeski (1966) reported hybrids involving the spring wheat variety PEMBINA to exhibit a degree of dominance for long mixing time from the PEMBINA parent. Hybrids from most other spring wheat crosses were for the most part intermediate in gluten strength. Wilson (1967) has reported that hybrids from crosses of weak gluten with strong gluten wheats exhibit better balance in overall physical dough properties than either parent. Thus, selections with unbalanced dough properties that would be unsatisfactory as varieties might be useful in hybrid combinations.

3. Loaf Characteristics Substantial variations in loaf characteristics of hybrids have been reported. Rodriguez et al. ( 1 966) found some experimental hybrids to produce larger loaves of bread with better texture and color than either of the parents. Also some wheats of inferior baking quality, notably PITIC 62 and PENJAMO 62, produced hybrids with baking quality as good as or better than the good quality parent. Similar variations in loaf volume and other baking characteristics have been reported by Shebeski (1 966) and Larrea (1966). Relatively meager available data indicate that quality considerations should not present an insurmountable obstacle to hybrid wheat development. Further study of the specific quality characteristics of F1 hybrids will provide much needed information on the inheritance of quality traits and should identify superior parent varieties. There will be a premium on genetic dominance or partial dominance of desirable quality traits. Wilson (1967) has suggested that hybrid breeding may simplify wheat improvement since many different quality types have potential commercial use. VI. Agronomic Considerations

To date, hybrid wheat research has concentrated on the study of cytoplasmic male sterility, fertility restoration, and measurements of heterosis. Agronomic traits that ultimately may determine the acceptability of hybrid wheat for the producer have received little attention. Presumably these traits can be built into parental stocks by conventional

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breeding methods and should not constitute a serious barrier to hybrid wheat development. A. PLANT HEIGHT

In most wheat crosses tall plant height is dominant or partially dominant (McNeal et al., 1965; Johnson et al., 1966; Larrea, 1966; Rodriguez et af., 1967; Briggle et al., 1967a,b). It is not uncommon for the FI hybrid from a cross of parental lines that are equal in height to be taller than the parents (Briggle et al., 1967a,b). When the parents differ in height the F1 hybrid generally is intermediate but more nearly like its taller parent. In those commercial wheat areas where highly productive dwarf wheats have become established, tall-growing hybrids would not be acceptable to wheat producers. Tall wheat hybrids would be susceptible to lodging when yields are maximized by the use of irrigation and heavy applications of fertilizers. The widely used productive Mexican dwarf varieties and United States variety GAINES possess genes for dwarfness from NORIN 10 that are inherited as recessives. When they are crossed with tall varieties, the resulting F1 hybrid is usually as tall as the tall parent (Briggle et al., 1967b; Rodriguez et af., 1967). Only when both parent varieties are short is the hybrid also short. Thus it appears that effective use of the NORIN genes will necessitate their introduction into the hybrid through both parents. Similarly, the genes for short plant height from the Korean variety SEU SEUN are transmitted as genetic recessives (Johnson et al., 1966). Unless genes for short straw in wheat that function as dominants are identified, the hybrid wheat breeder may be faced with the timeconsuming and expensive procedure of incorporating the recessive dwarf genes into all parental lines. Such a wheat may be the Tom Thumb dwarf variety that possesses a single dominant gene for dwarfness. There are some important wheat production areas where the climate usually does not support the development of tall straw. The high plains of the United States is such an area. It is possible that hybrids from standard height parental varieties or from tall by semidwarf crosses would be acceptable in production areas such as this. B. SHATTERING There is only a little information on the expression of this important trait in hybrids. According to Rodriguez et al. (1967) the Mexican dwarf varieties are resistant to shattering but tall United States varieties

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V. A. JOHNSON A N D J . W. SCHMIDT

when grown in western Mexico shatter excessively. Some of the most promising experimental hybrids studied in 1965 were from crosses between Mexican dwarf varieties and the tall United States and Canadian varieties CRIM, JUSTIN, and SELKIRK. All the hybrids shattered heavily under Sonora conditions. It has been observed that tight-glumed, shatter-resistant male sterile varieties do not set as much seed in field crossing blocks as do looseglumed, shatter-susceptible varieties (Johnson et al., 1967; Olson, 1966). It is postulated that loose-glumed varieties open wider and remain open longer than tight-glumed varieties. If so, seed production considerations may be at odds with the agronomic necessity for shattering resistance in wheat hybrids. C. DISEASE RESISTANCE In many areas of the world, stable wheat production requires varietal resistance to diseases. The use of high-cost hybrid seed will place an even greater premium on disease resistance than now exists. Where the rusts are a serious production hazard the use of resistant varieties is

x d V a r A" ( I ?

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FIG.4. Schematic development of a multilineal hybrid wheat variety with provisions for a broad base of stem rust resistance. (From Rodriguez et al., 1967.) Commercial variety with horizontal type stem rust resistance into which cytoplasmic sterility will be incorporated. Commercial variety into which genes for fertility restoration will be incorporated. Cytoplasmic male-sterile modification of variety A. Self-fertile nonrestoring multilineal lines ( I , 2, etc.) phenotypically similar to variety A""but genotypically different for resistance to stem rust. Male-sterile F1 lines derived from Cross 1. Possesses genes for fertility restoration and provides the heterotic yield effect when crossed with variety Arne.

HYBRID WHEAT

22 1

necessary to assure profitable stable production. The incorporation of genes for resistance to the rusts and other diseases into hybrids will be equally as important as their use in varieties. Where dominant genes are involved they can be utilized effectively from one parent of a hybrid. Recessive genes must be incorporated into both parents. The Mexican and Rockefeller Foundation Wheat Improvement Programs have pioneered the development of mu1tilineal wheat varieties as an approach to more effective and lasting control of rust. Two striperust-resistant multilineal varieties MIRAMAR 63 and BONZA 65 are commercially grown in Colombia and Ecuador. The multilineal approach to rust control appears to have excellent prospects for application in hybrid wheat (Rodriguez et af., 1967). A scheme for development of a multilineal hybrid wheat proposed by Rodriguez et af. (1967) is reproduced in Fig. 4.A similar approach could be followed for the control of other diseases in hybrid wheat.

D. ADAPTATION Many important wheat production areas of the world are characterized by highly variable climates. In these climates varieties with broad adaptation are preferred by producers to those that are narrowly adapted because their performance over a period of years is more stable. Recently developed Mexican dwarf varieties appear to possess outstanding adaptation and yield well in many areas of the world. Their broad adaptation is in part associated with photoperiod insensitivity that permits their use in many different latitudes and elevations (Krull et af., 1967). A single dominant gene conditions the insensititivy to photoperiod. Most United States and Canadian spring wheat varieties are photoperiod sensitive. Although the sensitivity to photoperiod is not known for winter wheats, they do exhibit wide differences in general adaptation. The recently released SCOUT variety appears to possess wide adaptation compared to many other currently grown hard red winter wheats in the United States (Johnson et al., 1968a). The problem for the hybrid wheat breeder is to “provide without loss of heterosis, a broad germplasm base population with high internal buffering and effective genetic checks against crop predators and other environmental hazards” (Jensen, 1966). Borlaug (1 965) has suggested seed blends of different, separately maintained, backcross-derived lines crossed to a common restorer line to accomplish this. An alternative procedure was proposed by Jensen ( 1 966). It involves crossing a highly heterogeneous A-line with one or a composite of separately maintained R-lines. The A-line would be developed and maintained in heterogeneous

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V. A. JOHNSON AND J. W. SCHMIDT

equilibrium in backcrossing through the mass pollination from a multiparent B-line composite. Some important advantages would accure from broad adaptation in wheat hybrids. They would perhaps exhibit more stable performance even in a restricted area of production. Such hybrids would be advantageous to companies or agencies engaged in hybrid wheat development. They could be produced and distributed more economically than a larger number of hybrids with restricted adaptation characteristics (Rodriguez et al., 1967). VII. Seed Production

The ratio of seed planted to the amount of seed harvested in wheat is relatively narrow. Seed cost then becomes a more critical factor than it is in hybrid corn or hybrid sorghum. Ultimately, the success of hybrid wheat as a commercial crop may be determined by the success of hybrid seed production. Low seed set on the male sterile parent in seed production fields could result in prohibitively costly hybrid seed. Some of the problems of seed production are related to the floral structures of the wheat plant. A. MORPHOLOGICAL CHARACTERISTICS

OF THE SPIKE

Wheat is a highly self-pollinating crop. It does not produce an abundance of pollen. Wheat pollen is similar in size to sorghum pollen. Although the floral structure of wheat is characterized by open florets with exposed stigmas and extruded anthers during anthesis, cross-pollination is less than 1 percent under normal conditions. Wilson (1967) has suggested that the nature of flowering in wheat indicates that adaptation to crosspollination was prevalent in its ancestors and may still be operating in the present-day cultivated forms. However, there probably has been selection pressure against floral structures that would promote crosspollination during the modern wheat breeding era (Johnson et al., 1967). Successful cross-pollination in wheat depends upon the receptivity of the stigmas, availability of pollen during the receptive period, and the viability of the pollen (Imrie, 1966). Although they are strongly influenced by environment, each attribute may exhibit significant genetic variation. 1 . Anther Size and Pollen Production Anthers of cultivated varieties of wheat are known to vary in size and extrusion (Joppa et al., 1967). A study of 45 varieties by Kherde et al.

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(1 967) showed varieties to differ in anther length and width. Anthers of soft red winter varieties tended to be both longer and wider than varieties in other market classes. Anther length probably is a reliable indicator of the amount of pollen produced (Cahn, 1925). Substantial variation in pollen shedding ability was found among 62 wheat varieties and lines in Washington (Olson, 1966). The amount of pollen released and spike morphology were significantly related. Varieties with lax awnless spikes released more pollen than did bearded club varieties. Semidwarfs as a group shed less pollen than taller varieties. Extrusion of anthers and shattering were found to be associated with high pollen-shedding ability. Comparisons of isogenic lines provided additional evidence for the association of plant height and head type with pollen shedding. Anther trapping from the lack of filament extension and boat-shaped flower glumes is suggested by Wilson (1967) as attributes that may seriously limit cross-pollination in wheat. The selection of wheats with large anthers and flat lemmas and paleas should be possible and would promote better cross-pollination. Rajki and Rajki (1966) observed that pollen viability in wheat is short compared to the receptive period of the stigmas. They suggest that warm, dry weather hastens anther development and pollen shedding.

2. The Stigma Rapid swelling of the lodicule located at the base of each flower in wheat forces the flower glumes to open when the stigma becomes receptive. Swelling of the ovary is important too. The flowers remain open until fertilized or until the stigma is no longer receptive. Cross-pollination of male-sterile wheat is dependent upon this flower-opening phenomenon. The separation of the lemma and palea at anthesis exposes the stigma. Varieties differ in their response to environmental factors with regard to the degree of flower opening (Rajki, 1962). She observed that some responded more adversely to rainy weather than others. Similarly, varietal differences in response to drought were noted. The first flowers of spikelets opened wider than did the second and third order of flowers. The stigma remains inside the open flower in male-sterile wheat. It receives the pollen entering the open flower. There is considerable exposure of the stigma along the sides of the flower when the angle of opening is wide. Stigmas tend to grow beyond the lips of the open glumes if pollination is delayed (Wilson, 1967). Wilson has suggested that the selection of wheats with large stigmas might reduce seed production problems on male-sterile wheat.

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V. A. JOHNSON AND J. W. SCHMIDT

Stigma receptivity in male-sterile wheat was studied by Imrie (1966). He concluded that stigma receptivity under average conditions of temperature (13-18°C.) and humidity (60-70%) in New South Wales would not be factors influencing seed set on male-sterile wheat. At moderate temperatures, stigmas remained receptive for 5 days under high humidity, and for 7 days or longer when the humidity was low. Imrie has suggested that stigma receptivity could become a limiting factor in seed set under irrigated summer seedings where high temperatures and high humidity are encountered. He believes that hot, drying winds also will shorten the period of stigma receptivity. Rajki and Rajki (1966) reported that stigmas may remain receptive for as long as 13 days if temperatures are cool during the flowering period. Stigma receptivity generally is of longer duration and is less critical than the period of pollen shedding and pollen viability. It follows then that the female should flower somewhat ahead of the male or pollen-donor parent. Rajki and Rajki (1966) and Wilson (1967) suggested that the male-sterile variety should flower 1 to 2 days ahead of the pollen-donor variety. This will assure receptive stigmas to receive pollen from the donor variety during its period of pollination. The proper nicking of the male-sterile and pollen-donor varieties is believed to be a primary factor determining seed set on the male sterile (Porter et al., 1967a).

B. SEEDSET ON MALE-STERILE VARIETIES Highly variable seed sets on male-sterile or on emasculated spikes of male-fertile varieties have been reported. Reported seed sets have ranged from less than 10 percent to more than 80 percent of normal (Wilson and Ross, 1962a; Livers, I964b; Kihara and Tsunewaki, 1964; Lacadena, 1966; Rajki and Rajki, 1966; Bitzer and Patterson, 1967; Johnson et al., 1967; Kherde et al., 1967; Porter et al., 1967a; Wilson, 1967). The experiments ranged in size from a few emasculated heads to field size. Pollen availability ranged from extremely sparse to highly concentrated. Environmental conditions were variable and in some studies unspecified. Some of the male-sterile lines utilized may have been partially self-fertile or contaminated with self-fertile plants. Most of the available information on seed setting may have only limited usefulness for commercial projections. Olson (1966) studied the relationship of airborne pollen and seed set in a series of crossing blocks in Washington. Substantial variation in seed set was associated with environmental conditions. Both the ratio of pollinator to sterile and pollinator varieties with high pollen-shedding ability were observed to contribute to high seed set.

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HYBRID WHEAT

Some of the most recent and extensive data on seed setting were reported by Wilson ( 1 967). The data are reproduced in Table 11. In fieldsize crossing blocks ranging in size from 5 to 36 acres, seed sets on the male sterile varieties varied from 46 to 82 percent. The ratios of female to male in the tests were from 2: 1 to 1 :2. Wilson refers to extreme conditions and poor seed set encountered one year in North Dakota as well as to hot, dry conditions in southern United States that adversely affected seed set. It is assumed that the data reported in Table I1 represent the highest seed sets obtained under field conditions by Wilson. The data, TABLE 11 Seed Yields of Steriles in Maintenance-Type Crossing Blocks in Various Regions of the United States“ Female Location

Field Crossing size block (acres) ratio*

Casselton, North Dakota Walla Walla, Washington Dumas, Texas Ulysses, Kansas Wichita, Kansas Deshler, Ohio Saginaw, Michigan ‘I

10 10

27 36 5 13

I5

2: 1 1:1

1:1

1:2 1:1

1:l I:I

Drill strip width (feet) 16 14 14 14 12 12 12

Kind

HRS SWW HRW HRW ForageC SRW

sww

Seed Yield yield (% of (bu./acre) pollinator)

28 20 34 24 32 36 22

67 82 57 66 80 72 46

Data from Wilson (1967). Female to male. F, crossing block.

therefore, indicate the genetic potential of each market class studied and something of the relative suitability of the production areas sampled. The seed-setting potential of the various classes appear to be about the same when serious environmental problems are not encountered. Wilson suggests that 70 percent seed set from 1 : I ratios of sterile and pollinator should be attainable in many varieties under favorable environmental conditions. Conditions that favor high yields and adequate moisture during anthesis to provide turgidity for flower opening and anther extrusion appear to be best for good cross-pollination in wheat. The level of seed set on male sterile wheat varieties that can be achieved commercially has not been adequately established. Rodriguez et al. (1967) state: “At the present time no one is able to accurately predict

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V. A. JOHNSON A N D J. W. SCHMIDT

what percentage of seed set can be obtained on cytoplasmic male sterile lines under field conditions. Neither is anyone able to foresee the width of strips of pollinator or restorer lines that must be sown in proportion to the cytoplasmic male sterile line. Moreover, no one is currently able to predict the climatic and geographic conditions under which a high seed set can be assured.” There has been speculation about procedures for producing hybrid seed wheat in commercial quantities. Most researchers have approached the question in terms of single-cross production. Crossing blocks composed of alternating drill width or larger strips would allow the harvest and marketing of pure single-cross hybrid seed. Wilson ( 1967) has suggested alternative approaches involving the blending of sterile and restorer stocks in the seed production field to lower seed costs. One possibility is mechanical blending of an Ff hybrid with the F1. Blending of variable female lines or restorer lines to produce multilineal hybrids (Borlaug, 1965; Jensen, 1966) could achieve greater stability of production. The use of nonrestored hybrids as females in 3way crosses (Pfeifer, 1966) could result in more economical hybrid seed production. C. DISEASEPROBLEMS A male-sterile variety that flowers 1-2 days earlier than the pollendonor variety is believed to produce the highest seed set in hybrid seed production fields. This means that many flowers of the male-sterile varieties will be open for one day or more before fertilization occurs. During this period the stigmas are exposed to infection from disease-producing organisms. The most serious of these in wheat are Claviceps purpurea (Fr.) Tul. that causes ergot and Ustilago tritici (Pers.) Rostr. that produces loose smut. Each could be a serious problem in hybrid wheat production fields. Recently developed systemic chemicals (oxathiins) may provide good control of loose smut. At present, there is no effective control for ergot. The ergot bodies which develop in the place of kernels have constituted as much as 65 percent of the harvest from some male-sterile plots in crossing fields (Reitz, 1967). The effect of flowering disparity on the incidence of ergot at Lincoln, Nebraska, is shown in Fig. 5. The black-appearing kernels are ergot fruiting bodies. Samples A to C were harvested from field-grown malesterile varieties, and Sample D is from a self-fertile restorer line grown in the same field. In Sample A there was a 3-day disparity in time of flowering of the sterile and its pollinator (female 3 days earlier than male); in B the disparity was 6 days; in C there was no disparity; and in D the seed is

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227

from a self-fertile variety. Clearly, the incidence of ergot increased with degree of disparity in flowering between the sterile and pollinator lines. It would appear that ergot has the potential to be a major hybrid seed production problem in some areas. Observations would indicate that it

FIG. 5. (A-C) Ergot (Claviceps purpurea) in field-grown male-sterile wheat varieties at Lincoln, Nebraska, in 1965. (D) Sample from a self-fertile restorer line grown in the same field.

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V. A. JOHNSON A N D J. W. SCHMIDT

may not be as potentially serious in the eastern soft winter wheat or southern hard winter wheat regions of the United States as in the northern hard spring wheat area (Wilson, 1967).

D. MALE-STERILE MAINTENANCE Hybrid wheat seed production involves two separate and distinct seed operations. Male-sterile maintenance is the first of these. It probably will be accomplished by growing the male-sterile A-line in alternating strips or blocks with its male-fertile but nonrestoring counterpart, the B-line. Seed harvested from the A-line will again be male sterile and will be utilized for further A-line increase or for hybrid seed production. Seed harvested from the self-fertile B-line (maintainer line) will be used, as needed, as the pollen donor for further A-line maintenance and increase. Strict isolation of male-sterile maintenance fields must be established to avoid contamination from unwanted self-fertile varieties grown in the area, some of which could carry genes for partial fertility restoration. The latter would be particularly disastrous in a commercial seed operation since it would result in partially self-fertile plants in an otherwise malesterile population and continuing recontamination in each subsequent seed increase generation. Several currently grown commercial wheat varieties are known to possess such genes for partial male fertility restoration. They represent a potential contamination problem for male-sterile maintenance fields grown in the same area. Similarly, commercial hybrid wheat fields in the area would constitute an even greater menace to Aline maintenance since pollen from the hybrid field could carry major genes for male fertility restoration. Isolation requirements for male-sterile wheat are not known. The direction and velocity of prevailing winds would be important factors in establishing minimal safe distances between fields.

E. HYBRIDSEEDPRODUCTION This is the second and last field seed operation leading to commercially available hybrid seed. It is similar to A-line maintenance except that a male fertility restorer line (R-line) selected as the male parent of a hybrid serves as the pollen-donor-parent for the male sterile A-line. Seed harvested from the A-line is hybrid seed since it must result from pollination of the male-sterile by the male-fertile R-line. This seed will be sold to farmers and will produce the next year a self-fertile hybrid crop. Seed harvested from the R-line is the result of self-fertilization and will again be utilized for hybrid seed production. Contamination from nonrestoring varieties in the vicinity of the hybrid

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seed production field would lead to male-sterile hybrid plants in the farmer’s hybrid field the next season. These would produce seed only by pollination from surrounding male-fertile plants. Such contamination, if extensive, could adversely affect performance of the hybrid. VIII. Economic Considerations

The price of commercially produced hybrid seed will reflect research and development expenditures as well as the costs of production, processing, and marketing. Of these, the actual cost of seed production will be the single most important price factor. Production costs cannot readily be projected for lack of precise information about several production factors although some estimates have been made. There is general agreement that the key to hybrid seed cost will be the level of seed set on the male-sterile parents in hybrid seed production fields as well as in malesterile maintainer fields. It has been suggested that seed set on the malesterile parent of less than 50% would result in prohibitively high cost of the hybrid seed (Reitz, 1967). The ratio of seed parent to pollinator parent in seed fields and the location of seed production in relation to areas of commercial seed use also will be reflected in the price of hybrid seed. The producer will grow hybrid wheat in preference to wheat varieties if it is profitable for him to do so. The choice will result from weighing costs against returns. It is not an easy choice since the relationship between costs and returns will change in different years and production areas. The cost of hybrid seed and the anticipated yield superiority of the hybrid in relation to the productiveness of the farmer’s land and the market price of wheat must be considered. Reitz ( 1 965) has provided a useful approach to the estimation of cost-return relationships. The calculations of Reitz as extended by Rodriguez et al. (1967) appear in Table 111. The amount of heterosis needed to recover the additional seed cost input at various yield levels and hybrid seed cost levels is projected. The heterosis percentages shown in Table I11 to be meaningful must be based on productivity of the hybrid in relation to the best commercially available variety. The line separating values in the table indicates the yield level necessary to compensate for differing levels of increased seed costs, assuming 20% heterosis. The 20% figure is considered reasonable on the basis of available heterosis estimates for wheat. It will be seen that hybrid production could be profitable with 20% heterosis if yield levels exceed 40 bushels per acre, even if the hybrid seed costs as much as $ I5 per bushel more than ordinary variety seed. Yields of wheat differ greatly throughout the world. Average yields in

230

V. A. JOHNSON A N D J. W. SCHMIDT

TABLE I l l The Percentage of Heterosis Necessary to Pay the Additional Cost of Hybrid Seed at Different Commercial Yield Levels" Additional* seed costs per acre Commercial yield (bu./acre)

$4

$5

$6

$8

$10

$15

10 I5 20 25 30 35 40 45 50 60 70 80 90 100 I20 150

20.0' 13.3 10.0 8.0 6.7 5.7 5.0 4.4 4.0 3.3 2.9 2.5 2.2 2.0 I .7 I .3

25.0

30.0

40.0 26.7 20.0

50.0 33.3 25.0

75.0 50.0 37.5

10.0 8.3 7.1 6.3 5.5 5.0 4.2 3.6 3.1 2.8 2.5 2.1 I .7

10.0 8.6 7.5 6.7 6.0 5.0 4.3 3.8 3.3 3.0 2.5 2.0

11.4 10.0 8.9 8.0 6.7 5.7 5.0 4.4 4.0 3.3 2.7

14.3 12.5 11.1 10.0 8.3 7.4 6.3 5.6 5.0 4.2 3.3

:;::

~~~~~~~~~

~

~

~~

I

~

21.0 18.8 16.7 15.0 12.5 10.7 9.4 8.3 7.5 6.3 5.0 ~

"Calculations of Reitz (1965) as extended by Rodriguez et a / . (1967). Refers only to the additional cost of hybrid seed above the amount now spent on conventional seed. Values are percentage of heterosis required when commercial grain value is calculated at $2.00 per bushel.

excess of 60 bushels per acre are achieved in the Netherlands and Denmark, in contrast to only 12 bushels per acre in Pakistan and 13 bushels in India. At the 20% heterosis level, hybrid wheat would be profitable at $15 per bushel additional cost in the Netherlands and Denmark; but in Pakistan and India hybrid production would be profitable only if the cost of hybrid seed were no more than $4.00 per bushel more than regular seed (Rodriguez el af., 1967). Average figures such as these may not be realistic in some countries where improved wheat varieties and production practices are rapidly achieving new yield levels. An example is Mexico where the average yield of wheat has now risen to 39 bushels per acre from 29 bushels in 1962-1963. IX. Outlook

The market potential of hybrid wheat is very large. It has been estimated that wheat hybrids could find a place on 30 million acres in the

HYBRID WHEAT

23 1

United States (Reitz, 1967). Production from as much as one million acres may be needed annually to supply the hybrid wheat seed market in the United States. An equally lucrative market exists in many other countries. The potential of hybrid wheat must be weighed against available research information. Optimism about the future role of hybrid wheat must be tempered by cognizance of the many technical problems that remain unsolved. The biological system that makes hybrid wheat possible exists. But is it adequate for general use in hybrid production? Cytoplasmic male sterility in wheat appears to be a relatively stable phenomenon. Male fertility restoration, on the other hand, is highly sensitive to environment and has not yet been demonstrated to be adequate for safe use in an array of environments. Heterosis in wheat equal to that in corn and sorghum has been demonstrated experimentally. Can the available data be projected safely to large-scale commercial production? This question too must be treated with a degree of caution. Heterosis as measured in limited space-planted seedings from handmade hybrids may not be equally demonstrable in large-scale commercial plantings. The quality of wheat is of major importance because of the role of wheat as a human food. Available information would indicate the maintenance of acceptable quality will not be an obstacle in hybrid wheat-if good quality parents are used. But what if the highest levels of heterosis are most easily achieved from poor quality parent varieties? A key to the future of hybrid wheat may be successful and stable seed production since it will determine hybrid seed cost. Available data reveal extreme variations in seed set on male-sterile varieties. The variation has been attributed to inherent varietal differences as well as to effects of environment and conditions of experimentation. The data can be viewed with optimism or pessimism according to the specific information one accepts for projection purposes. There is a paucity of information concerning the contribution of specific factors of environment to acceptable cross-pollination in wheat. Too little is known about genetic variation that could significantly contribute to stability of hybrid seed production. Finally, there is the question: Can hybrid wheat compete with improved wheat varieties in the major wheat production areas of the world? If one assumes an adequate stable male sterility-fertility restorer system, reasonable levels of heterosis as measured against the best commercial varieties, and stable hybrid seed production, then the response must be an enthusiastic Yes! If any one of the above conditions is not realized, the

232

V. A. JOHNSON AND J. W. SCHMIDT

future of hybrid wheat as a commercially significant crop is questionable. Time and the collective research efforts of agricultural scientists will tell.

REFERENCES Barnard, C. 1955. Australian J. Botany 3, 1-20. Bitzer, M. J. 1965. M. S. Thesis, Purdue University, Lafayette, Indiana. Bitzer, M. J., and Patterson, F. L. 1967. Crop Sci. 7,482-484. Borlaug, N. E. 1965. Ann. Rept. Rockefeller Found. Program Agr. Sci., 1964-1965, pp. 214-231. Briggle, L. W. 1963.CropSci. 3,407-412. Briggle, L. W., Daum, R. J.,and Stevens, H. 1964. Crop Sci. 4,220-223. Briggle, L. W., Cox, E. L., and Hayes, R.M. I967a. Crop Sci. 7,465-470. Briggle, L. W., Petersen, H. D., and Hayes, R. M. 1967b. Crop Sci. 7,485-490. Cahn, E. 1925.J . Am. SOC.Agron. 17,591-595. Fonseca Martinez, S. 1965. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Fonseca Martinez, S., and Patterson, F. L. 1965. Agron. Abstr. 57. 1 I . Fukasawa, H. 1953. Cytologia 18,167-175. Fukasawa, H. 1957. Cytologia 22,30-39. Fukasawa, H . 1958. Wheatlnform. Serv. 7,21. Fukasawa, H . 1959.Japan. J . Botany 17,55-9 I. Hermsen,J. G . Th. 1965. Euphytica 14,222-224. Imrie, B. C. 1966.Australian J . Exptl. Agr. Animal Husbandry 6, 175-1 78. Jensen, N . F. 1966. Crop Sci. 6,376-377. Johnson, V. A., Biever, K. J., Haunold, A., and Schmidt, J. W. 1966. CropSci. 6,336-338. Johnson, V. A., Schmidt, J. W., and Mattern, P. J. 1967. Qualitas Plant. Mater. Vegetabiles 14,193-21 1 . Johnson, V. A., Shafer, S., and Schmidt, J. W. I968a. Crop Sci. 8, 187- 191. Johnson, V. A., Schmidt, J. W., and Mattern, P. J. I968b. J . Econ. B o f . 22,I6-25. Joppa, L. R., McNeal, F. H., and Welsh, J. R. 1966. Crop Sci. 6,296-297. Joppa, L. R., McNeal, F. H., and Berg, M. A. 1967. Western SOC.Crop Sci. Abstr., p. 7. Kherde, M. K., Atkins, 1. M., Merkle, 0. G., and Porter, K. B. 1967. Crop Sci. 7,389-394. Kihara, H. 195 I . Cytologia 16,177- 193. Kihara, H. 1958. Proc. 10th Intern. Congr. Genet. 1,142-171. Kihara, H., and Tsunewaki, K. 196 I. Seiken Ziho 12, I - 10. Kihara, H., and Tsunewaki, K. 1964. SeikenZiho 16,l-14. Kronstad, W. E., and Foote, W. H. 1965. Crop Sci. 4,6 16-6 19. Krull, C. F., Narvaez, I., Borlaug, N . E., Ortega, J., Vazquez, G., Rodriguez, R., and Meza, C. 1967. Centro lnternacional de Mejoramiento de Maiz y Trigo Res. Bull. 7,1-27. Lacadena, J. R. 1966.Anales Esrac. Exptl. Aula Dei 8, 187- 192. Lacadena, J. R., Monteagudo, A., and Sanchez-Monge, E. 1966. Insr. Nacl. Invest. Agr. 5 4 , ~5-8. ~ . Larrea, P. E. 1966. M. S. Thesis, North Dakota State University, Fargo, North Dakota. Livers, R. W. 1964a. Science 144,420. Livers, R. W. 1964b.Agron.Abstr. 56,72.

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Livers, R. W., and Heyne, E. G. 1966.Agron. Abstr. 58, I 1. Maan, S. S., and Lucken, K. A. 1967a. Wheat Inform. Serv. 23-24,6-9. Maan, S. S., and Lucken, K. A. 1967b. Can. J. Genet. Cytol. 9, 147-153. McCuistion, W. L. 1968. Ph.D. Thesis, Oklahoma State University, Stillwater, Oklahoma. Mcllrath, W. 0. 1967. Ph.D. Thesis, Oklahoma State University, Stillwater, Oklahoma. McNeal, F. H., Baldridge, D. E., Berg, M. A., and Watson, C. A. 1965. Crop Sci. 5,399400. Merkle, 0. G., Porter, K. B., and Atkins, I. M. 1967. Texas Expt. Sta. Progr. Rept. 2468, 6-7. Nettevich, E . D., and Fodorova, T. N. 1966. Rept. Sci. Res. Inst. Agr. Central Regions Nonchernozem Zone. Moskovskaya Oblast, p p . 1 I I - I 14. Oehler, E.,and tngold, M. 1966. Wheat Inform. Serv. 22,l-3. Olson, R. J. 1966. M. S. Thesis, Washington State University, Pullman, Washington. Pal, B. P., and Nek Alam. 1938. Proc. Indian Acad. Sci. 7,109- 124. Patterson, F. L., and Bitzer, M. J . 1966. Purdue Univ. Res. Progr. Rept. 222,l-4. Pfeifer, R. P. 1966. Science Farmer 13. Porter, K . B., Lahr, K. A,, and Merkle, 0. G. 1967a. Texas Expt. Sta. Progr. Rept. 2467, 3-6. Porter, K. B., Merkle, 0. G., and Atkins, I. M. 1967b. Texas Expt. Sta. Progr. Rept. 2471, 13-15.

Rajki, E. 1962. Symp. Genet. Wheat Breeding, Hung.Acad. Sci. I962. Rdjki, E., and Rajki, S. 1966. Acta Agron. Acad. Sci. Hung. Tomus 15,199-214. Rajki, E., and Rajki. S. 1967. Acta Agron.Acad. Sci. H u n g . Tomus 16,240-246. Reitz, L . P. 1965. Rept. Crop Quality Council, Minneapolis, Minnesota. Reitz, L. P. 1967. Crops and Soils 19,6-7. Roberts, T. H., and Holland, R. F. 1962. CirculatedLetter. Robertson, L. D., and Curtis, B. C. 1967. Crop Sci. 7,493-495. Rodriguez, R., Quinones, M. A., Borlaug, N E., and Narvaez, I. 1967. Centro International de Mejoramiento de Maizy Trigo Res. Bull. 3,l-37. Rosenquist, C. E. 1931. J . Am. Soc.Agron. 23,81-105. Schmidt, J. W.. and Johnson. V. A. 1966. Proc. 2nd Intern. Wheat Genet. Symp., Hereditas Suppl. VOI. 2, I 19- 120. Schmidt, J. W., Johnson, V. A., and Maan, S. S. 1962. Nebraska Expt. Sta. Quart. 9,9. Shebeski, L. H. 1966. Canad. J. Gen. Cytol. 8,375-386. Widner, J. N.. and Lebsock, K. L. 1967. Agron. Abstr. 59,2 I . Wi1son.J. A. 1968. Euphytica 17,s~ppI.1. Wilson,J.A.,and ROSS,W. M. 1961.CropSci. 1,191-193. Wilson, J . A., and Ross, W. M. I962a. Crop Sci. 2,4 15-4 17. Wilson, J. A., and Ross, W. M. I962b. Wheat Inform. Serv. 14,29-30. Wilson, J. A., and Villegas, E. 1966. Cereal Sci. Today 11,306-309.

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AGRONOMIC CONTROLS OVER THE ENVIRONMENTAL CYCLING

OF TRACE ELEMENTS W. H. Allaway U.S. Plant, Soil and Nutrition laboratory, U S . Department of Agriculture, Ithaca. New York

1. Introduction ....................................................................................... 11. Environmental Cycles of Trace Elements ..................................... Ill. Agronomic Control Points in Trace Element Problems ..... A. Soil Selection ............................................... B. Trace Element Fertilization............................................................ C. Soil Management ...................................................... D. Crop Selection ..... E. Crop Management ............................. IV. Current Environmental c e Elements ............ A. Arsenic .......................................................................................

D. Chromium, E. Fluorine ...................................................................................... F. Lead ............................................................................... G . Molybdenum and Copper ....... .................... H. Selenium .... ....................................................... I . Vanadium .................................................................................... J . Zinc .............................................................. V. Some Potential Future Trends ....... ....................

...............................................

Page 235 236 239 239 244 245 246 247 249 249 25 I 25 1 253 255 257 258 262 266 268 270 27 I

I. Introduction

The objective of this review is to examine some developments that may point to needs for improved agronomic management practices that could regulate the concentrations of trace elements in crop plants. During the past ten years, several new trace elements have been added to the list of those directly or indirectly affecting human health. This list will un235

236

W. H. ALLAWAY

doubtedly become longer. Although the concentrations of trace elements in living tissues are ordinarily very low, these concentrations must often be maintained within narrow limits in order to permit optimum biological performance of plants, animals, and man. Trace elements may enter human bodies by many alternate routes. With the rise of modern industrial technology, the importance of the various routes of movement of trace elements through the environment is subject to abrupt changes. The route from soil, to plant, to animal or man remains the predominant one for many elements, however, and control of trace element movements along this route is an essential aspect of control over the quality of the human environment. As information concerning the role of trace elements in human and animal health is expanded, agronomists may be called upon to develop more sophisticated regulation of the concentration of an increasing number of trace elements in plants. In some instances, the required controls must be directed toward decreases, and in others, increases in the concentration of specific trace elements in food or feed crops. A wide variety of agronomic practices can be utilized in efforts to establish the controls required. II. Environmental Cycles of Trace Elements

A number of different elements, including trace elements, cycle from geochemical reserves through plants, animals, and man and then return to the reserves. Some of these cyclic movements, with accompanying chemical changes, are essential to all life. Each element moves through this cycle by pathways and mechanisms that are often specific for that particular element. For this reason, the scientific literature concerning trace elements has tended toward fragmentation, with those aspects concerned with soils or plants separated from those aspects concerned with animal nutrition or toxicity, or with human health. In addition, there has been fragmentation on the basis of the different elements involved. The concept of the total environmental cycle, as highlighted in the recent book by Bowen (1966), provides a useful viewpoint for unifying the varied aspects of trace elements in biology. Figure 1 shows some of the routes that may be involved in the environmental cycling of trace elements. In many instances, there is a strong interdependence among the different routes of movement, with agricultural and industrial processes combining to furnish the human body burden of each trace element. Zinc, for example, may be taken up from the soil by plants and enter the human body when these plants, or animals which have been fed these plants, become part of human diets. Zinc is also

TRACE ELEMENT CYCLING

237

mined and fabricated into metal products. It may then be dissolved o r abraded from these products through contact with food, animal feeds, o r potable water supplies, and enter human bodies when these are uitlized. Other potential routes, including the direct consumption of zinc mineral supplements or medications, may be important in supplying zinc to humans. For optimum human health, all these different pathways of environmental movement of zinc must be adjusted and combined so that some “normal” range of zinc concentrations is maintained in body tissues and fluids.

.

FIG.1 . Pathways of environmental movement of trace elements

Some of the different pathways of environmental cycling shown in Fig. I may appear to be of minor importance. It must be recognized, however, that very small amounts of trace elements are normally present in animal or human tissue. Any one of the pathways outlined in Fig. 1 may be important in supplying these small amounts. It could probably be demonstrated that each of the pathways outlined has, under certain circumstances, been responsible for the delivery of excessive amounts of one o r more trace elements to plants or animals, and possibly to man. The existence of alternate routes of movement may complicate the development and use of controls over the soil-to-plant movement of trace elements, but even so, control over the soil-to-plant movements of many of the trace elements remains as an important feature of the total control problem. This is emphasized by the current situation with respect

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W. H. ALLAWAY

to lead. Although there is widespread concern over the pollution of some environments by airborne lead from auto exhausts, it appears that most of the lead in the human body comes from foods (Working Group on Lead Contamination, 1965). Some of the typical concentrations and amounts of trace elements in different phases of their environmental cycles are shown in Table I. In order to condense the extensive literature concerning trace elements, Table I is based largely upon reviews (Bowen, 1966; Chapman, 1966a; Underwood, 1962) and summary complications that were in turn generalized from original data. In particular, the wide variations known to exist in the levels of trace elements in different soils, and in different plant species, have been smoothed in Table I. In this table, an attempt has been made to consider primarily those elements that have been shown to bear a direct or indirect relationship to human health, and which are usually present in living tissues at concentrations of less than one hundred parts per million. Even so, the selection of elements for Table I is somewhat arbitrary, and some elements that are of physiological importance are omitted. The estimates of the annual uptake of trace elements by plants growing on arable or grassland soils are the writer’s. These were based upon an estimated dry matter production of 25 x 10 l2 kg. [This is a rounded figure resulting from an estimate of about 3 tons per acre from 5,000,000,000 acres of arable land and 1 % tons per acre from 7,000,000,000 acres of grassland. This estimate is only one-fourth of that of Bowen (1966, p. 5 1 ) for the net dry matter production of arable and grasslands.] This dry matter production was then multiplied by a concentration selected to be about the midpoint of the “normal” range in plants. Thus, the estimated annual uptake of any one trace element may well be in error by one order of magnitude. The time required for depletion of the soil supply of the trace elements as shown in Table I is another arbitrary figure. This is calculated by comparing the annual uptake of each trace element per acre with the amount of that trace element in an acre of soil, assuming that the rooting zone of plants weighs 5 million pounds per acre. In spite of the extensive generalizations involved, the information in Table I contains the following indications. 1. It is possible for feed and food plants to grow “normally” at optimum or near-optimum rates, even though they contain insufficient cobalt, chromium, copper, iodine, manganese, selenium, and/or zinc to meet the dietary requirements of some animals for these elements. 2. It is possible for feed and food plants to grow at normal or near-

TRACE ELEMENT CYCLING

239

optimum rates and still contain sufficient selenium, cadmium, molybdenum, or lead to cause either direct toxicity of metabolic imbalance in animals that eat these plants. 3. The soil-plant system provides an effective barrier against toxicity from arsenic, iodine, beryllium, fluorine, nickel, and zinc. Plant growth will cease or be greatly depressed before these elements will be taken up from the soil, and accumulated in concentrations that would be dangerous for animals. 4. The soil-to-plant route is, for most trace elements, the major route of entry into the living tissues of plants, animals, and man. For fourteen of the twenty elements listed in Table I, the annual uptake of the element by crops and grassland plants is equal to (within an order of magnitude), or in excess of, the estimated amount mined. In those cases-copper, cobalt, chromium, nickel, and lead -where mining exceeds plant uptake, much of the mined output is fabricated into durable products that do not enter living tissues. 5 . The soil-plant system exerts an effective buffering action on the environmental cycling of trace elements. The amount of each trace element listed in Table I that is present in the soil within the rooting depth of plants is one hundred to one hundred thousand times the amount likely to be removed by any one crop. Ill. Agronomic Control Points in Trace Element Problems

In view of the importance of the soil-plant system to the overall control of environmental cycling of trace elements, it is perhaps fortunate that a number of different agronomic practices offer alternative opportunities toward achieving this control. These practices may be categorized as follows: (a) soil selection; (b) trace element fertilization; (c) soil management, or practices directed toward increasing or decreasing the availability to plants of the trace elements in the soil, including the use of competitive elements; (d) crop selection: and (e) crop management and utilization. Mechanisms involved in these practices are often complex and are different in principle for different trace elements. For this reason, only major features of these practices can be mentioned here, and citations will be primarily directed toward review articles which provide summaries of the very extensive literature involved. A. SOILSELECTION The concentration of trace elements in any one plant species may be markedly different, depending upon the character of the soil on which it is

TABLE I Concentrations and Amounts of Selected Trace Elements in Various Phases of the Environment Concentration in geochemical reserves" (ppm.)

Concentration in soils, (total)* (ppm.)

a: 2 b: 1-3 c: 0.003

6 (0.1-40)

Boron

a: 10 b: 20-100 c: 4.6

10 (2-100)

Deficiency, Not required 5-30; low toxicity toxicity + 75 (wide species differences)

Beryllium

a: 3 b: 1-3 c: 0.0000006

6 (1-40)

< 0.1; not

Element Arsenic

Bismuth

a: 0.17 b: 0.3-1 c: 0.000017

Bromine

a: 2.5 b: 1-6 c: 65

Concentration Critical levels in in plants" animal diets" (ppm.) (ppm.) I (0.1-5):

not required

5 (1-10)

Not required; certain compounds medicinal; As3, highly toxic; As5, moderately toxic

49

21

Annual mining' (kg.) 2.7

X

lo6

1OR

< 0.01

2.8 x 1 0 5

0.06; not required: toxic

Not required: moderate toxicity

< I

1.4

15; not required; low toxicity

Not required; may antagonize CI or I: Br-,

460

X

108

Annual uptake by grasses and cropsg

0%) 10'

108

Not required: highly toxic

required; toxic

-

Concentration in human bloodd*' (cCg.llO0 ml.)

N P 0

lo6

106

108

Soil depletion timeg (years) 102

10'

low toxicity, Brz, highly toxic a: 0.2 b: 0.03-3 c: 0.0001 1

0.06 (0.01-7)

Chromium

a: 100 b: 10-100 c: 0.00005

Cobalt

Copper

Cadmium

4;accumulates

(0.2-0.8). not required; toxic

Not required; moderate to high toxicity

100 (5-3000)

(0.2-1.0); not required; moderately toxic

Required at ?; low toxicity

a: 25 b: 1-20 c: 0.00027

8 (1-40)

(0.05-0.5); required at < 0.02 by legumes

Required at 0.07 0.03, high in by ruminants; liver low toxicity

a: 55 b: 5-45 c: 0.003

20 (2- 100)

Required at Required at 1-10, 2-4; normal dependent on 4-15; Mo; low toxicity at toxicity 20

lo7

107

102

2 x 109

107

105

1.5 X l o 7

106

104

4 x 109

108

103

108

104

107

104

in kidney

2

100

+

Fluorine

a: 625 b: 250-750 c: 1.3

Iodine

a: 0.5 b: 1-2 c: 0.06

200 (30-300)

5

Not required; Not required; 36f 1000 ppm. in bone normal 2-20; beneficial to bones and teeth: toxic at+ 50 moderate toxicity Not required; Required at normal 0.4; 0.1-1;low toxic at toxicity 10-20

8-12

8 x lo8

2X lo6

N

f: (Continued)

N

TABLE 1. (Continued) Concentration in geochemical reserves" (ppm.)

Concentration in soils, (total) * (ppm.)

Lead

a: 12.5 b: 5-20 c: 0.00003

10 (2-200)

Manganese

a: 1000 b: 50- 1100 c: 0.002

850 (100-4000) Required: Required at 10normal 154O;low 100; toxicity toxicity depends on Fe :Mn ratio

Molybdenum

a: 1.5 b: 0.2-3 c: 0.01

2 (0.2-5)

Required at
Nickel

a: 75 b: 2-70 c: 0.0054

40 (10- 1000)

Not required; Not required: normal 1; moderate to low toxicity toxic at 50

a: 0.05 b: 0.1-1.0 c: 0.00009

0.5 (0.1-2.0)

Not required: Required at normal 0.020.05-0.20; highly toxic 2.0: higher

Element

Selenium

Concentration Critical levels in in plants' animal dietsu (ppm.1 (ppm.)

i3 Concentration in human bloodd,' (pg.II00 ml.)

Not required: Not required: normal 0. Imoderate 10;toxic in toxicity culture soin.

Required at < 0.1 :moderate to high toxicity, dependent on Cu

Annual miningf (kg.)

Annual uptake by grasses and cropsg

(k.)

Soil depletion time9 (years)

27

2 x 109

107

104

3

6 X lo9

109

10s

3 x 107

107

103

1-8

3x

108

107

I04

20

9 x 105

107

103

2

+

in accumulators; toxic at 50- 100 Strontium

a: 375 b: 20-700 c: 8.1

300 (50- 1000)

Not required: beneficial to teeth and bones; gOSr hazard

1

4.8 x 1 0 7

I08

I 03

Required by some algae; normal 0.110; toxicity

Not required; may be beneficial; moderate toxicity

1-2

8 X 106

107

I04

Required at 8-15: toxic at 200

Required at 10-40; low toxicity

650

Not required: normal 53000;

nontoxic

Vanadium

a: 135 b: 20-150 c: 0.002

100 (20-500)

+ 10

Zinc

a: 70 b: 10-100 c: 0.01

50 ( 1 0-300)

+

m r 5x109

109

103

"Generalized from Bowen ( I 966). a = Value for igneous rocks; b = value for sedimentary rocks; c = value for seawater. bGeneralized from Bowen (1966). Generalized from Chapman (1966). Concentrations on dry basis. "Primarily from Underwood (1962), plus various sources. High toxicity indicates dietary level of < 10 ppm. may be detrimental. Moderate toxicity indicates level of 10-100 ppm. may be detrimental. Low toxicity means 100 ppm. required for detrimental effects. All concentrations on basis of entire diet (dry). Fluorine would be classified as highly toxic if in water, but moderately toxic in food. eFrom Bowen (1963). 'From Bowen (1966). QSeetext for method of estimation.

+

Em 3

2

6 cz 0

244

W. H. ALLAWAY

produced. This principle has been primarily utilized as a method of avoiding toxic concentrations of trace elements. A most formalized use of soil selection to avoid excessive concentrations of selenium in crops is described by Anderson (1961). In this instance, a large block of seleniferous soil was purchased by the government and removed from private use because of the danger of selenium toxicity if this land were to be used for food crops. Possibilities for the use of soil selection to avoid excessive concentrations of molybdenum in forage crops in western United States are evident from the work of Kubota et al. (1961, 1967b). Other instances of restricted use of certain areas for pasture production due to trace element excesses and deficiencies are described by Russell and Duncan (1 956). The geographic pattern of distribution of soils that produce crops having deficient amounts of selenium (Kubota et al., 1967a) indicates that soil selection to favor use of soils that produce crops having adequate levels of selenium for animals involves selection of broad geographic areas for feed crop production. This has been done inadvertently in the transfer of feed grains from the Plains States to the dairy and poultry farms of the Northeast. On the other hand, soils that produce plants having toxic concentrations of selenium may sometimes be avoided by modifying the boundaries of fields on an individual farm or ranch (Rosenfeld and Beath, 1964). It is possible that the movement of dairying away from certain parts of New England may have been partly due to a search for soils that would produce plants containing adequate concentrations of cobalt for cattle (Keener et al., 1954), even though cobalt deficiency was not recognized until long after these adjustments had taken place.

B. TRACE ELEMENT FERTILIZATION The most direct method of increasing the concentration of a trace element in a crop growing on a given soil is to apply an available form of the trace element to the soil, or directly to the plant, by means of fertilizers or sprays. The primary purpose of trace element fertilization has been to increase crop production. The practice has, therefore, been confined primarily to trace elements such as zinc, copper, iron, boron, molybdenum, or manganese, that are required for plant growth. Inasmuch as the use of trace element fertilizers and sprays is familiar to so many agronomists, and there are recent reviews of the subject (Berger and Pratt, 1963; Chapman, I966a; Wallace, 1962; Wittwer and Tuebner, (1959), only a few aspects that may be important in the movement of trace elements to animals and humans will be considered here.

TRACE ELEMENT CYCLING

245

The degree of control over the concentration in plants of some of the trace elements that are related to animal and human health varies markedly among the different elements, and is also dependent upon the soil. The concentrations of zinc in most plants is only moderately increased by applications of this element in fertilizers, even when increases in crop yield are obtained (Viets, 1966). On the other hand, the concentration of selenium in plants can be increased at least 10-fold by applications of selenium to the soil, even though no effect upon plant growth is obtained (Allaway et al., 1966). Although increased plant growth is the common primary objective of trace element fertilization, cobalt (Russell and Duncan, 1956), selenium (Allaway et af.,1966), and copper (Russell and Duncan, 1956) have been applied to soils in order to correct deficiencies of these elements in the animals that consumed the plants grown.

C. SOILMANAGEMENT From Table I, it can be inferred that the total amount of many of the trace elements that is present in the soil within the plant rooting zone is greatly in excess of the annual uptake by plants. Management practices that may affect the availability to plants of the reserve supplies of trace elements in soils may, therefore, have a great potential for control over the trace element concentrations in plants. However, the chemistry of trace elements in soils is dominated by reactions that lead to the formation of inert and insoluble compounds or complexes. The chemistry and availability to plants of trace elements in soils is covered by several recent general reviews (Hodgson, 1963; Mitchell, 1964; Viets, 1962). Soil management practices that affect the incidence of deficient and toxic levels of different trace elements in plants are generally discussed, element by element, in the book edited by Chapman ( 1 966a). The most widespread application of soil management to the control of trace element movement from the soil to the plant consists of the application of lime to overcome the toxicity of aluminum and manganese in acid soils. In general, liming tends to reduce the availability of the metallic cations and to increase the availability of the trace elements that are taken into the plant in the form of anions. Changes in soil reaction resulting from liming may affect the solubility of certain trace elements by favoring changes in the oxidation state of the element. Heavy applications of phosphorus fertilizers may reduce the availability of the metallic cation trace elements. Several practices that operate through predominantly physical mechanisms may have value in controlling the concentrations of trace elements

246

W. H. ALLAWAY

in plants. Kubota et al. (1963) found that increasing the soil moisture supply increased the concentration of cobalt and molybdenum in plants growing on some soils, but had little effect on other soils. Drainage of wet soils may affect the availability of trace elements in soils by favoring the formation of more highly oxidized forms of these elements. The maintenance of reducing conditions by flooding during rice production may be essential to the trace element nutrition of the plant. Leaching may offer practical opportunities for reducing detrimentally high concentrations of some of the more soluble trace elements in plants. Applications of organic matter to the soil may be used in an attempt to increase or decrease trace element concentrations in the plants grown. Zinc deficiencies in plants may be corrected by adding manures to certain soils that are low in organic matter, but old corral sites that have received large applications of manure for long periods of time are fequently deficient in available zinc (Chapman, 1966b). Although the use of competing elements to reduce the uptake and accumulation by plants of specific trace elements may offer promise in certain cases, one of the most celebrated attempts to utilize this effect resulted in failure. This was the attempt made to reduce the uptake of selenium by plants in the western United States by adding sulfate to the soil to compete with selenates in the plant uptake process. The seleniferous soils contained an abundance of native sulfates. Had the practice been tried on the seleniferous soils of Ireland, it might have been effective (Lakin, 1961).

D. CROPSELECTION Different plant species growing on the same soil may contain markedly different concentrations of the different trace elements. This has been used deliberately under field conditions to avoid either excesses or deficiencies of trace elements for animals. General aspects of trace element accumulation and metabolism in plants have been recently reviewed by Bollard and Butler (1966) and by Nicholas (1961). Plant species differences in relation to nutritional requirements have been reviewed by Gerloff ( 1963). There are some plant species that exhibit a marked tendency to accumulate certain trace elements. The concept of “accumulator” species was developed as a part of studies of selenium toxicity in livestock in the western United States (Rosenfeld and Beath, 1964). The known accumulator species are listed for each trace element by Bowen ( 1 966). Comparisons of trace element concentrations in different forage crop species, grown on the same soil, have been made by Loper and Smith (1961). Varietal differences in plant nutrition have been reviewed by Vose

TRACE ELEMENT CYCLING

247

(1 963). Within some crop o r pasture species, different varieties respond very differently to either excess or deficiency of certain trace elements. Butler and Johns ( 1 96 1) cite evidence that the ability of ryegrass plants to accumulate iodine is a strongly inherited characteristic, pointing to the possibility of developing strains that are very effective accumulators of this element. Differences in the ability of two strains of soybeans to accumulate iron and to grow on soils low in available iron are reviewed by Brown ( 1 96 I). T h e potential for regulating trace element concentrations in plants through programs of plant breeding is, however, generally unexplored. Crop selection for avoiding deficiencies o r excesses of trace elements in plants for animals (or humans) that consume these plants is most often effective on soils where the available supply of a trace element is on the margin of either deficiency or excess. Thus, the use of grass pastures instead of legumes o r legume grass mixtures may help to reduce the incidence of molybdenum toxicity on some soils, but even the grasses will contain potentially toxic concentrations of molybdenum where the available supply of this element is very high (Kubota et al., 1961). On soils where the supply of available cobalt is marginal, legumes may contain sufficient cobalt to meet the needs of ruminant animals, but grasses will generally be inadequate in this regard (Kubota, 1964). Some potential opportunities for use of crop selection to control environmental cycling of trace elements may merit study in connection with future problems. One of these is the use of “accumulator” plants, with as much of the growth as possible removed, to deplete critical areas of potentially toxic levels of available trace elements in the soil. Precautions to prevent the trace elements contained in the harvest from reentering biological systems would, of course, be required. In some cases, the harvested trace elements may be used for industrial purposes in lieu of conventionally mined supplies. This process was explored for some of the selenium accumulators during a period when prices for industrial selenium were quite high. Another opportunity may be the use of crops that are grown for a specific constituent-for example, sugar cane or sugar beets-on areas that have excess levels of potentially toxic elements, providing that these elements are removed during the refining processes. This is done to some extent whenever wheat is produced on seleniferous soils. The refined white flour is lower in selenium than is the unprocessed wheat.

E.

CROP

MANAGEMENT

T h e use of crop management to control the movement of trace elements from plants to animals is based on changes in trace element concentra-

248

W. H. ALLAWAY

tions in crops at different stages of maturity, differential concentration of trace elements in different parts of the plant, or differences in the availability to animals of the trace elements in plants as the plants mature and are cured or otherwise processed. The concentrations of several trace elements in forage species at different stages of maturity have been measured by Beeson and MacDonald ( 1 95 I ) and by Loper and Smith (1 96 1). Effects of maturity on trace element content are not highly consistent, and the data of Beeson and MacDonald for cobalt in alfalfa (Medicago sativu) show increasing concentrations with advancing maturity, whereas Loper and Smith show an opposite trend. Ehlig el al. (1968) show that the trend in selenium concentration with stage of maturity varies with the supply of available selenium in the soil. On soils with moderate supplies of available selenium, the selenium concentration in alfalfa is higher in young plants than in plants at early bloom stage. On soils with a low supply of available selenium, the selenium concentration in the plant tended to be uniformly low at all stages of growth. In spite of the variable relationship between trace element concentrations and stage of growth, it may be possible to establish trends that are consistent for a given trace element in one forage species on a particular soil. Then it may be possible to avoid deficiencies of this trace element for animals by utilizing the forage at the stage when the concentration of the element is highest. Similarly, potential toxicity may be avoided by utilizing the forage at a stage when the concentration of a potentially toxic element is least. Russell and Duncan (1956) describe a situation in New Zealand where “bush sickness” or cobalt deficiency in animals on certain pastures could be minimized if the pasture were kept closely grazed. They also describe instances where copper deficiency in animals showed seasonal trends related to seasonal changes in copper concentrations in the plants. Range management techniques for minimizing selenium toxicity are described by Rosenfeld and Beath ( I 964). These techniques are designed to favor consumption of grasses on ranges where grasses and selenium accumulator plants are both present. Hartmans and Van Der Grift ( 1 964) describe a copper deficiency in grazing cattle in Holland that becomes worse as the grazing season progresses. During the winter period, the cattle tend to recover, even though they are fed on cured hay from swards very similar to those grazed during the summer. The suggestion is made that the copper in cured hay is more available to the animal than is copper from fresh, uncured pasture. Farmers in certain parts of Great Britain are of the opinion that certain

TRACE ELEMENT CYCLING

249

fields on their farms can be used safely for hay production, but molybdenum toxicity will occur in cattle if these fields are used for pasture (Russell and Duncan, 1956). IV. C u r r e n t Environmental Problems Involving Specific T r a c e Elements

In this section, some current problems involving the environmental cycling of specific trace elements are described. Many of these problems have become more important in the last decade or so, and they may become even more critical in the future. The changing importance of these problems has arisen from recent findings in nutrition or from the recognition of changes in the total environment. The application of agronomic information offers attractive possibilities for achieving improved control over the cycling of some of the trace elements discussed. In other cases, information essential to development of the necessary controls is deficient at this time, but may be obtained by agronomic research. A. ARSENIC Arsenic is one of the few trace elements for which recent developments may indicate decreasing rather than increasing concern over concentrations in human food. A primary reason for this situation rests on the decreasing use of arsenical pesticides, and their replacment by organics. Thus, the concern over potentially toxic concentrations of arsenic in plants has been transferred (and multiplied in the process) to concern over residues of the organic pesticides. The environmental movement of arsenic has been recently reviewed by Shroeder and Balassa ( 1 966). According to this review, the toxicity of arsenic is dependent upon its oxidation state and the pentavalent form, which is most common in aerated soils, is much less toxic than the trivalent form. Arsenic is ubiquitous in nature, and is found in forest plants and wild animals that have supposedly never been exposed to residues of arsenical pesticides. The levels of arsenic in sea foods are frequently in excess of established tolerance limits for this element. Plants or processed foods prepared from plants rarely contain more than 1 ppm. of arsenic. Arsenic added to poultry and livestock diets as arsanilic acid is quite rapidly removed from edible tissues. Frost ( 1 965) has pointed out that other elements, especially selenium, may have been the real cause of some of the historical cases of arsenic poisoning. The alleged carcinogenicity of arsenic has been strongly disputed (Frost, 1965; Shroeder and Balassa, 1966). A recent survey of residues in randomly selected “total diets” in the United States indicates

250

W. H. ALLAWAY

that the daily human intake of arsenic in this country is very low (Duggan and Weatherwax, 1967). Arsenic has not been shown to be an essential element for either plants or animals (Liebig, 1966; Underwood, 1962). Specific arsenic compounds are useful in the control of certain parasites, and these are frequently added to poultry and livestock diets for antibiotic purposes. Growth stimulation in animals has been reported from the use of these compounds, but the mechanisms involved are not clear (Underwood, 1962). An additional beneficial function of arsenic in animal diets may reside in the counteraction of selenium toxicity by certain arsenic compounds (Underwood, 1962: Frost, 1965). Inasmuch as the beneficial effects of arsenic in animal nutrition are dominated by the actions of specific arsenic compounds used in specific situations of animal nutrition and management, there does not appear to be a need to develop agronomic practices that would result in a controlled increase in the levels of arsenic in food or feed crops. The primary agronomic problem related to arsenic at the present time consists of the need to restore to optimum levels of plant production certain soils that have been contaminated with arsenic. This problem has been reviewed by Liebig (1966). Crop yields are frequently reduced on fields that have accumulated arsenic from pesticides. Under these conditions, the levels of arsenic in the plant roots may be very high, but the levels in the tops are only moderately elevated in comparison to plants grown on soils untreated with arsenic, The primary danger of arsenic toxicity to humans or animals from these crops is due to soil and dust adhering to the plants as consumed. A classification of vegetable and fruit crops according to their tolerance to arsenic has been prepared. Applications of zinc sulfate to the soils, or zinc EDTA to the foliage, have helped to improve plant growth on soils that have been contaminated with arsenic. Soil applications of ferrous sulfate, aluminum sulfate, and lime have also been found to alleviate some yield depressions due to arsenic residues in soils. In view of the similarity in chemical behavior of arsenates and phosphates, it may be difficult to develop soil treatments that will reduce arsenic solubility in soils without causing a coincident decrease in levels of available phosphorus. There are apparently few recorded instances where naturally occurring arsenic is a primary cause of low productivity. Arsenic in water, even where no arsenicals have been applied to the drainage area, has caused toxicity in animals (Russell and Duncan, 1956).

TRACE ELEMENT CYCLING

25 1

B. BROMINE Concentrations of bromine in human diets in the United States have probably increased due to the use of organic bromides as fumigants for soils and stored grains, as herbicides, and in auto fuels (Duggan and Weatherwax, 1967; Heywood, 1966). Bromine is almost universally present in plants and animals, and both plants and animals have high tolerance levels for bromides (Martin, 1966; Underwood, 1962). Bromine has not been established as an essential element for either plants or animals, but bromide may substitute for a part of the chloride requirement of chicks (Leach and Nesheim, 1963). Inasmuch as chloride deficiency in plants or animals is very rare, this substitution has little practical value. Under conditions of low iodide and high bromide intake, bromides may interfere with iodine metabolism and accentuate iodine deficiency in animals. Under conditions of excessive iodine intake, bromides may reduce hyperthyroid effects (Underwood, 1962). The increased use of bromine compounds as pesticides does not appear to create critical new agronomic problems due to the bromine in these compounds. As pointed out by Martin (1966), bromine is readily removed from soils by leaching, and so any bromine excesses in soils would probably be temporary. C. CADMIUM Concern over the concentrations of cadmium in human food stems largely from the recent work of Shroeder and his associates at Dartmouth Medical School (Shroeder and Balassa, 196 1; Shroeder et al., 1963a,b), showing that a low, but prolonged, intake of cadmium leads to hypertension and reduced longevity in laboratory animals. Studies by this same group have indicated that the level of cadmium in humans who die from hypertensive diseases is higher than that in humans whose deaths are due to other causes (Shroeder, 1965a). There is a positive correlation between the levels of cadmium in air in U.S. cities and the incidence of cardiovascular diseases in those cities (Carroll, 1966). It is not certain that levels of cadmium that would be detrimental to humans are moving from soil to plant and on into the food chain. Shroeder and Balassa (1 96 1) and Shroeder et al. ( 1967) have shown that cadmium is present at low levels in many plants, but other sources, including the atmosphere and water supplies may make significant contributions to the human body burden of this element. Sea foods and meats are normally higher in cadmium than are food grains and vegetables. In an earlier study

252

W. H. ALLAWAY

Shroeder and Balassa ( 1 963) implicated cadmium present as an impurity in superphosphates as a factor contributing to increased levels of cadmium in food plants, but more recent work (Shroeder et al., 1967) shows that the cadmium present in superphosphate probably makes little contribution to the cadmium content of plants. The levels of dietary cadmium required to produce detrimental effects on humans are not known. Much of the work with laboratory animals has been done with levels of 2 and 5 ppm. of cadmium added to the drinking water. Any attempt to relate cadmium levels in water to cadmium levels in food must be based on information on the degree of absorption and retention in the animal of cadmium from the two sources. At the present time, there is no information on the extent to which cadmium contained in different foods is absorbed from the gastrointestinal tract. Although food plants normally contain less than 0.5 ppm. of cadmium, preliminary studies by the writer indicate that plants may accumulate up to about 3 ppm. of cadmium before severe plant growth depression occurs. In the work of Shroeder and his associates ( I 963a,b), it has been shown that cadmium tends to accumulate in rats and mice, and detrimental effects are noted only in older animals. Because humans are normally long-lived in comparison with rats and mice, a low level of dietary cadmium may be sufficient to cause undesirable accumulation in older people. One of the first problems needing attention by agronomists will be to determine, in cooperation with nutritionists and the medical profession, whether or not significant amounts of cadmium are moving from soil-toplant-to-man. This will involve studies of the degree to which cadmium in different plants is absorbed by animals. If it should be determined that the soil-to-plant segment of the food chain is an important factor contributing to excessive human body burden of cadmium, the specific soils and soil management systems associated with high levels of cadmium in specific crops can be identified, even though this may require extensive research. Although little is known about the factors influencing uptake of cadmium by plants, the similarity of cadmium and zinc suggests possible approaches to reducing the cadmium content of plants in the event that such a reduction appeared to br desirable. Heavy applications of cadmium-free phosphate fertilizers may tend to reduce cadmium concentrations in the tops of plants if cadmium, phosphorus interactions resemble those of zinc and phosphorus (Burleson and Page, 1967). Increasing the level of available zinc in soils may tend to reduce cadmium uptake by plants, and the resulting increased zinc in foods may also help to minimize detrimental effects of cadmium (Shroeder el al., 1967;

TRACE ELEMENT CYCLING

253

Shroeder and Buckman, 1967; Mason and Young, 1967). It may also be possible to minimize detrimental effects of dietary cadmium by increasin the levels of dietary cobalt (Gabbiani et al., 1967), or the levels of dietary selenium (Mason and Young, 1967). T o summarize the current situation with regard to cadmium, it seems quite certain that agronomists must be concerned over the cadmium content of food and feed crops. It is possible that a need to decrease the concentration of cadmium in these crops will become evident.

D. CHROMIUM The most recent addition to the list of elements essential to man and animals is chromium. The work resulting in the identification of chromium as an essential part of a “glucose tolerance factor” has been reviewed by Mertz (1967). Human patients with a diabetes-like state of abnormal glucose metabolism have, in some cases, responded to chromium treatment. A syndrome simulating the disturbed glucose metabolism in adult diabetes millitus has been produced in rats by dietary chromium deficiency (Shroeder, 1965b). There is some evidence that, in a significant number of residents of the United States, tissue and serum chromium concentrations may be lower than optimal (Tipton and Cook, 1963; Mertz, 1967; Feldman and Knoblock, 1967). Geographic variations in the chromium concentrations in human tissues have been noted (Shroeder et al., 1962). Chromium, especially in the hexavalent form, is toxic at high levels to animals, but the margin between essentiality and toxicity is substantial (Mertz, 1967). It would therefore seem to be quite possible that a controlled increase in the amount of chromium moving from soils to plants to man would be desirable and might result in decreased incidence of diabetes. On the basis of present knowledge, it is by no means certain that a controlled increase in the chromium content of plants is practical, or even possible. Chromium is one of the few essential elements for which no accumulation against a concentration gradient is evident a t any point in the biological cycle from soil to plant to animal. According to Goldschmidt ( 1 954), most of the chromium in the earth’s mantle is in the form of chromite ( F e C r r 0 4 ) ;in mixed oxides of chromium, iron, and aluminum; or in silicate lattices. In all these forms, it is insoluble and of low availability to plants. Chromates are rare in nature and would be stable only in alkaline, oxidizing situations. Even so, excessive levels of native chromium have been implicated as a cause of infertility of certain soils, especially those formed from serpentine rocks (Robinson et al., 1935).

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The uptake of chromium by plants has been reviewed by Pratt ( 1 966a). Although some stirnulatory effects of chromium salts on plant growth have been noted, chromium cannot be considered an essential element for plants on the basis of present evidence. In many cases where naturally occurring chromium toxicity to plants has been suspected, it is by no means certain that chromium is the only potentially toxic element involved. Plants suffering from induced chromium toxicity frequently are found to contain about the same concentrations of chromium in the tops as are found in unaffected plants. Normal concentrations of chromium in feed and food plants, or food products prepared from plants, are generally in the range of 0.03 to 1 ppm. The diet used by Shroeder (1965b) to develop chromium deficiency in rats contained 0.15 to 0.20 ppm. of chromium, and his chromium-adequate or control diet contained an additional 2 ppm. of chromium added as chromic acetate. Therefore, it may be that a majority of the food and feed crops now produced contain too little chromium to meet the requirements of man and animals. There is little information on the degree to which the chromium contained in plants is absorbed and retained by animals. One form of trivalent chromium, calcined chromic oxide, is so inert in the digestive tract of animals that it is widely used as a “marker” in studies of the digestibility of forages. It seems reasonable to expect a fairly low retention of plant chromium. Attempts to increase the amount of chromium in the food chain by soil and crop management practices must, then, overcome some formidable obstacles. Soluble chromium added to the soil is likely to revert to very insoluble oxides. Chelated forms of trivalent chromium are frequently less stable than are the iron complexes of the same ligands, and therefore the addition of chelated chromium to the soil may result in only a temporary increase in the chromium in the soil solution. A large increase in available chromium in the soil may result in only a modest increase in the chromium concentration in plants and an even smaller increase in digestible chromium. It may prove to be worthwhile to reinvestigate the areas where high levels of native chromium were implicated as a cause of infertility. Perhaps under some systems of management, these areas can be used to produce food crops of high chromium content for use as valuable components of blended foods or animals feeds. None of the food or feed crops at the present time has been identified as an effective chromium accumulator. In view of the difficulties involved in the soil-plant system, the use of chromium additions to processed foods or domestic water supplies may be the most practical means of increasing human chromium intake.

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E. FLUORINE On the basis of studies of the incidence of osteoporosis in residents of different towns, Hegsted (1 967) has stated that “fluoride deficiency is probably the primary nutritional deficiency in United States.” At the same time, toxicity due to excess fluorides has been a serious problem in plants, animals, and people. Most of the attempts to increase the fluoride intake of humans have been directed toward addition of fluorides to domestic water supplies. Attempts to decrease the incidence of fluoride toxicity have centered on decreasing airborne fluoride fumes and dusts, restricting the use of waters that are naturally high in fluorides, or removal of fluorides from minerals used as feed sources of calcium and phosphorus. Much less attention has been directed toward control over the concentration of fluorides in foods, although Underwood (1 962) states “at low overall intakes of fluorine . . . food sources of fluorine may become significant.” The importance of fluoride deficiency and fluoride excess to animals and humans, and the relatively narrow range between beneficial and detrimental concentrations of fluorides, indicate a need for evaluation of the fluorides taken up from the soil by food and feed plants. The effects of fluorides on humans and animals have been reviewed by Underwood (1 962). Fluoride injury to farm animals has usually been due to excessive levels of naturally occurring fluorides in the drinking water, injudicious supplementation with fluoride-bearing minerals, or exposure to fluoride fumes. The fluoride fumes may be inhaled directly or may be ingested as residues from aerial deposition on food plants. Fluoride toxicity that can be ascribed to fluorides taken up by the roots of plants, translocated to the tops, and then eaten by animals has not been reported. Most food and feed crops contain 1 to 3 ppm. of fluoride, unless contaminated by aerial deposition of fluorides. Tea is a noted exception, and levels of 100 ppm. or more of fluoride in tea have been reported. Fluorides contained in dry feeds, even when added as soluble sodium fluoride, are less toxic than the same amount of fluoride contained in drinking water. At low levels of fluoride intake, differences between soluble and insoluble sources of fluoride tend to disappear. There is little information on the relative value of fluorides from food in comparison to the same amount of fluorides from water under conditions of low fluoride intake. Underwood (1962) reports that “normal mixed North American diets, which contain very little tea, contribute about 0.3-0.5 mg fluoride per day. . . . Where water is artifically fluorided to contain 1 ppm. fluoride a consumption of 1200 to 1500 ml/day supplies 1.2- 1.5 mg. fluoride daily.” Research on fluorides in plants and soils has been reviewed by Brewer

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(1 966a). Most of this work has been concerned with the injurious effects of airborne fumes of hydrogen fluoride. Injury to plants from fluorides taken up by the roots from the soil is very rare, but under certain conditions plants growing on acid soils will accumulate fluorides and exhibit injury symptoms identical to those caused by hydrogen fluoride fumes. Almost all the published data on fluoride content of plants is for leaf samples, with relatively few data on seeds and fruits. Leaf samples generally contain 2 to 20 ppm. of fluoride in areas where there is little or no airborne fluoride contamination and no evidence of fluoride injury. If fluoride concentrations in the fruits and seeds are similar to those of leaves, it would seem possible, at least from the standpoint of plant physiology, to produce food plants that would contain sufficient fluorides to make a very significant contribution to the total fluoride intake of humans. Although soils frequently contain substantial amounts of fluoride, this is generally insoluble and not taken up by plant roots. Addition of soluble fluorides to unlimed, acid soils will result in increased concentrations of fluorides in plants, but these same additions made to limed soils have little effect upon the fluoride concentration in plants. The addition of calcium fluoride to either acid or neutral soils is ineffective in increasing the fluoride concentration in plants. Substantial additions of insoluble fluorides have been made to many soils coincident with the application of phosphatic fertilizers. These additions have not measurably affected the concentrations of fluoride in the plants produced. The concentration of fluorides in solution in agricultural soils is apparently controlled by the solubility of calcium fluoride, aluminum silicofluoride, fluorapatite, and adsorbed fluorides. It may be impossible to increase the level of available fluoride in soils and still provide an adequate supply of calcium to permit optimum plant growth. A controlled increase in fluoride concentrations in plants might result from the use of organic compounds of fluorine as sources of this element for soil addition. The requirements for these organic sources, with respect to stability in soils, lack of toxicity, uptake and translocation by plants, and nutritional value as sources of fluoride for animals, are rigid. Crop selection and plant breeding may offer potential routes toward controlled increase in the fluorine concentration in foods. As evidenced by tea, some plants apparently can accumulate fluorides from soils on which other plant species contain very low concentrations of this element. A screening of the different varieties of major food and feed crops to see whether or not any are effective in taking up fluoride from neutral and alkaline soils might be an initial step in this approach.

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The difficulties involved in achieving a controlled increase in the fluoride concentration in food plants, and uncertainty concerning the nutritional value of fluorides in plants, may indicate that it is impractical to consider increasing human fluoride intake through increase in the soilto-plant movement of fluorides. And, it must be recognized that the same factors that make it difficult to manipulate the fluoride concentration in plants have effectively prevented fluoride toxicity in spite of high levels of total fluoride in soils. Although the levels of fluoride in food plants may be too low to supply all of the fluoride required by humans, these levels are quite constant. The constancy of fluoride levels in foods is essential to the safe addition of fluorides to domestic waters. F. LEAD

Lead toxicity in humans has been well documented and is a prob!ein of long standing (Working Group on Lead Contamination, 1965). Most of the current interest centers around chronic effects due to long exposure to low levels of lead. Airborne lead from auto exhausts is a particular point of concern. I t is estimated, however, that over half of the normal body burden of lead in residents of the United States results from lead in the food supply (Goldsmith and Hexter, 1967). It is not known how much of the lead in the food supply results from deposition of airborne lead onto the tops of food plants plus the inhalation of lead by farm animals, and how much arises from uptake of lead from the soil by food plants. The isotopic concentration of terrestrial sources of lead is not constant, and lead-210 plus its daughter polonium-210 are more abundant where lead has arisen by decomposition of uranium. Alpha emissions from lead210 and polonium-210 may constitute a health hazard. Tso et al. (1966) have found that most of the lead-210 and polonium-210 in tobacco appears to arise from that taken up from the soil. Uranium in the phosphate rock used in fertilizers is a possible origin of the lead-210 and polonium-2 10 found. The deposition of lead on soils and plants along highways and in urban areas is well established (Cannon and Bowles, 1962; Purves, 1967), but even so, there are areas that are remote from highways and have never been treated with lead insecticides where the lead concentration in the plants is high (Mitchell and Reith, 1966). There have been no well confirmed beneficial effects of lead on either plants or animals. In view of the widespread concern over environmental lead in urbanized areas, it seems quite likely that a concerted effort may be made to reduce the average body burden of lead in the residents of

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the United States and other countries. This effort must include attempts to reduce the lead content of food plants, along with attempts to reduce the amount of airborne lead from auto exhausts. At the present time, there are no well established practices for use in reducing the lead content of food crops. Information on the uptake of lead by plants from culture solutions and soils has been summarized by Brewer (1966b). It is difficult to explain the observation that additions of fairly large amounts of soluble lead salts to soils have little effect on the concentration of lead in the tops of the plants, whereas at the same time different soils, even where deposition of airborne lead is not a factor, produce plants with varying concentrations of lead in the tops. Part of the differences noted in the lead concentrations in plants growing on different soils may be due to differences in stage of maturity of the plants sampled. Mitchell and Reith (1 966) have shown large increases in the lead concentrations of the same pasture species growing on the same soil as the plants mature, and continuing while they remain dormant over the winter. The work of Mitchell and Reith also suggests that in attempts to reduce the lead content of plants, the soils that produce plants of high lead content should be used for crops that are harvested at an immature stage, such as closely grazed pasture. Crops that grow for a long time prior to harvest, such as winter range, might be best suited to soils that are known to produce crops of low lead content. An exhaustive study of the chemistry of lead in soils and the mechanisms of uptake and translocation of lead in plants may point the way for development of management practices that will decrease the amount of lead in the food chain.

G . MOLYBDENUM AND COPPER Some of the current interest in environmental levels of molybdenum can be traced directly to an earthquake which elevated certain coastal areas at Hawkes Bay, New Zealand in 1931. One of the results of this uplift was the draining of a lagoon of about 5 square miles in the vicinity of the city of Napier. In the ensuing years, the soil from the bottom of the former lagoon was used for agricultural production and then for residential development. In 1955 it was discovered that the children in the city of Napier had a lower prevalence of dental caries than did children in the nearby city of Hastings, even though the water supplies were similar for the two cities. An investigation into the probable causes of the decreased caries incidence in Napierchildren indicated that elevated levels of molybdenum in the home garden vegetables grown on the former lagoon

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bottom near Napier might be responsible. This exciting investigation has been reported by Ludwig et al. (1960) and Healy et al. (1961). In controlled experiments, additions of molybdenum to diets has decreased the incidence of dental caries in rats. The New Zealand findings on relation of molybdenum to human dental caries have been confirmed by studies of the teeth of children living in high-molybdenum areas of the British Isles (Anderson, 1966). Much of the research on the role of molybdenum in animal nutrition has been concerned with effects due to excess molybdenum. This research is reviewed in detail by Underwood ( 1962). Although molybdenum is listed as one of the elements required by animals, the required levels have been considered to be less than 1.0 ppm. of the dry diet for many species. Higher levels of molybdenum produce a disorder called molybdenosis or molybdenum-induced copper deficiency. The dietary concentration of molybdenum required to produce this disorder varies according to the copper and sulfate concentrations in the diet, but where copper is low (< 4 ppm.), as little as 5 ppm. of molybdenum may have some detrimental effects. Dietary levels of 10 to 20 ppm. of molybdenum are nearly always associated with some evidence of disturbed copper metabolism in ruminant animals. Nonruminants, especially horses, are less subject to molybdenum toxicity. In the New Zealand studies of molybdenum in relation to dental caries, some of the edible beans produced in home gardens in Napier contained as much as 9 ppm. of molybdenum. It is therefore possible that the level of dietary molybdenum required for optimum dental health for humans is high enough to cause conditioned copper deficiency in ruminants if the diet is low in copper. Diets that are quite high in molybdenum may be readily utilized without harmful effects if the diets are supplemented with extra copper or if the animals are injected with a suitable form of copper (Gray and Daniel, 1964; Dye and O’Harra, 1959). T h e concentration of molybdenum in the blood of residents of different parts of the United States has recently been reported (Allaway et al., 1967). Molybdenum concentrations in excess of 0.5 pg/ 100 ml. were found in less than one-fifth of the blood samples examined, but about 3 percent of the blood samples in this study contained more than 10 pg. of molybdenum per 100 ml. of blood. Although the relation of molybdenum intake to levels of molybdenum in the blood is not well established for humans, it has been shown that in animals the levels of molybdenum in the blood are closely related to dietary molybdenum intake. If the relation of blood molybdenum to dietary molybdenum intake is similar in animals and humans, it is possible that a substantial part of the popula-

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tion of the United States may be receiving less molybdenum than would be desirable for optimum dental health. At the same time, a small fraction of this population may be receiving sufficient molybdenum to cause some interference with copper metabolism. As additional information concerning the role of molybdenum in human dental health is developed, more deliberate controls over the molybdenum content of foods may be suggested. These controls may involve increasing the concentration of both molybdenum and copper in the foods produced in many areas, and at the same time directing efforts toward reduction of the molybdenum in foods produced in certain other areas that are naturally very high in available molybdenum. If it should be desirable to control the levels of molybdenum in foods, there are several promising agronomic approaches to this problem, and agronomists have a considerable background of experience in the use of these approaches. The recent review by Johnson ( 1966) summarizes much of the extensive literature on molybdenum in the soil-plant system. Other reviews of molybdenum in plant nutrition have been provided by Bear (1 956) and Anderson ( 1 956). Much of the work on molybdenum done by soil and plant scientists has been directed toward the correction of molybdenum deficiencies in plants. The concentration of molybdenum in molybdenum-deficient plants are frequently less than 0.1 ppm., with substantial differences in the concentration in different parts of the plant. When the leaves of the plants contain as much as 0.5 to 1.0 ppm. of molybdenum, it is unlikely that plant growth responses will result from the use of molybdenum fertilizers. Thus, the molybdenum concentrations desired in food plants for optimum dental health of humans may turn out to be 5 or 10 times the concentrations required for optimum plant growth. The molybdenum concentration in the plant can be increased beyond the minimum levels required by plants through practices that increase the level of available molybdenum in the soil. N o injury to most plants is seen even where the molybdenum concentration exceeds 100 ppm., and many suggestions for use of molybdenum fertilizers are accompanied by caution against excessive use that may result in feed crops containing concentrations of molybdenum that will interfere with copper metabolism in animals. In addition to the use of molybdenum fertilizers, the application of lime to acid soils is frequently an effective method of increasing the availability of soil molybdenum and the concentration of moiybdenum in plants. The addition of lime to certain acid soils of high total molybdenum content may result in the production of feed crops containing excessive concentrations of molybdenum. A decrease in molybdenum concentration in plants growing on soils

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naturally high in available molybdenum may be more difficult to achieve than an increase in molybdenum in the plants of low-molybdenum areas. The use of sulfate or manganese fertilizers may help to reduce molybdenum uptake (Johnson, 1966). Many of the soils that produce highmolybdenum plants are wet and poorly drained, and the concentration of molybdenum in crops growing on these soils can be expected to decrease if soil drainage and aeration are improved (Kubota et al., 1963). Increasing the acidity of some of the alkaline, high-molybdenum soils would also decrease molybdenum uptake by plants, but this change might be difficult to achieve in highly buffered soils, and might also result in lower production of crops. A major route toward improvement in the use of soils that produce high-molybdenum crops consists of identification of the maximum number of these areas, followed by blending the crops produced there with crops produced on low-molybdenum areas. These areas can also be used as feed sources for animals, provided that the animals also receive supplements or injections of effective forms of copper. Any general increase in the molybdenum concentration in food or feed crops must be accompanied by a concurrent increase in the copper concentration in these crops, or else by a general use of copper supplements or medications for humans and animals. Research on the role of copper in plant nutrition and in fertilizers has been reviewed by Reuther and Labanauskas ( I 966). To quote directly from this review, “In a wide variety of plants copper deficiency is characterized by levels of less than 4 ppm in the dry matter of leaves. The range for normal growth in most plants usually falls between 5 and 20 ppm. . . . relative to excess levels. . . the data indicate that amounts in excess of 20 ppm in tissue material should be looked upon with suspicion.” The concentration of copper in plants is generally less responsive to additions of copper to the soil than is the concentration of molybdenum in plants to molybdenum additions. Furthermore, the possibility of reduced plant growth due to excessive additions is greater for copper than for molybdenum. Even so, addition of copper to the soil has been successfully used to correct copper deficiencies in grazing livestock under circumstances where any increased plant growth as a result of copper fertilization was of secondary importance (Underwood, 1962). On alkaline soils of high molybdenum content in Nevada, however, soil applications of copper sulfate have not increased the copper concentrations in forages produced, nor have they resulted in decreased molybdenum concentrations in forages (Spencer el al., 1958). In spite of the obvious difficulties involved, it appears to be possible, at least in theory, to establish a reasonable degree of control over the

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molybdenum and the copper concentrations in food and feed plants growing on most soils. If future research in nutrition and dental health should indicate a need to maintain the molybdenum concentration in feed and forage plants at 3 to 5 ppm., and the copper concentration in these plants at 6 to 12 ppm., for example, existing information concerning copper and molybdenum in soil-plant systems may provide an excellent basis for the development of the required control measures. However, the procedures required to monitor control efforts and ensure that the majority of crops do, indeed, contain the desired concentrations of these elements may be very laborious. The availability of soil molybdenum and copper may be quite different in different parts of the same farm. The concentrations of molybdenum and copper in plants may depart significantly from the desired ranges without visible effects upon plant growth. The monitoring procedures required, such as soil tests and plant analyses, must then be simple, rapid, and adapted for use on a field-byfield basis.

H. SELENIUM Prior to 1957, selenium was primarily noted for its toxicity to animals, and areas where range and crop plants might contain dangerously high levels of this element were recognized. Since 1957, selenium has emerged as an essential element, the livestock industry of certain areas has been rescued from disaster by injecting animals with selenium, and some evidence has appeared indicating that humans may at times be deficient in this element. Much of the current knowledge concerning selenium in biological systems is summarized in the recent monograph edited by Muth ( 1 967). The reviews by Rosenfeld and Beath (1964) and by Anderson et al. ( I 96 1) cover many of the investigations of selenium toxicity. Concentrations of selenium in animal diets in excess of 4 to 5 ppm. have generally resulted in depressed growth rates, infertility of eggs, or other undesirable effects upon animals. In one instance (Tinsley el al., 1967) reduced growth was evident when 1 ppm. of selenium was fed to rats over extended periods of time. Selenium has been considered to be carcinogenic, but this is in dispute at the present (Harr et al., 1967; Volgarev and Tscherkes, 1967). Due to earlier indications of carcinogenicity, however, deliberate additions of selenium to foods or animal feeds are prohibited in the United States. Studies of selenium deficiency in animals have often centered around interactions between selenium and vitamin E, and a pronounced sparing effect of selenium upon vitamin E requirements and vice versa has been

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frequently observed. In fact, controversy has existed concerning the absolute necessity for selenium if the diet contains an abundance of vitamin E or other antioxidants. Many students of the problem, however, have concluded that selenium is an essential element in its own right, irrespective of the vitamin E supply, and this conclusion is reinforced by the very recent findings of Thompson and Scott (1967). The concentrations of selenium required in the diets of various animals to prevent selenium deficiency have ranged from 0.04 to about 0.20 ppm. of selenium, dependent upon the kind of animal and type of diet, including the vitamin E content. Injections of selenium, usually accompanied by vitamin E, are routinely used in certain areas to prevent “white muscle disease” in lambs and calves. Several million individual injections have been used since this type of selenium therapy was licensed in the United States. Deficiency of selenium in humans is apparently rare, but there are indications that selenium deficiency may be one of the factors involved in certain instances of malnutrition in children. Hopkins and Majaj (1967) report responses to selenium therapy in Jordanian children suffering from malnutrition. Burk er al. ( 1 967) have shown that the level of selenium in the blood of Guatemalan children suffering from kwashiorkor averaged about 10 pg. per 100 ml. of blood; whereas normal Guatemalan children and residents of the United States showed about 22 pg. of selenium per 100 ml. of blood. I n a recent study of trace element levels in blood of residents of the United States (Allaway et al., 19671, 210 donors from 19 different locations had an average level of 2 1 pg. of selenium per 100 ml. of blood. Eight of these 210 donors had blood selenium levels less than 13 pg. per 100 ml., with none less than 10 pg. per 100 ml. Thus it would appear that the blood selenium levels of residents of the United States are nearly always in excess of those exhibited by malnourished children. There was a distinct geographic pattern in the blood selenium levels of residents of the United States with those donors from Rapid City, South Dakota, in a “high-selenium area,” showing an average level of 26 pg. of selenium per 100 ml. of blood. Even the Rapid City residents showed blood selenium levels less than one-fifth of those exhibited by sheep suffering from selenium toxicity. So, by extrapolation from animals to humans, selenium toxicity in humans would probably be extremely rare in the United States. It may, then, be quite desirable to control the selenium concentration in food and feed crops at levels somewhere between 0.1 and 1.0 ppm. of selenium. Such a control would almost certainly increase the efficiency of animal and poultry production in some areas, and the possibility of

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benefit to humans may be greater than the danger to humans. In the United States, attempts to increase the concentration of selenium in plants of the low-selenium areas would, however, require reevaluation of the legal restrictions against additions of selenium to the food supply. In a recent survey (Kubota et al., 1967a) of selenium in crops in the United States, about 44% of the forage crop and wheat (grain) samples analyzed contained between 0.1 and 1.0 ppm. of selenium, 20% contained more than 1 ppm., and 36% contained less than 0.1 ppm. There were 1788 samples of these crops considered in this survey. The problem of adjusting selenium levels in crops in the United States to the 0. I to 1.O ppm. range is, thus, one of increasing the selenium levels in part of the crops produced and decreasing the selenium levels in another part. Even though the desired range of selenium concentrations in plants appears to be narrow, there is reason to believe that the problems of increasing the concentration in low-selenium plants to a level within this desired range may be surmountable. In fact, this has been done in the field under experimental conditions both in New Zealand (Hartley, 1967) and in the United States (Allaway et al., 1966). In both cases, addition of selenium to low-selenium soils resulted in increased selenium concentrations in the forage and in improved animal health. However, in both cases the first harvests following selenium applications contained selenium concentrations near to, or perhaps above, the minimum levels that might cause some toxicity symptoms in animals. In the United States, soils that produce plants containing less than 0.1 ppm. of selenium tend to occur in board areas, and within these broad areas a high percentage (+80%) of the area is occupied by low-selenium soils (Kubota er al., 1967a). Therefore, if practical methods for using soil applications of selenium to increase the selenium concentration in crops produced can be developed, the areas where these practices will be of value are already substantially identified, and it may be possible to develop recommendations applicable to nearly all the farms in these areas. This may simplify the organization of technical assistance programs needed to guide the on-farm application of selenium additions. Plant species differences in respect to accumulation of selenium from low-selenium soils have been investigated by Davies and Watkinson (1966) and by Ehlig et af. (1968). Although plant species differences in selenium accumulation may be of nutritional importance when plants are near the margin of selenium adequacy or toxicity to animals, these species differences do not appear to present a serious obstacle to the use of selenium additions to soils that naturally produce low-selenium plants. If the concentration of selenium in one crop species is adjusted to a level

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of approximately 0.5 ppm. of selenium by means of selenium fertilizers, it is very unlikely that any other crop that might be used on the same field will contain a toxic level of selenium. It is also unlikely that any crop species will contain inadequate concentrations of selenium when grown on this field. One of the major obstacles to the use of selenium fertilizers on soils that produce low-selenium crops is the current imperfect knowledge of the chemistry of selenium in soils. In most experiments where selenium additions to low-selenium soils have been followed by repeated measurements of selenium untake by plants, it appears that over 90 percent of the added selenium remains in the soil even after two or three years of continuous cropping and plant removal. A rather precise knowledge of the nature of the residual selenium, and of the reactions which might change it availability to plants, is essential to any confident use of selenium fertilizers. The studies of Cary et al. (1967) indicate that an adsorbed or isotopically exchangeable form of selenite is a major form of residual selenium in selenium-treated acid, and neutral soils. Some form of hydrous iron oxide has been suggested as the adsorbing solid phase. The rates of equilibrium of soluble selenite with the soil adsorbent and the availability of the resulting adsorbed selenite to plants differ in different soils. The reasons for these differences are obscure. Elemental selenium also appears to be one of the forms of residual selenium in selenium-treated soils. Although elemental selenium is theoretically unstable in aerated soils, the rate at which elemental selenium is oxidized varies in different soils. In some soils, added elemental selenium is converted to forms available to plants at a rate that is adequate to supply plants with sufficient selenium to meet the needs of the animals that eat these plants (Watkinson and Davies, 1967; E. E. Cary, U.S. Plant, Soil and Nutrition Laboratory, Ithaca, New York, unpublished). Factors influencing the rate of oxidation of elemental selenium in soils and the reasons for differences in the stability of elemental selenium in different soils are not well understood at present. The problem of decreasing the selenium concentration in plants growing on soils that naturally produce crops of toxic or near-toxic concentrations of this element is quite formidable. Many of these soils are alkaline, and under alkaline, well-aerated conditions, the selenium in the soil can be expected to become oxidized to selenates. Selenates are not strongly adsorbed or fixed in alkaline soils and they are generally quite available to plants. Ravikovitch and Margolin ( 1959) have shown that additions of barium chloride to a high-selenium soil result in decreased selenium con-

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centrations in plants. The effect is presumably due to the formation of sparingly soluble barium selenates in the soil. Although theoretically attractive, the field use of barium chloride to reduce the plant availability of soil selenium may be practical only on certain potentially, highly valuable agricultural lands. The use of sulfate additions to provide an ion that will compete with selenates in the plant uptake process has, as mentioned earlier, not been effective on the seleniferous soils of the United States. Soil selection, crop selection, and crop management practices are currently used in meeting the high selenium problem in the United States. A group of plants called “selenium accumulators” are often a major part of the vegetation on highly seleniferous areas (Rosenfeld and Beath, 1964). The occurrence of these plants is a certain indication of danger to grazing livestock and provides a convenient means for identifying areas to be avoided in the movement and distribution of range livestock. Where these selenium-accumulator plants occur in mixed communities with desirable range grasses, the grazing practices are adjusted insofar as possible to utilize the grasses but not the accumulators. Food and feed crops produced on seleniferous areas are usually blended with similar crops from areas low in selenium, and may become valuable sources of selenium in the mixed feeds imported into low-selenium areas. In the United States, the very localized occurrence of high-selenium crop land and the system of centralized milling and feed mixing plants leads almost automatically to the necessary dilution of high-selenium crops. It is possible, however, that these high-selenium crops could be more effectively used if a scheme providing for deliberate monitoring of the selenium levels in food and feed crops, coupled with controlled blending of crops from high-selenium areas with those from selenium-deficient areas, were developed. To summarize, it appears quite possible that improved controls over selenium concentration of plants may become generally recognized as desirable in the fairly near future. Any attempt to control selenium concentrations in plants by agronomic practices must be considered premature at this time, due to unresolved technical and legal problems. However, the possibilities for developing the required agronomic practices, and the potential benefits from their use, are sufficiently attractive to merit future research.

I. VANADIUM From the standpoint of human nutrition, recent interest in vanadium stems from the possibility that this element may be useful in controlling

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levels of cholesterol in the blood, and it may also have a role in the prevention of dental caries. Research leading to indications of these roles for vanadium has been reviewed by Hudson (1964), Shroeder et al. 1963c), and Soremark (1967). Although vanadium is toxic to man and animals, there is uncertainty concerning the dietary concentrations required to cause toxicity in different species. Shroeder and Balassa ( 1967) consider vanadium to be of low toxicity. Mice have been maintained for long periods with 5 ppm. of vanadium (added as vanadyl sulfate) in their drinking water. The basal diet used contained 1.4 ppm. vanadium for part of this period and 3.2 ppm. for the remainder. These mice showed no ill effects due to vanadium (Shroeder and Balassa, 1967). Hathcock et al. (1964) found that 25 ppm. dietary vanadium, either in the form of vanadyl sulfate or ammonium vanadate, was toxic to chicks. The concentrations of vanadium in foods have been reported by Shroeder et al. ( I 9 6 3 4 and by Soremark (1967). Most foods contain less than 1 ppm. of vanadium. Processed fats are higher in vanadium than most other foods. Animals tend to concentrate vanadium in their bones, and thus meat does not appear to be a good source of vanadium. On the basis of the data reported by Shroeder et al., and by Soremark, it appears that the danger of vanadium toxicity to humans from food supplies is slight. However, Cannon (1963) has shown that certain plant species are vanadium accumulators and may contain concentrations of this element that would be toxic to grazing livestock. These vanadium accumulators are generally shrubs native to semiarid regions, although some mushrooms and mosses also appear to be vanadium accumulators. Cannon ( 1 963) reports some evidence to indicate an inverse relationship between calcium and vanadium in plants, and that vanadium may substitute for molybdenum in the nitrogen fixation process in low-calcium leguminous plants. The discovery of the essential role of vanadium for certain blue-green algae has been described by Arnon (1958). The response of these algae to vanadium is dependent upon light intensity. Vanadium will not replace molybdenum in nitrogen fixation by Anabaena, but earlier reports indicate that vanadium will replace molybdenum in nitrogen fixation by Azotobacter. The uptake of vanadium by higher plants has been studied by Cannon ( 1 963) and Soremark ( 1 967), and earlier work on stimulatory and toxic effects of vanadium in plants has been reviewed by Pratt (1966b). In all plant species studied, vanadium added to the soil was taken up by plants. Some stimulatory effects of vanadium on higher plants have been noted,

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but these have been very erratic and may have been due to indirect effects upon soil microorganisms. When very high levels of vanadium are added to soils or culture solutions, plant growth is depressed. Under these conditions, high concentrations of vanadium are found in the roots, but the tops of the plants usually contain vanadium concentrations less than those likely to be toxic to animals that eat these plants. Thus the soil-plant system may provide animals and humans with protection from vanadium toxicity. It seems possible, on the basis of the meager information on the environmental cycling of vanadium, that control over the movement of vanadium from soils to plants might be beneficial for the protection of humans from dental decay and from elevated levels of blood cholesterol. The danger of vanadium toxicity does not appear to be serious. An increase in the level of available vanadium in soils might increase the activity of certain nitrogen-fixing microorganisms. Information on how to control the availability of vanadium in soils and the uptake of this element by food plants is deficient. Research designed to remedy this deficiency may prove to be worthwhile. J.

ZINC

One of the earliest trace elements to be recognized as essential to plants and animals was zinc, and yet concern over deficiencies of zinc in plants, animals, and man is probably greater now that at any former period. Many of the reasons for this current concern have been discussed at a recent symposium (Prasad, 1966). These may be summarized as follows: ( 1 ) Zinc deficiency in plants is becoming more widespread, and it seems likely that the concentration of zinc in food and feed crops is, on the average, declining. (2) The use of galvanized metals in plumbing systems is becoming less common, and a decreased contribution of zinc from metals to the food and water supply is probable. (3) Plant proteins in human foods are coming into wider use as a means of alleviating protein deficiencies, especially in underdeveloped countries. Zinc is less available to animals from diets containing plant protein than it is from diets based on animal protein. (4)As mentioned earlier, increased levels of zinc in human diets may be beneficial in counteracting detrimental effects of cadmium. ( 5 ) Zinc deficiencies in a number of species, including man, have been observed under so-called “field” or “practical” conditions during the past ten years. Impaired healing of wounds in humans has also been attributed to zinc deficiency. Since the danger of toxicity from excess dietary zinc is minimal, it would seem evident that a general increase in the concentration of zinc

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in human and animal foods would be desirable. It is not at all certain, however, that an increase in the soil-to-plant movement of zinc to levels in excess of the amounts needed to sustain optimum crop yields is a feasible method of increasing the level of zinc in human diets. The zinc nutrition of plants has been recently reviewed by Viets ( I 966) and Chapman ( 1 96613). Although the zinc concentration in plants often reflects the level of available zinc in the soil, the changes in zinc concentration in plants brought about by the use of zinc fertilizers are more evident in the leaves than in the seeds. In fact, Viets reports cases where the yields of field beans were substantially increased by use of zinc sprays or fertilizers, with no change in the zinc content of the seeds. Since seeds and fruits are major sources of plant material in human diets, it may be that a very substantial increase in the levels of available zinc in soils would have only a slight effect upon the level of zinc in human diets. The low availability of the zinc in plants to the animal that eats these plants constitutes another barrier to improvement of the zinc status of humans by use of zinc fertilizers. The low availability to animals of zinc in plants has been attributed to the presence of insoluble complexes of zinc with calcium and phytic acid in plants (Oberleas et al., 1966). In animal proteins, phytate is not present, and the zinc in diets based on animal protein is more digestible and effective in meeting the zinc requirements of animals. The use of high levels of animal protein in human diets undoubtedly is of great value in protecting people from zinc deficiency in countries where adequate supplies of animal protein are available. If agronomic practices are to have a substantial impact upon the zinc status of humans, it would appear that research efforts designed to increase the concentration and availability to animals of zinc in seeds and fruits should be emphasized. There may be some important food plants in which the zinc concentration of the edible portion can be substantially increased by the use of zinc fertilizers. Chelated sources of zinc in fertilizers may be more effective than ionic sources of zinc in controlling the zinc content of seeds and fruits. The timing of zinc application to the soil or plant may offer opportunity for selectively increasing the zinc content of certain parts of the plant. Another substantially unknown area concerns the effect of zinc concentration in plants upon the availability of zinc to animals. If zinc concentrations can be increased without concurrent increases in calcium and phytate in plants, the increased zinc may be much more available to animals. It would seem that there are attractive opportunities for agronomic research directed toward improvement of the zinc status of humans. Until

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this research has been done, however, it would appear that the use of zinc fertilizers should be directed toward providing the plant with adequate supplies of zinc to permit optimum growth, and that the supplementation or fortification of human diets with zinc salts or complexes will have to be used as a primary method of correcting zinc deficiencies in humans. V. Some Potential Future Trends

In the author’s opinion, the use of information on trace elements will become a routine part of the diagnostic techniques used by the medical profession. When a patient is examined by a diagnostician, a sample of some tissue or fluid, probably blood, will be taken and analyzed for a number of different trace elements. Then, by means of prescribed supplements and dietary recommendations, the patient will be adjusted to some “desired range” of blood or tissue concentration of each of these elements. At the present time, current research effort on the role of trace elements in human and animal nutrition is at a high level, as evidenced by the number of symposia and the number of scientific papers in this area. An increasing number of clinical tests of the efficacy of trace element supplementation in human disease problems are under way. The information developed in these experiments will be applied to human health problems as rapidly as techniques for making the necessary measurements become available to clinicians. Widespread availability to clinicians of the analytical techniques essential to the application of trace element information in problems of human health seems almost assured. Atomic absorption spectrophotometers are coming into use in clinical laboratories. Services providing quantitative measurement of very low concentrations of trace elements in biological specimens by neutron activation analysis are becoming more sensitive, more accurate, and more generally available to clinicians. The review edited by Morrison (1 965) describes current trends in the development of instrumental procedures for trace element analysis. In retrospect, each new advance in analytical chemistry has been followed by a surge of new information concerning the essentiality, function, or toxicity of certain elements in biological systems. Recent and current advances in atomic absorption spectroscopy, fluorometry, and neutron activation analysis are generating another new surge. Any attempt by physicians to adjust the levels of trace elements in the bodies of their patients will be most effective if trace element concentrations in the food supply are uniform or standardized for each major type of food. In some cases, it may be possible to control trace element levels in food, by agronomic practices, to the point where most people will

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normally have the desired or ideal body burden of these elements. In other cases, the trace element levels in food plants must become an unvarying base upon which supplements or injections can be superimposed with confidence that overdosage is unlikely. From this point of view, agronomic work with trace elements may show the following. 1. Agronomists must keep informed on the progress of medical and nutritional research with trace elements, and as nutritionists and physicians establish desired ranges of trace element intake by humans, agronomists must develop soil and plant management practices to accommodate to these desired ranges. 2. Where the desired level of a given trace element in food plants exceeds the level required for optimum plant growth, agronomists, nutritionists, public health officials, and others must jointly decide whether the soil-to-plant route, or some alternate route, or both, should be used to supply human requirements for this element. 3. Agronomists must develop soil and plant practices that will provide both the desired concentrations of trace elements in food and feed crops, and a high degree of uniformity of these concentrations in almost all of the major food and feed crops produced. Agronomists must also guide the on-farm application of these practices, and assist with development of necessary procedures for monitoring trace element concentrations in foods. It is difficult to predict the exact routes that may be followed toward these objectives and to establish a timetable for progress toward effective control over the environmental cycling of trace elements. A t the very least, it would appear that the efforts of agronomists to develop procedures that will control the trace element content of plants will contribute to the improvement of human health. REFERENCES Allaway, W. H., Moore, D. P., Oldfield, J . E., and Muth, 0. H. 1966. J . Nutr. 88,4 I 1-4 18. Allaway, W. H., Kubota, J . , Losee, F. L., and Roth, M. 1967. Arch. Environ. Health 16, 342-348. Anderson, A . J . 1956. Advan. Agron. 8, 163-202. Anderson, M. S. 1961. U.S. Dept. Agr., Agr. Handbook 200,53-55. Anderson, M. S., Lakin, H . W., Beeson, K. C., Smith, F. F., and Thacker, E. 1961. U S . Dept. Agr., Agr. Handbook 200. Anderson, R. J . 1966. Brit. Dental J . 120, 271-274. Arnon, D. 1. 1958. In “Trace Elements” ( G . A . Lamb, 0. H. Bentley, and J. M. Beattie, eds.), pp. 1-32. Academic Press, New York. Bear, F. E. (ed.). 1956. Soil Sci. 83, 159-258.

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Beeson, K. C., and MacDonald, H. A. 1951. Agron. J. 43, 589-593. Berger, K. C., and Pratt, P. F. 1963. In “Fertilizer Technology and Usage” (M. H. MacVicker, G . L. Bridger, and L. B. Nelson, eds.), pp. 278-340. Soil Sci. SOC.Am., Madison, Wisconsin. Bollard, E. G.,and Butler, G. W. 1966.Ann. Rev. Plant Physiol. 17,77-112. Bowen, H. J. M. 1963. Atomic Energy Res. Estab. R 4196, H.M. Stationary Office, London. Bowen, H. J. M. 1966. “Trace Elements in Biochemistry.” Academic Press, New York. Brewer, R. F. 1966a. In “Diagnostic Criteria for Plants and Soils” (H. G . Chapman, ed.), pp. 180-1 96. Univ. California, Div. of Agr. Sci., Riverside, California. Brewer, R. F. 1966b. In “Diagnostic Criteria for Plants and Soils” (H. G . Chapman, ed.), pp. 2 13-2 17. Univ. California, Div. of Agr. Sci., Riverside, California. Brown, J . C. 1961. Advan. Agron. 13,329-369. Burk, R. F., Pearson, W. N., Wood, R. F., and Viteri, F. 1967. Am. J. Clin. Nutr. 20, 723-773. Burleson, C. A., and Page, N. R. 1967. Soil Sci. Soc. Am. Proc. 31,5 10-5 13. Butler, G. W., and Johns, A. T . 196I . J.Australian Inst. Agr. Sci. 27,123- 133. Cannon, H. L. 1963. Soil Sci. 96, 196-204. Cannon, H. L., and Bowles, J. M. 1962. Science 137,765-766. Carroll, R. L. 1966.J.Arn. Med. Assoc. 198,267-269. Cary, E. E., Wiezorek, G. A,, and Allaway, W. H. 1967. Soil Sci. Soc.Am. Proc. 31,2 1-26. Chapman, H. G., ed. 1966a. “Diagnostic Criteria for Plants and Soils.” Univ. California, Div. of Agr. Sci., Riverside, California. Chapman, H. G . 1966b. In “Diagnostic Criteria for Plants and Soils” (H. G. Chapman, ed.), pp. 484-499. Univ. California, Div. of Agr. Sci., Riverside, California. Davies, E. B., and Watkinson, J. H. 1966. New Zealand J. Agr. Res. 9, 317-327. Duggan, R. E., and Weatherwax, J. R. 1967. Science 157, 1006-1010. Dye, W. B.,and O’Harra, J. L. 1959. Bull. Univ. Nevada, Agr. Expt. Sta. 208. Ehlig, C. F., Allaway, W. H., Cary, E. E., and Kubota, J. 1968.Agron. J . 60,43-47. Feldman, F. J., and Knoblock, E. C. 1967.Anal. Chirn. Acta 38,489-497. Frost, D. V. 1965. Worlds Poultry Sci. J . 21, 139-156. Gahhiani. G . . Bais. D., and Deziel, C . 1967. Canurl. J. Physiol. Phormucol. 45, 443-450. Gerloff, G . C. 1963. Ann. Rev. Plant Physiol. 14, 107-124. Goldschmidt, V. M. 1954. “Geochemistry” (A. Muir, ed.). Oxford Univ. Press (Clarendon), Oxford. Goldsmith, J. R., and Hexter, A. C. 1967. Science 158, 132-1 34. Gray, L. F . , and Daniel, L. 1964. J. Nutr. 84, 3 1-37. Ham, J. R., Bone, J. F., Tinsley, I. J., Weswig, P. H., and Yamarnoto, R. S. 1967. In “Selenium in Biomedicine” (0.H. Muth, ed.) pp. 153-178. A.V.I. Publ., Westport, Connecticut. Hartley, W. J. 1967. In “Selenium in Biomedicine” (0.H. Muth, ed.), pp. 79-96. A.V.I. Publ., Westport, Connecticut. Hartmans, J.,and Van Der Grift, J. 1964. Inst. Biol. Scheck. OnderzoekLandbouwgewasser Wageringen Jaarboek pp. 145-155. Hathcock, J. N., Hill, C. R., and Matrone. G. 1964. J . Nutr. 82, 106-1 10. Healy, W. B., Ludwig, T. G., and Losee, F. L. 1961. Soil Sci. 92,359-366. Hegsted, D. M. 1967. Postgrad. Med. 41, A49-A53. Heywood, B. J. 1966. Science 152, 1408. Hodgson, J. F. 1963.Advan. Agron. 15, 119-159. Hopkins, L. L., and Majaj, A. S. 1967. I n ”Selenium in Biomedicine” (0.H. Muth, ed.), pp. 203-214. A.V.I. Publ., Westport, Connecticut.

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Hudson. T . G. F. 1964. “Vanadium: Toxicology and Biological Significance.” Elsevier, Amsterdam. Johnson. C. M. 1966. In ”Di:ignostic Criteria for Plants and Soils” (H. G . Chapman, ed.), pp. 286-30 I . Univ. California. Div. of Agr. Sci.. Berkeley, California. Kcenet. H. A . . Percival, C ; . P..and Morrow, K. S. 1954. Bull. New HampshireAgr. Expt. Sta., 411. Kubota, J . 1964. Soil Sci. Soc. Am. Proc. 28,246-25 1. Kubota, J., Lazar, V. A., Langan, L. N., and Beeson, K. C. I96 I . Soil Sci. SOC.Am. Proc. 25,227-232. Kubota, J., Lemon, E. R., and Allaway, W. H. 1963. Soil Sci. SOC. Am. Proc. 27,679-683. Kubota, J., Allaway, W. H., Carter, D . L., Cary, E. E., and Lazar, V. A. 1967a. J . Agr. Food Chem. 15,448-453. Kubota, J., Lazar, V. A., Simonsen, G . H., and Hill, W. W. 1967b. Soil Sci. Soc. Am. Proc. 31,667-67 1. Lakin, H. W. 1961. U . S . Dept. Agr. A g r . Handbook 200, 27-37. Leach, R. M., Jr.. and Nesheim, M. C. 1963. J . Nutr. 81, 193-199. Liebig, G. J. 1966. I n “Diagnostic Criteria for Plants and Soils” (H. G. Chapman, ed.) pp. 13-23. Univ. California, Div. of Agr. Sci., Riverside, California. Loper, G. M., and Smith, D. 1961. Univ. WisconsinAgr. Expt. Sta., Res. Repr. 8. Ludwig, T. G., Healy, W. B., and Losee, F. L. 1960. Nature 186,695-696. Martin, J. P. 1966. In “Diagnostic Criteria for Plants and Soils” (H. G. Chapman, ed.), pp. 62-64. Univ. California, Div. of Agr. Sci., Riverside, California. Mason, K. E., and Young, J. 0. 1967. In “Selenium in Biomedicine” (0. H. Muth, ed.) pp. 383-394. A.V.I. Publ., Westport, Connecticut. Mertz, W. 1967. Federation Proc. 26, 186-193. Mitchell, R. L. 1964. In “Chemistry of the Soil” (F. E. Bears, ed.), 2nd ed., pp. 320-368. Reinhold, New York. Mitchell, R. L., and Reith, J. W. S. 1966. J . Sci. FoodAgr. 17,437-440. Morrison, G . H., ed. 1965. “Trace Analysis: Physical Methods.” Wiley (Interscience), New York. Muth, 0. H., ed. 1967. “Selenium in Biomedicine.” A.V.I. Publ., Westport, Connecticut. Nicholas, D. J. D. 1961. Ann. Rev. Plant Physiol. 12,63-90. Oberleas, D., Muhrer, M. E., and Odell, B. L. 1966. In “Zinc Metabolism’’ (A. S. Prasad, ed.), pp. 225-238. Thomas, Springfield, Illinois. Prasad, A. D., ed. 1966. “Zinc Metabolism.” Thomas, Springfield, Illinois. Pratt, P. F. 1966a. I n “Diagnostic Criteria for Plants and Soils” (H. G. Chapman, ed.), pp. 136- I4 I . Univ. California, Div. of Agr. Sci., Riverside, California. Pratt, P. F. 1966b. In “Diagnostic Criteria for Plants and Soils” (H. G. Chapman, ed.), pp. 480-483. Univ. California, Div. of Agr. Sci., Riverside. California. Purves, D. 1967. Plant Soil 26,380-381. Ravikovitch, S.,and Margolin, M. 1959. EmpireJ. Exptl.Agr. 27,235-240. Reuther, W., and Labdnauskas, C. K . 1966. In “Diagnostic Criteria for Plants and Soils” (H. G . Chapman, ed.), pp. 157-179. Univ. California, Div. of Agr. Sci., Riverside, California. Robinson, W. O., Edgington, G . , and Byers, H . G . 1935. U S .Dept. Agr. Tech. Bull. 471. Rosenfeld, I . , and Beath, 0. A. 1964. “Selenium.” Academic Press, New York. Russell, F. C., and Duncan, D. L. 1956. Commonwealth Bureau Animal Nutr. Bucksburn, Aberdeenshire, Scotland Tech. Comm. 15. Shroeder, H. A. 1965a. J . Chron. Diseases 18, 647-656. Shroeder, H . A. 196%. Life Sci. 4, 2057-2062.

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Shroeder, H. A., and Balassa, J . J . 1961. J . Chron. Diseases 14, 236-258. Shroeder, H. A., and Balassa, J . J. 1963. Science 140, 819. Shroeder, H. A., and Balassa, J. J . 1966. J . Chron. Diseases 19, 85-106. Shroeder, H. A., and Balassa, J. J. 1967. J . Nurr. 92, 245-252. Shroeder, H. A., and Buckman, J . 1967. Arch. Environ. Health 14, 693-697. Shroeder, H. A., Balassa, J . J . , and Tipton, 1. H. 1962. J . Chron. Diseases 15,941-964. Shroeder, H . A., Vinton, W. H. and Balassa, J . J . 1963a.J. Nurr. 80,39-47. Shoreder, H. A., Vinton, W. H., and Balassa, J . J. 1963b.J. Nutr. 80,48-54. Shroeder, H. A., Balassa, J. J . , and Tipton, I. H. 1963c. J . Chron. Diseases 16, 10471071. Shroeder, H . A., Nason, A. P., Tipton, I. H., and Balassa, J. J. 1967. J . Chron. Diseases 20, 179-210. Soremark, R . 1967. J . Nutr. 92,183- 190. Spencer, V. E., Reading, R. E., and Thran, L. W. 1958. Univ. Nevada Agr. Expr. Sta. Bull. 202. Thompson, J . N., and Scott, M. L. 1967. Proc. Cornell Nutr. Con$ 1967, pp. 130-136. Tinsley, 1. J., Harr, J. R., Bone, J. F., Weswig, P. H., and Yamamoto, R. S. 1967. I n “Selenium in Biomedicine” (0.H. Muth, ed.), pp. 141-152. A.V.I. Publ., Westport, Connecticut. Tipton, I. H., and Cook, M . J . 1963. Healrh Phys. 9, 103-145. Tso, T. C., Harley, N., and Alexander, L. T. 1966. Science 153,880-882. Underwood, E. J . 1962. “Trace Elements in Human and Animal Nutrition.” Academic Press, New York. Viets, F. G. 1962. J . Agr. Food Chrrn. 10, 174-178. Viets, F. G . 1966. I n “Zinc Metabolism” (A. S. Prasad, ed.), pp. 90-128. Thomas, Spring. field. Illinois. Volgarev, M. W., and Tscherkes, L. A. 1967. I n “Selenium in Biomedicine” (0.H. Muth, ed.)pp. 179-184. A.V.I. Publ., Westport, Connecticut. Vose, P. B. 1963. HerbageAbstr. 33, I-B. Wallace, A,, ed. 1962. “ A Decade of Synthetic Chelating Agents in Inorganic Plant Nlltrition.” Wallace, Los Angeles, California. Watkinson,J. H.,and Davies, E. B. 1967. New 2ealandJ.Agr. Res. 10,116-121. Wittwer, S . H., and Teubner, F. G . 1959.Ann. Rev. Plant Physiol. 10,13-32. Working Group on Lead Contamination. 1965. “Survey of Lead in the Atmosphere of Three Urban Communities.” Environ. Health Series: Air Pollution. U. S. Dept. of Health, Education and Welfare, Cincinnati, Ohio.

CARBOHYDRATE SYNTHESIS IN MAIZE Roy G. Creech The Pennsylvania State University University Park, Pennsylvania

Page 275 ................. 276 276 278 B. Recent Developments .................................................................... General Carbohydrate Content and Transformations ................. 302 during Kernel Development 304 Nature of the Carbohydrates .................................................... 304 A. Mono- and Oligosaccharides ........................................................... 306 B. Sugar Nucleotides ........... ................... C. Polysaccharides . ................. 308 Enzymes in Starch Synthesis ... ................. 3 10 3 I7 General Statement ................. 318 References ..........................................................................

1. 11. Genetic Mutations

111.

IV.

V. VI.

I. Introduction

The investigations of the effects of genetic mutations on the regulation of metabolic processes in maize promise to increase our understanding of gene action in higher plants. Maturing maize endosperm is especially appropriate for studies of the effects of genetic interactions on carbohydrate metabolism because of the many gene mutations available that influence carbohydrate properties, and because of the relatively high carbohydrate levels found in maize kernels. The goals of such studies have been to reveal the metabolic effects of such genes and to elucidate the metabolic processes themselves. This review will cover some of the gene mutations associated with carbohydrate changes in maize endosperm and will also present some of the current evidence on the nature of carbohydrate metabolism, especially starch synthesis, in maize endosperm. For more thorough reviews of general carbohydrate metabolism in both plants and animals, the reader is referred to articles by Whelan (196 l ) , Porter ( 1 962), Badenhuizen ( 1 963), Manners ( 1 962), Northcote (1964), Nordin and Kirkwood (1963, 275

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Akazawa ( 1 9 6 3 , Pazur ( I 9 6 3 , Hassid ( 1 967), and Caputto et al. ( I 967). For a discussion of genetic mechanisms and gene action the reader is referred to the review entitled “A Biochemical Approach to Corn Breeding” by Hageman et af. which appears in Volume 19, Advances in Agronomy ( 1967). II. Genetic Mutations

A. EARLYRESEARCH There are many genetic mutations in maize which have been shown to affect endosperm components. Several of these mutations have been shown to alter the type and quantity of carbohydrates, including starch, in the kernels. Collins (1909) described a waxy mutant, possessing the gene designated as wx, that was different from normal dent corn in sugar and starch content. Weatherwax ( 1 922) demonstrated by iodine staining that the starch in waxy endosperm consisted solely of a “rare” form of carbohydrate called “erythrodextrin,” currently known as amylopectin. This starch stained red with iodine, in contrast to “amylodextrin,” currently termed amylose, which stained blue. Bates et al. (1943) and Sprague et af. (1943) confirmed that waxy endosperm of maize contained starch consisting of nearly all amylopectin, a branched-chain polysaccharide. The presence of amylopectin in cereals was demonstrated previously by Parnell (1921). Other waxy mutants have been reported by Bear (1944) and Mangelsdorf (1947). The wx mutants produce starch that is practically all amylopectin (Bates et af., 1943). Andres and Bascialli (1941) reported two waxy mutations in a variety of Argentine flint that possessed a small amount of amylose. These waxy genes are allelic to the other series. It is now generally accepted that the starch granule is usually a mixture of two polysaccharides (Greenwood, 1956; Whelan, 1961). Amylopectin is the major component in most starches, ranging from 75 to 85%, and is of high molecular weight, in the magnitude of 1 X 10’. Amylopectin consists of chains of a - ~ -1-4) ( and a - ~ -1-6)-glucosidic ( linkages to form a ( treelike structure. Amylose is primarily linear with a - ~ -1-4)-linked glucose residues. The structure of amylopectin and amylose will be discussed in more detail in Section 111. Two sugary genes, designated sul and su2, were described by East and Hayes ( 1 9 1 1) and Eryster (1 934). Mangelsdorf ( 1947) reported two gene that also mutations, designated dull (du) and amylaceous sugary (warn), altered the carbohydrate properties of maize endosperm. Amylaceous sugary was later found to be allelic to the s u l gene on chromosome 4,

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which had been previously described by East and Hayes ( 19 1 1). Andrew et al. (1944) reported that the gene wx increased sugars and watersoluble polysaccharides (WSP) in a sul background and alone. Cameron ( 1 947) reported that the two genes, sul and du, interact with one another to increase the amylose content of endosperm starch to about 65%, compared to the amylose content of standard dent corn of about 25%. These two genes were also observed to increase the WSP fraction and decrease the total starch synthesized. Kramer and Whistler ( 1 949) established that the su2 gene increased the amylose content to 35%. Dvonch et al. ( I 95 1) and Dunn et al. (1 953) reported that the genes du, sul, and suz interact to increase the amylose content of starch to 77%; however, no net increase in absolute yield of amylose was obtained because of a net decrease in total endosperm starch. Cameron and Cole ( 1959) reported that the gene combinations sul du and sul su2 reduced the accumulation of starch, compared to suIalone. Vineyard and Bear (1 952) described a gene mutation, termed amyloseextender (ae), which was the first endosperm mutation isolated that substantially increased the amylose content without drastically decreasing the total starch content. The ae endosperm produces starch with about 60% amylose. Deatherage et ul. (1954) reported the development of a hybrid, using the ae mutation, that was high in total starch yield with more than 60% amylose. Kramer et al. ( 1 958), using various combinations of the genes ae, du, sul, suz, and wx, reported that specific genotypes possessed amylose contents that ranged from none to over 70%. Zuber et al. ( I 960) suggested that the range in amylose content of from about 54 to 7 1% between certain maize inbreds possessing the ae gene was due to modifier genes. As the amylose content increased the total starch content decreased. The gene shrunken-1, shl,is an endosperm mutation that reduces the total starch to a very significant degree, causing the formation of a collapsed or shrunken kernel. This mutation was first described by Hutchinson ( 1 92 1). Burnham (1 944) described a mutant with a phenotype somewhat similar to shrunken-1, which was shown by Mains (1949) to be a distinct mutation from sh,. This gene was designated shrunken-2, shz. Laughnan (1953) studied the effects of the genes sh2 and sul in the distribution of endosperm carbohydrate reserves. It was found that almost 20% of the dry weight of the shrunken kernels was composed of sugars, which was about a I0-fold increase over standard dent kernels. Most of the sugar consisted of sucrose, which was 16% of the dry weight. A corresponding decrease in starch was observed. Total carbohydrate

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production was lowest in the double recessive, shz sul.In comparison with suI kernels, the shPsuI kernels showed a decrease in the accumulation of WSP. Laughnan concluded that shz precedes sul in the synthesis of starch and WSP. This conclusion was based on the assumption that each gene controls a specific biochemical step in starch and WSP synthesis. Laughnan pointed out that by using additional mutations in specific combinations it should be possible to gain valuable information about starch synthesis in maize. Cameron and Teas (1953) have shown that two other mutations, brittle- 1 ( b t l )and brittle-2 (btr),reduce endosperm starch substantially without the accumulation of WSP. Both genes caused increases in reducing sugars and sucrose and reductions in total starch. B. RECENTDEVELOPMENTS

I . General Carbohydrate Changes Creech et al. ( 1963) and Creech ( 1 965) reported the effects of the gene mutations ae, du, sh2, suI,s@, su2, and wx, singly and in combination, on qualitative and quantitative changes in carbohydrates in maize endosperm during kernel development from 16 through 28 days after pollination. Part of these data are presented in Table I. The more interesting interactions will be discussed. Significant differences between genotypes and stages of kernel development were noted for all carbohydrate and dry matter analyses. The gene su66is a new mutation that is allelic to sul and has an opaque wrinkled phenotype. It is dominant to the standard allele sul. The shz suz genotype reported by Creech (1 965) was actually su66suz as verified by allele tests (R. G. Creech, unpublished data). The dry matter content of normal increased from 15.7% at 16 days to 43.8% at 28 days after pollination. All the recessive genotypes except suz and su2 wx (not shown here) were significantly less than normal at most stages of development. The genotypes that were extremely low in dry matter were shP1ae wx, du shz, shz sul, ae du sull ae du wx, ae sulwx, and ae suz wx. The other genotypes were intermediate between these and normal. It is important to keep these dry matter differences in mind when comparing the quantities of particular carbohydrates reported here as percentages of dry matter. The reducing sugars content of normal decreased from 9.4% at 16 days to 0.8% at 28 days. All other genotypes possessed about the same amount of reducing sugars as normal except shz, sul, ae wx, du wx,

CARBOHYDRATE SYNTHESIS IN MAIZE

279

sh2 SUI,ae du sul, ae du su2, and ae du wx, which were higher in reducing sugars than normal. Of these, shz, sul, ae du sul, and ae du su2 were exceptionally high, especially at the later stages of development. Sucrose content in normal decreased from 8.2% at 16 days to 2.2% at 28 days. The genotypes shz, du shp,sh2 sul,and ae du wx were exceptionally high in sucrose (7 to 10 times normal) at about all stages of kernel development. The genotypes ae, du, suI,ae du, ae suI,ae su2, du wx, su6'j suz, sul wx, ae sul su2, du sul wx, and du su2 wx had 2 to 4 times as much sucrose as normal. The genotypes ae wx, suI suz, ae du sul,ae du su2, ae suI wx, ae suz wx, and suIsu2 wx had 5 to 6 times as much sucrose as normal. The gene ae appeared to be completely epistatic to sul for sucrose content. The gene shz seemed to be partially epistatic to du, sul, and wx. The WSP content of normal was 3.7% at 16 days and 2.2% at 28 days. Apparently, WSP was not accumulating during kernel development. Significant increases over normal were noted for sul, du sul, suz, sul su2, sul wx, ae du suI,ae sul su2, du sul su2,du sul wx, du su2 wx, and suI suz wx. The gene sul was associated with a dramatic increase in WSP at all 4 stages of kernel development. The genes ae and shz were apparently epistatic or partially epistatic; however, this epistatic effect was partially bypassed in the triple recessives. The genes du, su2, and wx appeared to intensify the accumulation of WSP in combination with suland other genes. The SPgene appeared to produce only about one-third as much WSP in combination with su2 as did the suI allele. This corresponds to the fact that SPkernels are more normal in appearance than those of suI. The starch content of normal increased from 39.2% at 16 days to 73.4% at 28 days. Extreme starch reduction (about one-half or less of normal) was associated with the genotypes shp, suI, ae suI, du suI, du sh2, sh2 sul, ma, sul su2, sul wx, ae du wx, du su1 S U ~ du , sul wx, and suI su2 wx. I n general, kernels of genotypes high in sugars are low in starch. Total sugar content is the sum of the reducing sugars and sucrose contents. Total carbohydrates content is the sum of all the carbohydrates analyzed. It is important to note that these data do not include the other oligosaccharides that may have been present. These data were presented for reference. There seemed to be a general decrease in total sugar with kernel development. A general increase in total carbohydrates with kernel development was observed in all cases except those that were medium to high in sugars and low in WSP and starch. Symmetric correlation data for all variables at all 4 stages of kernel

TABLE I The Quantities of Various Carbohydrates" and Total Dry Matterb in Entire Kernels of Several Maize Genotypes at Four Stages of Development"*d Kernel age (days)

Reducing sugars (%)

Sucrose (%)

normal

16 20 24 28

9.4 2.4 1.6 0.8

ae

16 20 24 28

Genotype

drc

Total sugar

Total carbohydrates

t4 00

0

Dry matter (%)

(%)

WSP (%Y

8.2 3.5 2.6 2.2

17.6 5.9 4.8 3.0

3.7 2.8 2.8 2.2

39.2 66.2 69.2 73.4

60.5 74.9 76.1 78.6

15.7 27.1 37.2 43.8

8.6 4.8 3.1 1.9

21.9 13.9 8.3 7.4

30.6 18.7 11.4 9.4

5.7 4.2 3.7 4.4

20.8 37.6 48.9 49.3

57.2 60.5 64.0 62.9

18.4 26.0 34.0 37.5

16 20 24 28

8.8 4.8 2.8 1.3

15.5

10.5 6.1 6.7

24.2 15.3 9.0 8.0

4.1 2.7 2.4 1.9

25.1 44.6 56.5 59.9

53.4 62.6 67.9 69.8

16.2 25.6 33.5 38.9

16 20 24 28

6.9 4.9 4.4 3.6

21.4 29.9 24.9 22. I

28.3 34.8 29.4 25.7

5.6 4.4 2.4 5. I

22.3 18.4 19.6 21.9

56.1 57.6 51.4 52.8

16.8 20.3 22.9 26.3

16 20 24 28

9.2 5.4 3.6 3.9

16.5 10.2 9.5 4.4

25.7 15.6 13.1 8.3

14.3 22.8 28.5 24.2

23.3 28.0 29.2 35.4

65.3 66.5 70.8 69.6

19.9 25.6 30.5 37.6

Starch (%)

(%Y

.e

? 0

m m

8 3:

16 20 24 28

7.4 3.5 I .9 I .4

10.5 9.2 2.6 1.9

16.7 12.7 4.5 3.3

3.6 3. I 2.5 1.9

39.3 50.7 63.9 64.6

59.6 61.8 70.9 69.8

17.5 24.9 34.9 43.6

16 20 24 28

10.1

3.5 2.5 1.6

9.6 5.2 4.5 1.7

19.7 8.7 7.0 3.3

3.5 2.3 2.8 2.2

34.1 53.3 61.9 69.0

57.2 64.6 71.5 74.5

14.9 23.9 33.1 37.3

16 20 24 28

6.9 3.7 2.2 2.1

12.6 8.3 5.3 5.3

19.6 12.0 7.6 7.4

3.7 3.6 3.6 3.2

18.3 29.3 37.2 34.4

41.5 44.9 48.4 45.1

19.3 24.8 31.5 33.9

ae wx

16 20 24 28

6. I 3.8 3.9 3.2

23.8 23.2 17.9 12.3

29.9 27.0 22.4 15.4

4.2 4.6 5.6 4.6

19.7 26.6 37.1 39.5

53.9 58.2 64.9 59.5

18.3 23.5 25.0 28.3

du wx

16 20 24 28

7.3 4.1 3.8 3.0

32.8 19.9 15.4 12.5

59.6 66.4 64.7 69.5

21.1 25.7 30.4 34.8

16 20 24 28

8.9 8.1 7. I 5.7

5.5 12.2 11.4 11.6 5.0 4.9 4.6 4.9

21.3 34.3 37.9 45.4

shz

25.5 15.8 11.6 9.5 24.1 25.4 19.1 20.1

7.2 11.7 14.4 15.7

47.3 50.1 46.9 45.4

20.5 23.8 25.2 24.6

S U ~

33. I 33.5 27.8 24.5

~

(Continued)

TABLE 1. (Continued) Kernel age (days)

Reducing sugars (%)

Sucrose (%)

Total sugar (% )

WSP (%Y

Starch (%)

16 20 24 28

10.4 4.0 3.3 2.5

14.6 8.5 7.6 6.8

25. I 12.6 10.9 9.3

6.3 9.5 10.0 13.6

26.8 38.3 38.6 35.1

58.1 60.3 59.5 57.9

18.8 28.3 33.8 38.3

16

20 24 28

4.9 2.8 2.4 2.5

16.8 11.2 9.6 10.4

21.8 14. I 12.0 12.8

33.7 31.5 31.0 36.9

11.9 20.1 20.5 18.9

67.5 65.6 63.5 68.6

20.1 28.5 31.1 35.4

ae du sul

16 20 24 28

12.8 9.2 4.7 4.6

24.6 18.0 15.5 10.6

37.3 27.2 21.3 15.3

9.6 12.4 16.1 18.2

23.6 30.9 32.7 38.0

70.5 70.5 70.0 71.5

17.3 22.6 25.8 27.6

ae du wx

16 20 24 28

6.8

39.9 34.6 30.7 23.7

46.7 38.7 34.3 28.1

4.2 3.6 4.5 4.9

15.9 26.6 31.1 32.0

66.7 68.9 69.9 65.1

18.5 24.6 25.8 24.5

Genotype

su, su:

4.1

3.6 4.4

Total carbohydrates (%)'

Dry

matter (%)

TI

'

? 0

6

R

f

ae su, su2

ae su, wx

du su, wx

16 20 24 28

8.5 3.5 2.7 2.4

23.2 9.7 7.9 8.6

31.7 13.2 10.6 11.0

16 20 24 28

8.0 5.2 3.s 2.8

28.2 21.9 15.0

16 20 24 28

5.9 3.2 2.9 2.3

6.6 10.4 10.6 11.0

23.8 41.6 39.6 41.0

62.0 65.3 61.1 65.9

20.3 27.1 31.5 34. I

11.1

36.2 27.0 18.5 13.9

4.5 8.4 12.2 12.4

22.0 30.7 38.5 38.3

62.7 66.0 69.1 64.5

16.3 21.7 25.8 26.2

16.8 10.2 7.8 6.7

21.7 13.4 10.7 9.0

24.4 36. I 38.4 47.5

14.7 21.4 17.5 15.9

60.8 70.9 66.6 72.3

22.0 27.9 33.4 35.3

%

E0 4

P 4

h

du

S U ~w x

16 20 24 28

9.2 5.2 3.O 2.7

25.7 19.5 10.6 8.9

34.9 24.1 13.3 11.6

4.6 14.8 14.3 16.7

17.2 24.7 33.9 38.1

53.4 64.2 61.5 64.5

15.2 20.8 27.9 30.7

v)

<

5 X E v)

Golden Cross Bantam (su,) Sweet Corn

16 20 24 28

8. I 3.2 1.9 I .6

15.4 5.5 3.9 1.9

23.6 8.7 5.9 3.6

7.8 27.0 33.3 34.8

28.7 35.5 38.5 33.9

60.1 71.3 77.7 72.3

16.1 26.8 33.5 37.0 ~~

(Continued)

? I

2

2

TABLE I. (Continued)

Genotype Least significant difference, genotypes within ages 5% 1%

Kernel age (days)

Reducing sugars (%)

Sucrose (%)

Total sugar

(7%)

WSP (%Y

Starch (%)

Total carbohydrates

(%Y

Dry matter (%)

w

0 4

3.2 4.2

10.4 13.9

10.9 14.5

10.4 13.9

14.2 18.8

15.3 20.4

2.9 3.9 0

!a

Least significant differences, ages within genotypes 5% 1%

m

R

s 2.4 3.2

6.0 1.9

5.8 7.7

Percent of dry matter. Percent of fresh weight. Three replications. Adapted from Creech ( 1 965). WSP, water-soluble polysaccharides. ’Sum of weights of reducing sugar, sucrose, WSP, and starch/dry matter weight a

”

4.8 6.3

7.6 10. I

1.5 9.9

2.9 3.8

285

CARBOHYDRATE SYNTHESIS IN MAIZE

development for 3 1 genotypes are presented in Table 11. All correlations except one (alcohol insolubles/WSP) were highly significant. Total sugars, reducing sugars, and sucrose contents were negatively correlated with dry matter and starch contents. This indicated that these sugars are precursors of starch, which has been well established by other workers (see Section IV). A correlation value of -0.8 1 between sucrose content and AIS content indicated, as previous workers have shown, that one may obtain increases in sugar content by selecting types with low AIS. AIS determinations are relatively inexpensive as compared with sugar determinations. This is of value in sweet corn breeding. TABLE 11 Symmetric Correlation Matrix of Seven Variables" Expressed as Percentages on the Basis of Dry Weightsb Variable 1. 2. 3. 4. 5. 6. 7.

Dry matter Reducing sugar Sucrose Total sugars AIS"

WSP Starch

1

-0.76**' -0.62** -0.72** 0.73** 0.17** 0.56**

2

3

4

5

6

0.12*' 0.72**

-0.47**

0.46** -

4.60** 0.18** -0.45**

-

-0.81**

0.22** -0,58**

-0.87** 4.24** -0.61**

Individual data of 3 1 genotypes at four maturities in three replications ( N = 372). *Adapted from Creech (1965). *Significant at 5% level (r>O.l 1). **Significant at 1 % level (r>O.lS). Alcohol insolubles (80 percent ethyl alcohol). a

Creech and McArdle ( 1966) reported the results of analyses for carbohydrates in mature maize kernels of the same genotypes used in the earlier work (Creech, 1965), which is discussed above. In general, data for the carbohydrates in mature kernels were similar to the findings for 28-day-old kernels for all the genotypes investigated. Anderson et al. (1962) reported that high-amylose maize (ae) had more protein and oil and less endosperm starch than standard dent corn. Less starch in ae kernels is probably associated with the lower test weight of high-amylose kernels. The significance of the above data lies in the elucidation of the interactions between specific genes as reflected in the carbohydrate properties of the kernels. The gene shz apparently caused a block between the sugars and the polysaccharides. These data supported the findings of Laughnan (1 953). The suIgene is associated with a significant production

286

ROY G. CREECH

of WSP, as reported by Culpepper and Magoon (1924); however, the gene ae is epistatic to sul,which results in the absence of WSP in the double recessive ae suI. The nature of these interactions at the biochemical level is not known at this time. The data of Kramer et al. (1958),Zuber et al. (1958),Creech et al. ( 1 963), Creech (I 965) and of earlier workers, previously cited, on the roles of the genes ae, du, shz, sul, su2, and wx indicate that starch synthesis involves multiple systems and that the picture is indeed complex. The ae gene, in addition to changing the amylose content (Vineyard and Bear, 1952) also caused a substantial increase in sugars (Creech, 1965) and reduction in total starch (Kramer et al., 1958;Zuber et al., 1960; Creech et al., 1963;Creech, 1965;Creech and McArdle, 1966). In addition, ae combined with wx and du wx caused dramatic increases in sugars and reductions in starch. This supported the view that ae, du, and wx may be involved in separate pathways of starch synthesis. The amylose content data of Kramer et a/. (1 958),shown in Table 111, combined with the effects of ae and wx on sugars and polysaccharides contents (Creech, 1965),also suggested that the normal allele A e may be involved with the formation of branched-chain polysaccharides (amylopectin) and that the normal allele W x may be involved in the synthesis of straight-chain polysaccharides (amylose). Nelson and Rines (1 962) reached a similar conclusion concerning Wx. They reported that starch granules from wx endosperm were deficient in uridine diphosphoglucose (UDPG) transferase activity, which was present in starch granules of Wx endosperm. Since M’X endosperm lacked both amylose and starch granule-bound UDPG transferase activity, Nelson and Rines suggested that UDPG transferase is necessary for amylose synthesis. This enzyme catalyzes the transfer of glucose from UDPG to an a-~-1-4 linkage on the nonreducing end of the polysaccharide acceptor. Nelson and Tsai (1 964) later reported findings that indicated that 17 different waxy mutants transferred glucose to starch at about one-tenth the rate of similar preparations from kernels of nonwaxy maize (Table IV). The source of the activity was indicated to be in starch granules from the embryo and maternal tissue. The endosperm, which is the site for the major amount of starch synthesis, was apparently devoid of starch granule bound transferase activity. Badenhuizen and Chandorkar ( 1965) suggested that the low UDPG transferase in waxy maize starch granules may be due to the absence of straight-chain or amylose starch. Starch granules varying in amylose content were isolated and assayed for amylose content and UDPG transferase activity (Table V). Use was made of the natural increase in

CARBOHYDRATE SYNTHESIS IN MAIZE

TABLE 111 Amylose Content of Mature Kernels of 24 Genotypes of Maize" Gene combination

Amylose in starch (%)

normal ae

27 61 38 29 40 0 57 60 54 15 63 47 0 55 0

du SUI su2

wx ae du

ae suI

ae su2 ae wx du sul du S U ~ du wx su,

su2

SUI

wx

0

su2 wx ae du suI ae du su2 ae suI su2

41 48 54 13 73

ae sul wx du sui S

U ~

du

S U ~wx

0

du

S U ~w x

0 0

su1

su2 wx

'' Adapted from Kramer ef al. ( 1958).

TABLE IV The Release of A D P (Millimicromoles per Milligram of Starch Granules) from ADPG in Preparations of Starch Gtpnules of Waxy and Nonwaxy Kernels at 16 Days of Agen,* ~

~~

~

~

Preparation

Nonwaxy

Waxy

Whole kernels Kernels without embryos Embryos alone

29 26 84

3.4 2.4 128.0

Adapted from Nelson and Tsai (1964). Each value is the average of 4 determinations of 2 separate preparations.

287

288

ROY G. CREECH

amylose content during the development of the kernel. There seemed to be some association between amylose content and UDPG transferase activity in kernels of the two high amylose strains. TABLE V Starch Content and UDPG-Transferase Activity in Kernels of High-Amylose (ae) Cornn

Tissue Endosperm

Pericarp

Transferase activity (mpmoles UDPI 5 mg starch/l5 min.)

Post pollination time (days)

Var. 1026

Var. 1027

Var. 1026

Var. 1027

14 16 18 20 26 30 12 26

17.0 20.0 24.0 49.0 54.6 7.0 20.0

9.0 25.0 35.0 43.0 47.5 52.0 11.0 -

5 .O 10.0 38.0 50.0 70.0 36.5 52.5

18.0 40.0 30.0 65.0 80.0 50.0 -

Amylose (%)

-

Adapted from Badenhuizen and Chandorkar (1965).

2. Phytoglycogen The influence and interactions of the ae, du, sul, and wx mutations on the synthesis and structure of phytoglycogen in maize endosperm were reported by Black et al. (1 966). Phytoglycogen is a highly branched water-soluble polysaccharide that accumulates in sweet corn (sul) endosperm. A literature review related to the characterization of phytoglycogen has been presented in an article by Peat et al. ( I 956). Black and co-workers analyzed several mutant and normal kernels for phytoglycogen content (Table VI). Phytoglycogen was found in kernels of sul, du sul, du wx, sul wx, ae du sul, ae du wx, ae sul wx, and du sul wx. This was the first evidence that phytoglycogen is present in kernels of other genotypes besides ml, although Creech (1965) reported that WSP was accumulating in genotypes possessing the genes du and wx (see Table I). The ae gene appeared to be epistatic to sul in that no phytoglycogen was produced in the double recessive ae sul. It also sharply reduced the amount of phytoglycogen in combinations with du and wx. The results of periodate oxidation and P-amylolysis of the phytoglycogen isolated from kernels of specific genotypes are shown in Table VII. These data agreed very closely with those reported for phytoglycogen in

CARBOHYDRATE SYNTHESIS IN MAIZE

289

T A B L E VI The Quantities of Phytoglycogen in Entire Kernels of 15 Maize Genotypes at 24-Day Maturityusb Ph ytogl ycogen (%)

Genotype, homozygous

normal ae

0.0 0.0 0.0 24.9 0.0 0.0 0.0 0.0 17.9 1.9 19.0 6.6 Traced 7.3 27.9

du SUle

wx ae du ue suI ae wx du suI du wx SU1 wx ae du suI ae du wx ae suI wx du SU, wx

Least significant difference

0.05

= 3.0

Adapted from Black el al. (1966). *Three replications (three randomly selected ears). Percent of dry matter. Insufficient material for quantitative analysis. Golden Cross Bantam background.

TABLE VII The P-Amylolysis Limits and Mean Overall, Internal and External Chain Lengths of Phytoglycogen from Maize Mutants"** Genotype, homozygous

P - limits (%)

su 1

dU S U ~

wx ae du su, ue suI wx du S U , wx su1

I'

40.5 39.7

14.5

40.6

14.1 14. I

40.1 41.0 38.4

13.5 14.7 14.1

Adapted from Black et al. ( 1 966). of duplicate determinations.

" Mean

Mean chain length, glucose units Overall External Internal 8.4 8.1 8.2 7.9 8.5 7.9

6.1 6.0 5.9 5.6 6.2 6.2

2 90

ROY G. CREECH

sweet corn by Greenwood and Das Gupta (1 958) and indicate the lack of structural differences between phytoglycogen preparations from different genotypes. The interactions of ae with du suI and suI wx caused a significant decrease in phytoglycogen, but there were no apparent qualitative changes in phytoglycogen structure. The results of the assays for phytoglycogen, the phytoglycogenforming branching enzyme (previously reported by Lavintman and Krisman, 1964), Q-enzyme, and amylose degrading enzymes are summarized in Table V 111. Branching enzyme produces phytoglycogen from amylose and amylopectin. Q-enzyme produces amylopectin, but not TABLE VIII Interactions of Maize Endosperm Mutations on the Synthesis of Phytoglycogen and Related Enzymes in Various Genotypes".* Genotype, homozygous

Phytoglycogen

Branching enzyme

Q-enzyme

Degradative enzyme

normal ae du su I

wx ae suI du wx su, wx ae su, wx Adapted from Black er al. (1966). The symbol (+) denotes the presence of phytoglycogen or the corresponding enzyme ; (-) denotes its absence. a

phytoglycogen, from amylose. The degradative enzyme, which was later demonstrated to be an a-amylase (Jaynes, 1966), degrades amylose, amylopectin, and phytoglycogen to maltose and maltotriose. The branching enzyme was isolated from kernels of all the genotypes that possessed phytoglycogen. The genotypes normal, ae, and ae sul did not appear to contain either the branching enzyme or phytoglycogen. Q-enzyme and the degradative enzyme were present in kernels of all the genotypes investigated. The results obtained with du and wx were somewhat contradictory in that the branching enzyme was present but phytoglycogen was not detected; however, the double mutant du wx possessed both. It was suggested that either the branching enzyme concentration was too low in du and wx to produce a detectable amount of phytoglycogen or

CARBOHYDRATE SYNTHESIS IN MAIZE

29 1

that the degradative enzyme degraded the phytoglycogen as it was produced. The authors suggested that the presence of the phytoglycogen-forming enzyme in all the mutants in which phytoglycogen was found indicated that the enzyme may be involved in the in vivo synthesis of phytoglycogen and that it may be inducible. Erlander (1958) has proposed that phytoglycogen is synthesized before starch; partial debranching then occurs to yield arnylose and amylopectin. It would appear, however, that a substantial portion of the phytoglycogen may be produced from starch by the action of the branching enzyme (Black et al., 1966). 3 . Sugars

Jordan ( 1 965) and W. S. Jordan and R. G. Creech (unpublished data) conducted a series of paper chromatography separations of sugars in kernels of normal, ae, du, shz, sulrsu2,and wxgenotypes that were taken at several stages of kernel development and quick-frozen in liquid nitrogen in 1964. Thirteen double and 10 triple recessive combinations were also investigated. Only part of the data are presented in Table IX. The glucose and fructose contents between certain genotypes at 20 days post pollination were significantly different; however, the most striking difTABLE IX The Quantities of Sugars in Kernels of Several Genotypes of Maize at 20 Days after Pollination as Determined by Paper Chromatography"

Genotype

Glucose

Sugars (mg. sugar/g. dry weight)* Ratio,' Fructose fructose:glucose Sucrose

normal ae du

88.3 a b 89.8 ab 114.9 a 61.9 bc 53.5 c 75.2 bc 83.1 bc 54.8 c 55.9 c 51.6 c 71.5 bc

66.6 a b 58.4 a b 85.1 a 44.9 b 36.6 b 59.5 a b 63.1 a b 46.8 b 33.4 b 38.1 b 56.2 a

Sh2

su 1 SUI

wx ae du ae du su2 ae du wx ae suI suz

0.75 0.65 0.74 0.73 0.68 0.79 0.76 0.85 0.60 0.74 0.79

119.9 g 266.5 bc 224.6 bcde 343.2 a 169.2 defg 145.3 g 146.4 fg 2 16.5 cdf 233.1 bd 293.8 ab 250.6 b

Maltose 1.5 cd 1.8 cd 2.6 cd 1.5 cd 0.0 d 0.0 d 0.0 d 9.8 bc 19.2 a 4.2 bcd 11.3 b

From Jordan (1965). Data within carbohydrate classes followed by the same letter are not significantly different at the 5% level (Duncan's Multiple Range). 'Average ratios, fructose:glucose = 0.70. a Ir

292

ROY G . CREECH

ferences between mutants occurred with sucrose and maltose. There seemed to be a fructose:glucose ratio of about 0.7. The kernels of shz were high in sucrose as Laughnan (1 953) had previously reported. The sucrose contents were in general agreement with those reported by Creech (1965) for kernels at 20 days after pollination. The maltose data were striking in that ae du sue kernels were about 13 times higher in maltose than normal, indicating rather significant interactions between the 3 genes. The nature of these interactions is unknown: however, Jordan discussed possible roles of maltose in starch synthesis. It is also possible that the maltose is due to a-amylase action on starch and oligosaccharides (Jaynes, 1966). Jordan (1 965) also presented data on freshly harvested ears taken at 20 days in 1965 and found general agreement with the data shown in Table IX. Attempts were made in the latter study to stop activities of enzymes within 5 minutes after removing the ears from the plants.

4 . Starch Granules Kernels of standard dent corn and several recessive genotypes were studied at 12, 18, and 24 days after pollination to determine the changes effected by specific gene mutations (ae, btt, bt2, du,& h, 0 2 , shl, suam, sul, sue, and wx) on starch granule characteristics during development of the endosperm (Brown, 1966; R. P. Brown and R. G. Creech, unpublished data). Differences between genotypes were found for starch granule birefringence end-point temperature (BEPT), size, count per unit weight, iodine stain absorption, and granule X-ray diffraction patterns. Table X shows the BEPT data for starch granules of different genotypes at 12, 18, and 24 days post pollination. The genes floury-1 (R1), horny (h),and opaque-2 ( 0 2 )are genes that modify endosperm texture but appear not to be directly related to starch synthesis (Mertz et al., 1964). The BEPT between genotypes ranged from 68.3 to 72.7"C. for granules from 12-day kernels, 65.7 to 96.7"C. for granules from 18-day kernels, and 63.7 to 97.7"C. for granules from 24-day kernels. The genotypes were not significantly different in BEPT for 12-day granules but were very significantly different at 18 and 24 days. This evidence provided additional information that starch synthesis may be different in early and late stages of endosperm development. The genes effecting differences in starch granule structure did not appear to express their effects prior to 12 days after pollination, but these effects were measurable at 18 and 24 days. The ae, ae suI, ae su2, and ae wx starch granules exhibited higher BEPT than normal at both 18 and 24 days. These data are in agreement

293

CARBOHYDRATE SYNTHESIS IN MAIZE

TABLE X Mean Birefringence End-Point Temperatures (BEPT) ( " C . )for 23 Genotypes at 12, 18, and 24 Days after Pollination"

Genotype

12-day

normal ae bt, btP du

68.3 a 69.0 a 68.3 a 71.7 a 68.0 a 70.0 a 69.0 a 71.0 a 68.0 a 68.7 a 68.0 a 69.0 a 71.3 a 70.7 a 68.3 a 69.7 a 70.0 a 70.7 a 71.3 a 69.3 a 72.7 a 71.7 a 68.0 a

P1 h 02

shi SUam

su I su:! wx

ae du ae su, ae su2 ae wx du sh:! du S U I dU S U ~ du wx Shy

wx

SUI suy

BEPTh Kernel age 18-day 73.3 efg 96.7 a 73.0 fgh 70.7 ghi 72.7 fgh 74.3 def 73.0 fgh 73.0 fgh 72.3 fgh 65.7 k 67.3 j k 66.3 jk 73.3 efg 87.3 c 91.3 b 87.3 c 91.3 b 69.0 ij 7 I.O ghi 70.3 hi 75.5 de 7 I .O ghi 67.7 jk

24-day 70.3 efghi 97.7 a 69.3 fghij 68.3 hijkl 70.7 efgh 69.3 fghij 71.3 efgh 69.0 ghijk 71.3 efgh 65.7 ijklm 69.0 hijk 63.7 Im 74.3 de 73.3 defg 88.0 b 87.7 b 82.3 c 64.7 jklm 73.7 defg 63.3 rn 76.7 d 74.0 def 67.3 hijklm

Adapted from Brown ( 1966). within kernel age classes followed by the same letter are not significantly different at the 5 % level (Duncan's Multiple Range Test).

* Data

with the data for starch granules from mature kernels (Pfahler et al., 1957; Kramer et al., 1958) (Table XI). In Table X, it can be observed that the gene su2, alone and in combination with du, depressed the BEPT below normal a t 18 and 24 days: the maximum depression occurred at 24 days. Combinations of s h ~ with du and suz also possessed low BEPT. The granules from ae du, which were not significantly different from du and normal a t 12 and 24 days, showed a high BEPT a t 18 days. This suggested that the gene ae was effective in producing high BEPT starch between 12 and somewhere around 18 days but du masked o r inhibited the expression of ae after that time. These data strongly suggest that the mechanisms of starch granule deposition which influence BEPT vary as

2 94

ROY G. CREECH

the kernel develops and that these mechanisms are under genetic control. Brown was careful to determine that the starch from the endosperm was the same as the starch from intact kernels. TABLE XI Birefringence End-Point Temperature (BEPT) of Starch Granules from Mature Kernels of 14 Genotypes of Maize" Genotype normal ae du su I

suz wx

ae du a e su, a e sun a e wx du S U I

du

S U ~

du wx su, su2

BEPT ("C.)

69 92 69 65 58 71 71 80 87 83 72 56 74 66

~~

"Adapted from Kramer et a / . (1958).

The mean width of the starch granules from the same samples described above were determined by Brown (1966). Part of these data are shown in Table XII. As can be observed, there is a general increase in granule size with age except with sul. The su1 granules seemed to be about the same size at all three stages of development. The genes ae, htl, btz, du, PI, sh,, mam,and sul appeared to be associated with small granule size, which indicated that these mutations alter starch granule deposition. Wolf et al. (1964) reported that ae reduced the size of the granule and caused the development of granules abnormal in shape. Creech (1 965) also reported that the genotypes ae, du, sh2, and sul had smaller granules than normal in kernels at 28 days after pollination. Creech also reported that the ae and du kernels possessed granules that were abnormal in shape and that the granules from sulwere aggregated in clusters and some very small unstained (iodine) granules were present. Brown (1 966) also presented photomicrographs of all the genotypes that

CARBOHYDRATE SYNTHESIS IN MAIZE

295

he investigated and calculated the width:length ratios of starch granules of each genotype, which showed significant differences. TABLE XI1 Starch Granule Size Data for 13 Genotypes at Three Stages of Kernel Developmenta

Genotype normal ae bti btz

du

PI h 0 2

sh I SU"" su1

sup wx

Starch granule size Kernel age (days post pollination) 12-day 18-day 24-day Width Length Width Length Width Length 2.95 bc 2.85 c 3.25 ab 3.29 ab 3.22 ab 3.37 ab 3.57 a 3.23 ab 3.06 ab 2.78 c 2.87 c 3.34 ab 3.05 ab

3.33 bcd 3.13 d 3.57 abcd 3.73 abcd 3.57 abc 3.78 abcd 3.96 ab 3.50 abcd 3.44 bcd 3.17 cd 3.15 cd 3.67 abcd 3.36 bcd

5.65 b 5.05 c 3.80 fgh 3.65 gh 3.35 hi 4.56 cde 6.13 ab 6.49 a 4.68 cd 3.01 i 2.14 j 5.76 b 6.57 a

5.96 bc 5.78 c 4.17 hij 3.91 ijk 3.74 jk 4.82 efgh 6.54 ab 6.90 a 4.94 defg 3.37 k 2.56 1 6.64 a 7.02 a

7.99 bc 5.56 efgh 5.1 I ghi 5.1 1 ghi 5.19 fghi 7.11 d 8.96 a 7.34 cd 4.89 h 6.04 ef 3.06 j 7.68 cd 8.61 a b

8.53 bc 6.32 fg 5.64 ghi 5.62 ghi 5.98 gh 7.66 de 9.45 a 8.07 cd 5.28 hi 6.29 f 3.52 j 9.14 ab 9.41 ab

Adapted from Brown ( I 966); R. P. Brown and R. G . Creech (unpublished data). Data within size class within ages followed by the same letter are not significantly different at the 5% level (Duncan's Multiple Range Test). 'I

Brown (1 966) also presented data on the number of starch granules per unit weight of endosperm starch from kernels at 18 and 24 days post pollination for 23 maize genotypes. Only the data for the single recessives are shown in Table XIII. Significant differences in numbers of granules per unit weight were observed between maturities and between genotypes. The order of the number of starch granules per microgram of starch showed a general relationship to the order of granule width and length as shown in Table XII. Low counts per unit weight were related to large granule sizes as expected and high counts per unit weight were related to small granule sizes; however, an exception did occur. For example, sul suz granules were significantly smaller than du sul granules at 24 days but du sul had more granules per microgram of starch than sul suz. These differences suggested that starch granule densities varied between genotypes. Knowledge of the number of granules per unit weight may be important in consideration of the activities of starch granulebound enzyme systems.

296

ROY G. CREECH

Brown (1 966) determined the infrared absorption patterns of the starch granules of 25 genotypes at 24 days post pollination and found them to be similar. Apparently the functional groups are similar in the starch granules of normal and the mutant genotypes. TABLE XI11 The Estimated Number of Starch Granules per Microgram of Starch in Kernels at I8 and 24 Days Post Pollination for 13 Genotypes" Number of granules per pg. starchb Kernel age (days post pollination) 18-day 24-day

Genotype

normal ae bti bt,

6825 d 9309 cd 14749 cd 26473 b 44581 a 8244 d 5532 d 4219 d 1 1996 cd 32412 b 52721 a 8661 cd 7352 d

du

PI h 02

sh i SPm

SU I SUr

wx ~

2447 efg 5845 cdefg 9089 bc 9854 bc 7105 cdef 3765 defg 1647 g 2552 efg 1743 cde 6387 cdefg 19954 a 2997 defg 2313 fg _____~

_______

Adapted from Brown ( I 966). *Data within kernel ages followed by the same letter are not significantly different at the 5% level (Duncan's Multiple Range Test).

X-ray diffraction patterns of starch granules from kernels at IS and 24 days of normal and 23 mutants were obtained by Brown (1 966). The X-ray patterns were classified according to Zobel (1964). These data are presented in Table XIV. Some of the starch X-ray diffraction patterns as compiled by Zobel are presented in Table XV. The A pattern was shown by normal corn starch and the B pattern was shown by potato starch. Maize amylose was shown to be amorphous, while maize amylopectin produced an A pattern. This was added evidence that the crystalline make-up of starch granules is mostly due to the branched-chain fractions. The A patterns of su2 and du su2 kernels at 18 and 24 days post

2 97

CARBOHYDRATE SYNTHESIS IN MAIZE

T A B L E XIV X-Ray Diffraction Patterns of Commercial Maize Amylose, Maize Amylopectin, and Potato Starch and Starch Granules from Kernels of Different Genotypes at 18 and 24 Days after Pollination Classified as Similar to the A and B Patterns Described by Zobel ( I 964)"s" Kernel age Genotype or material Amylose Amylopec tin Potato starch normal ae br, btz du

PI h 02

sh I su I suz

wx ae ae ae ae du

du suI

dU

SUI

su2

wx shz

du su2 du wx Sh2

24-day

Amorphous

Amorphous

A B A B A A A A A A -

SUam

SLP

18-day

suy

wx

A AC A B B B B A A* A" A Z* Ab

A

B A B A A A A A A A A A A" A B B B B A AD A' A Zd A

Adapted from Brown (1966). Pattern determined from small amount of starch. Weak crystalline A pattern. "Classified as nearly amorphous. a

pollination were in conflict with the data of Dvonch et al. (1951) and Badenhuizen ( 1959), which indicated that starch preparations from mature kernels produced B patterns. A second starch sample from su2 kernels at 24 days post pollination (Creech, 1965) demonstrated an A pattern, also. The starch granules of su2 and du suz produced a less crystalline pattern than that of normal but were definitely of the A type.

ROY G . CREECH

298

TABLE XV Starch X-Ray Powder Diffraction Patterns as Compiled by Zobel (1964)

B"

A" d-Spacing, A 8.72 1.70 5.78 5.17 4.86 4.37 3.78 3.30 2.88

Int

20

d-Spacing, A 15.8 8.90 7.94 6.14

W-

10.1

W-

11.5

S

1.3

S

17.1

S-

18.2 20.3 23.5 27.0 31.0

m S

W+ W

Intb

20

m

5.59 9.93

WW-

m

5.16

S

4.54 4.00 3.70 3.38 2.60

W+

m

mW W

11.1 14.4

17.2 19.5 22.2 24.0 26.3 34.4

"The A and B patterns were given by corn and potato starches, respectively, at moisture contents of 10 percent. A CuK, radiation source was used. * Intensity scale: strong (s), medium (m), weak (w), less than (-), more than (+).

The S U ~granules may change from A to B during the development of the kernel to maturity. Hizukuri et al. (1961) report changes from B to A patterns for starch granules in germinating soybean seedings under controlled temperature. The genotype s~~~ suz produced an amorphous pattern. From these data it would appear gene mutations can influence the degree of crystalline makeup within the granules. The weak and broad diffraction peaks of starch from certain mutants suggested relatively unorganized structure as compared to that of normal. The B pattern was related to the gene ae and was observed for starch from kernels at both 18 and 24 days. In general, Brown (1966) and R. P. Brown and R. G . Creech (unpublished data) have shown that 12-day starch granules from kernels of several maize mutants and normal appear to be similar for BEPT, size and shape. During the kernel development after 12 days the specific mutant genes under study seemed to alter the formation of the starch granule. Additional investigations need to be made to establish whether different mechanisms of starch synthesis are in effect during early and late endosperm development.

5 . Ultrastructure In an electron microscope study of normal and several mutant types of corn endosperm, Williams ( 1966) found that the carbohydrate-storing

CARBOHYDRATE SYNTHESIS IN MAIZE

299

organelles of sul and ae suI varied morphologically from the organelles in normal, ae, and wx genotypes. At 20 days post pollination the normal organelles were observed to be about 5-8 p in diameter, contained one or a few starch granules, stroma, and stroma lamellae. These were limited by an intact membrane. The organelles of suI contained small starch granules 1-2 p in diameter that were surrounded by phytoglycogen. N o lamellae were observed. T h e organelles from suI were about 20 p in diameter and were limited by a labile membrane. Samples of ae sul contained what appeared to be two starch granule types and phytoglycogen was not observed as in suI.

6 . Pyrophosphorylases Tsai and Nelson (1966) reported that the maize mutant shz completely lacked adenosine diphosphoglucose (ADPG) pyrophosphorylase activity in both endosperm and embryo tissues (Table XVI). ADPG pyrophosphorylase activity was present in normal tissues. This indicated that starch synthesis in normal proceeds principally with ADPG as substrate and that ADPG is chiefly synthesized by ADPG pyrophosphorylase in maize tissues. These data also indicated that only limited amounts of starch are formed with UDPG a s substrate. Tsai and Nelson rule out the possibility that any major portion of starch synthesis proceeds by the route of glucose 1-phosphate (G-1-P) and starch phosphorylase. TABLE XVI Specific Activities of ADPG-Pyrophosphorylase, UDPG-Pyrophosphorylase, and Starch Phosphorylase in Embryo and Endosperm Preparations from shg and normal Maize, Measured as Incorporation of G l u ~ o s e - ' ~from C ADPG-I4C and UDPG-I4C in the Coupled Reactions of Pyrophosphorylase and Starch Granule-Bound Glucosyl Transferase"

Embryo Phosphorylase

Sh2

A DPG -pyroU DPG-pyroStarch

0 364 -

Incorporation (countslmin.) Endosperm normal shz normal 10 255 -

0 131 6

300 127 6

~~~~~

I'

Adapted from Tsai and Nelson ( 1966).

T h e ability of maize endosperm to synthesize ADPG, UDPG, cytidine diphosphoglucose (CDPG) and guanosine diphosphoglucose (GDPG) was investigated by Vidra (1967). Table XVII illustrates the results of

300

ROY G . CREECH

assays for ADPG-, UDPG-, CDPG-, and GDPG-pyrophosphorylase activities in 3 maize endosperm genotypes, normal, shz, and su2. One of the more interesting discoveries in this investigation by Vidra was that a relatively high amount of CDPG-pyrophosphorylase activity was TABLE XVII Pyrophosphorylase Activities in the 25 to 65 Percent Ammonium Sulfate Fraction of Kernels of 3 Maize Genotypes"

Genotype normal shr su2

ADPG

+ +

Types of pyrophosphorylase activity" UDPG CDPG

+

+ +

+ +

+

GDPG -

-

Adapted from Vidra ( 1 967). (+) Denotes presence of activity; (-) denotes absence. In all cases, UDPG-pyrophosphorylase activity was much greater than any of the others.

present in each genotype, which may mean that CDPG is important in carbohydrate metabolism in maize endosperm. CDPG has been primarily associated with animal and bacterial organisms, and only recently discovered in trace amounts in peas and parsley (Verachtert et al., 1964). The CDPG-pyrophosphorylase activity in maize endosperm was comparable to or somewhat higher than the ADPG-pyrophosphorylase activity and appeared to be independent. These data are shown in Table XVIII. Differences were noted between genotypes for the three pyrophosphorylase activities, which may contribute to the causes for the differences between mutants for amounts of specific carbohydrates (Creech, 1965). Vidra also suggested that these differences indicate that the pyrophosphorylases may function as key points of metabolic control in maize endosperm. The absence of ADPG-pyrophosphorylase activity in shz agreed with the findings of Tsai and Nelson (1966). Vidra presented evidence that the ADPG-, CDPG-, and UDPG-pyrophosphorylase activities were due to three separate enzymes (Vidra and Loerch, unpublished data).

7 . Starch Granule Digestibility Sandstedt et al. (1962) studied the pancreatic digestibility of maize starches compared to that of other starches. These data for the starches from different genotypes of maize are presented in Table XIX. The ae gene appeared to be associated with both high amylose (Vineyard and

30 1

CARBOHYDRATE SYNTHESIS IN MAIZE

TABLE XVlIl The Activities of Specific Pyrophosphorylases in Preparations of Maize Endosperm from Kernels at 20 Days Post Pollination from 3 Genotypes of Maize" Genotype* (nucleoside)

(x

Specific activity cpm./mg. protein)"

Total activity (cpm./5000 cpm. Rx. mix.)

normal ( A DPG) ~ h z (ADPG) S U ~ (ADPG)

18.9k 2.8 0.0 9 . 3 1 0.6

0 1 9 8 1 12

normal ( U D P G ) shr (UDPG) suz (UDPG)

279.5 k 11.5 158.91 7.6 175.7*1 1.2

2992 k 142 2 164& 28 1 21181372

normal (CDPG) shs (CDPG) S U ~ (CDPG)

l l . 7 k 1.7 14.1 5 2.5 17.95 2.1

l 9 6 i 32 2 7 6 1 53 2 5 4 1 20

1 5 8 k 26

Adapted from Vidra ( 1967). 'Average number of kernels/5 g. was: normal ( 3 9 , shZ (36) and suL,(45). k standard error. 'I

TABLE XIX A Comparison of the Susceptibility of Various Starches to Pancreatic Digestion" Starch source normal ae du SUI

SUL,

wx

ae du a e su2 du S U ~

Digestion (%) 69 24 80 71 88 85 42 22 76

"Adapted from Sandstedt et al. ( 1962).

Bear, 1952) and high resistance to enzyme action. Kramer el al. ( 1958) and Brown ( 1 966) reported that ae starch had a very high BEPT (Tables X and XI). However, the genes du and su2, though associated with higher

3 02

ROY G . CREECH

amylose than normal, seemed to be associated with high digestibility. Sandstedt and co-workers concluded that amylose was not associated with digestibility. They suggested that the answer may lie in the structure of the starch granule, i.e., in differences in the bonding of the starch molecules and/or in possible anomalous linkages between molecules. Brown (1966) suggested that su2 starch was less crystalline than normal starch, but this does not account for the results with wx. Note also that su2 starch granules had a lower BEPT than normal granules. Sandstedt has suggested that improving the digestibility of the starch of commercial strains of maize may be important in improving the nutritional value of corn, especially to nonruminants. 8. Other Mutations

In Weijer’s (1 952) catalog of maize genes, there are descriptions of several other endosperm mutations involving different genes from those previously discussed, and there are undoubtedly many such mutations that have never been reported or preserved. However, most of the research has been conducted with the ones described above and these studies have established that genetic variability exists for carbohydrate synthesis in maize endosperm. Ill. General Carbohydrate Content and Transformations during Kernel Development

Many experiments have been made on the type and quantity of carbohydrates in maize kernels. Much of the early work was conducted on open-pollinated varieties and was concerned with sugar contents during kernel development. Postharvest transformations in sweet corn kernels were also investigated extensively because of their significance in the palatability of the processed product. Straughn (1 907) reported that the sugar contents of mature kernels of different varieties of sweet corn were similar; however, he noted that the degree of wrinkling of the kernels was associated with the amount of sugar. Highly wrinkled kernels were slightly higher in sugar than less wrinkled kernels. The finding that the sugar content of corn in the edible stage was altered rapidly during the first few hours after removal of the ears from the plant was of special interest. Straughn and Church (1909) and Appleman and Arthur (1919) indicated that the loss in sugar described earlier was caused by both respiration and its transformation into polysaccharides, chiefly starch. Doty et al. (1945) conducted similar experiments on 39 inbred and hybrid strains of sweet corn over a four-year period. They concluded

CARBOHYDRATE SYNTHESIS IN MAIZE

303

that the genetic constitution of the plant affected the conversion of sucrose to polysaccharides during storage. Appleman and Eaton (1921) found a progressive decrease in total sugars, an increase in fat, and a very high increase in starch during the development of kernels from open-pollinations. Culpepper and Magoon ( 1 924, 1927) found that total sugars increased up to 15 days post pollination and then decreased slowly during the latter stages of kernel development. Reducing sugars were highest at the initial stages and decreased steadily throughout the growth and development period. Sucrose increased rapidly to about the fifteenth day and then decreased slowly during the next 15 days. In this case, sucrose comprised the great majority of the total sugars content. Total polysaccharides were observed to increase continuously during the development of the ear. The sweet corn varieties, unlike standard dent corn, contained a high water-soluble polysaccharides (WSP) content. Hassid (1 945) presented a review of the chemical structure and properties of the two components of starch, amylose and amylopectin, and glycogen (from animals). Parker (19 3 9 , Hassid and McCready ( 1 941), and Sumner and Somers (1943) described the water-soluble polysaccharide that had been observed in sweet corn. Although problems occurred in sample preparations (apparently degradative enzymes), they were able to characterize the WSP fraction of sweet corn and demonstrated that it possessed a branched structure similar to but more highly branched than amylopectin. This highly branched fraction was termed phytoglycogen because of its similarity to animal glycogen. Lampe and Meyers ( I 9 2 3 , Evans ( I 941), Andrew el al. ( 1944), Earley (1952), and Ingle et al. (1965) conducted studies on the changes which occur in the carbohydrate constituents and other constituents during kernel development. These findings were essentially the same as those reported by Culpepper and Magoon (1 924), even though the materials and techniques were different. These data indicated that there was a relationship between moisture content, reducing sugars, sucrose, starch, and total dry weight, which followed a definite pattern during kernel development. Moisture decreased constantly and starch and total dry weight showed a constant increase, beginning about 2 weeks after fertilization. Reducing sugars content was high in unfertilized ovules and remained high until about 10 days after fertilization, at which time it began to decrease slowly. Sucrose content decreased slightly immediately after fertilization and then increased rapidly, reaching its highest concentration about 18 days post fertilization. After 18 days sucrose began to constantly decrease, reaching a very low level in mature kernels. Earley ( I 952) demonstrated that starch was present in ovules prior to

3 04

ROY G . CREECH

fertilization. Bernstein (1943) suggested that the small amount of starch present in corn kernels 1 week post fertilization was in the pericarp. Peat e l al. (1956) extracted phytoglycogen from sweet corn kernels with 0.01 M mercuric chloride to inhibit carbohydrases likely to degrade phytoglycogen. When mercuric chloride was used the sugars present in the extract were glucose, fructose, sucrose, raffinose and a trace quantity of maltose. Without the inhibitor the fraction contained glucose, fructose, maltose, maltotriose, and higher maltodextrins, but no sucrose or raffinose. These results showed the importance of avoiding autolytic changes during extraction. Bond and Glass (1 963) identified seven low molecular weight carbohydrates at various stages of germination of maize kernels. Raffinose was present during the 2-day steeping period but disappeared after 24 hours in the germination chamber. Fructose, glucose, and sucrose were present throughout the experiment. The glucose concentration increased during germination to a level about 57 times that at the beginning, which was almost as great as sucrose. Myo-inositol and glycerol were present at the beginning but disappeared early during germination. Maltose was present after 48 hours. Additional studies on carbohydrate types and quantities were reported by Creech et al. (1963), Creech (1965), and Jordan (1965). These data were presented in Section 11. The hydrolysis of translocate sugar has recently been shown to be necessary prior to its movement into the storage cells of corn endosperm (J. C. Shannon, personal communication). This was shown by the relatively high proportion of kernel radioactivity in the monosaccharides at an early time after treatment of the whole plant with l4COz(Fig. 1). Hydrolysis appeared to occur in the “pedicel region.” One hour after 14C02 treatment started (treatment time 0 hours) almost all the I4C-sugar of the cob was in sucrose. However, in the endosperm it was in the monosaccharide fraction. The pedicel region contained radioactivity in both sucrose and the monosaccharides (Fig. 2). After entry into the endosperm tissue, sucrose was rapidly resynthesized from the monosaccharides prior to its utilization in starch synthesis.

+

IV. Nature of the Carbohydrates in Plants

A. MONO- A N D OLIGOSACCHARIDES The principal monosaccharides according to Axelrod (1 9 6 9 , are presented in Table XX. In general the monosaccharides do not occur free in

305

CARBOHYDRATE SYNTHESIS IN MAIZE

the plant. D-Glucose and D-fructose are notable exceptions, and they often occur in fairly high concentrations.

Normal Total

6(

' 4c

0 3 6

o/o of

12

24

36

Time (hours)

total

4(

2(

r

12 Time

24

36

a f t e r t r e a t m e n t (hours)

FIG. 1. The proportions of radioactivity in the total kernel and in the saccharides glucose, fructose, sucrose, and starch after treatment of the whole plant with T O 2 . Key to the symbols: G , glucose; F, fructose; S, sucrose; ST, starch.

The oligosaccharides are polymers formed by linking of several monosaccharide units. They are generally arbitrarily limited to molecules containing fewer than 10 units. Sucrose is the most abundant and widespread oligosaccharide among photosynthetic plants, including maize. Maltose is found in many plants, however, generally in small amounts. Its presence has generally been attributed to autolysis of starch or similar polysaccharides; however, there is a possibility that it may also be formed by transglucosidation. Generally, one expects to find along with maltose the higher oligosaccharides derived from starch by autolysis.

306

ROY G. CREECH

These oligosaccharides have been observed in maize (see previous sections). B. SUGARNUCLEOTIDES Uridine diphosphoglucose (UDPG) was first isolated from plants by Pedicel

Endosperm

Cob

100

A

Y S '4c

010 of sum

I

0

I

2

I I L J L 6 0 2 6 0 Time a f t e r t r e a t m e n t ( h o u r s )

I

L

2

6

FIG.2. 'The proportions of radioactivity in glucose (G), fructose (F), and sucrose (S) in the endosperm, pedicel, and cob after treatment of the whole plant with 14COr. TABLE XX The Principal Monosaccharides of Plants" Trioses D-GI yceraldehyde Dihydroxyacetone Tetroses D-Erythrose D-Erythruclose

Pentoses L- Arabinose D-Xylose D-Xylulose D-Ribose D-Ribulose D-Deoxydbose

" From Axedrod ( I 965).

H exoses D-Glucose D-FruCtOSe D-Mannose D-Galactose L-Sorbose L-Rhamnose L-Fucose Heptoses D-Sedoheptulose D-Mannoheptulose

CARBOHYDRATE SYNTHESIS IN MAIZE

3 07

Leloir and his group in 1950 (Caputto et al., 1950). This significant discovery opened the door to the vast amount of research and interest in the roles of sugar-linked nucleotides in the synthesis of the oligosaccharides and polysaccharides. Some of the sugar nucleotides commonly found in higher plants are listed in Table XXI. The plant contains sugar nucleotides other than UDPG, such as ADPG, GDPG, TDPG, and deoxyuridine-DPG. CDPG has been detected in bacteria (Okuda et al., 1964). TABLE XXI Some Sugar Nucleotides Found in Higher Plants'' ~

Sugar nucleotide ADP-glucose

AD P-galactose ADP-rnannose GDP-glucose GDP-mannose GDP-fucose TDP-glucose TDP-galactose TDP-rhamnose U DP-glucose

U DP-galactose UDP-rhamnose Deox yuridinedip hosphoglucose Deoxyuridinedip hosphogalactose

Reference Baddiley et a[. (1961) Recondo et al. ( I 963) Murata et al. (1963) Murata ei al. ( 1964a) Dankert et al. (1964) Dankert et al. ( 1 964) Elbein et al. (1964) Munch-Petersen (1956) Ginsburg (1960, 1961) Pazur and Shuey (1961) Neufeld ( 1962) Neufeld ( 1962) Caputto et al. ( 1950) Munch-Petersen et at. (1953) Munch-Petersen (1955) Kurahashi ( 1 957) Burma and Mortimer ( 1 956) Neufeld ef al. ( 1 957) Ginsburg (1958) Turner and Turner (1958) Ganguli ( 1958) Su (1965) Neufeld (1962) Neufeld (1962) Neufeld ( 1 962) Neufeld ( I 962)

"Modified from Axelrod ( 1965).

CDPG-pyrophosphorylase has been found in maize endosperm so it will not be surprising to find CDPG present also (Vidra, 1967; J. D. Vidra and J. D. Loerch, unpublished data).

308

ROY G . CREECH

C. POLYSACCHARIDES 1 . Starch Starch is the most abundant and important energy-yielding reserve polysaccharide in the plant world. It is the most important energy source for animals, including man. It is also an important raw material for numerous industrial products. In maize, starch is synthesized in the chloroplasts and in nonphotosynthetic organs, especially endosperm. The cellular organelle in which starch is synthesized is termed an amyloplast (Buttrose, 1962; Badenhuizen, 1959, 1963). Starch has been shown to comprise 86.4% of the endosperm, 8.2% of the germ, and 7.3% of the bran (mostly pericarp) in mature maize kernels (Earle et al., 1946). It has been established, however, that the starch type and content in maize kernels is under genetic control (see Sections II,A and 11,B). a. The Starch Granule. Over 250 years have elapsed since Leeuwenhoek observed starch granules with his primitive microscope (Kirchoff, 18 1 1). His impression that the starch granule possessed an outer membrane was later disproved; however, most of his observations were accurate. In early studies many workers considered starch as a homogeneous substance, and it was not until the 1930’s that these discrepancies were partially corrected. Badenhuizen (1 939) described the formation of starch granules by apposition. Starch granule enlargement was described as the deposition of starch molecules in layers on the surface of the granule. Meyer et al. (1940) were the first to show the complicated structure of starch by the discovery of two heterogeneous starch fractions, amylose and amylopectin, in maize kernels. Distribution of amylose and amylopectin within the starch granule has been an area of disagreement. Badenhuizen (1955a, 1963), in an experiment designed to elucidate the distribution of amylose, found that in most starches amylose appeared to be evenly distributed throughout the layers of the granules. In wx maize starch the amylose fraction, which comprised only a small amount, was localized in the center of the granule. Observations of starch granules with an intact system of layers led Badenhuizen (1955b) to describe each layer as possessing an inner part, which was crystalline, birefringent, and resistant to degradation, and an outer part, which was loosely packed, amorphous, and more susceptible to degradation. The structural concept proposed by Meyer (1942) pictured the amylose and amylopectin molecules intermingled in a radial fashion within the granule. This revived the classical micellar theory of Nageli (1 858). By

CARBOHYDRATE SYNTHESIS IN MAIZE

309

electron microscopy, the layered structure of starch granules in maize and other plants has been conclusively shown (Whistler et al., 1955; Buttrose, 1960). A concept of layer formation by the storing of starch molecules in a different manner alternately by day and night was supported by the findings of Bakhuyzen ( 1925). H e demonstrated that under constant environment the layers were not formed in the granules. Buttrose ( 1 960, 1962) confirmed this finding by electron microscopy studies of starch granule formation in barley and wheat endosperm grown under constant environment. Conflicting evidence was obtained in potato tubers, however (Roberts and Proctor, 1954; Buttrose, 1962). Layers were present in starch granules of potato tubers grown under constant environment. Bunning and Hess (1954) apparently observed the same thing in Pellionia and proposed that a diurnal endogenous enzyme rhythm was responsible. Badenhuizen and Dutton ( 1 956) and Yoshida et al. (1 958) found that when plants were exposed to I4CO2most of the radioactivity was on the peripheral layer of the newly synthesized granules. This supported the proposal that starch granules grow by apposition from an external source of glucose; however, it is not known how synthesis is regulated so that a discontinuous shell structure is formed. Starch granules from different sources exhibit differences in crystal structure as evidenced by different starch granule X-ray diffraction patterns, classified as A , B, and C types. Bear and French (1941) and Zobel ( 1 964) have discussed these patterns in reviews. Cereal grains, including normal and wx maize, yield the A pattern (Tables XIV and XV). Potato starch and some of the high amylose maize starches produce B patterns. The C pattern was exhibited by tapioca and horsechestnut starches. Starch granule structure was shown to be interconvertible by physical or chemical treatment to produce A, B, o r C patterns. For a review of the crystalline nature of starch, the reader is referred to Zaslow (1965). Starch granules exhibit a dark cross when viewed by polarized light. Meyer (1952) suggested that the cross was typical of spherical aggregates of radially oriented crystalline micelles. Badenhuizen ( 1 959) suggested that birefringence results from the parallel arrangement of the outer branched molecules. Neither of these suggestions has been established as fact. Starch granules from various species were shown to have different gelatinization temperatues (Schoch and Maywald, 1956). Initial and terminal gelatinization temperatures were recorded. Terminal gelatinization (birefringence end-point temperature) was 70" to 73°C. for

3 10

ROY G. CREECH

standard dent corn starch, 56” to 67°C. for potato starch, and 72°C. for waxy (wx) starch granules. Genetic effects were previously discussed in Section 11. For a thorough review of the occurrence and development of starch granules in plants the reader is referred to an article by Badenhuizen (1 965). b. The Characteristics of Amylose and Amylopectin. Amylose stains blue with iodine and has been shown to have an X-ray pattern indicative of a helical structure (Bear, 1942; Rundle and French, 1943). There is evidence that the helix contains 6 D-glucose residues per turn. This allows the iodine molecule to be located within the helix (previously suggested by Hanes, 1937; and Freudenberg et al., 1939). Amylopectin stains purple with iodine, not blue. It is a branched polymer and the currently accepted view is that it has the “bush” form proposed by Meyer and Bernfeld ( 1 940). Amylopectin resembles animal glycogen but is not as highly branched. The average number of D-glucose residues per branch is 25-26 (Potter and Hassid, 1951); however, individual chain-length studies are currently in progress.

2 . Phytoglycogen Several workers have described the characteristics of the WSP fraction in sweet corn (Culpepper and Magoon, 1924; Parker, 1935; Morris and Morris, 1939; Hassid and McCready, 1941; Sumner and Somers, 1943; Dvonch and Whistler, 1949; Peat et al., 1956). By careful isolation, using mercuric chloride to prevent enzyme activity, Greenwood and Das Gupta (1958) and Black et al. (1966) demonstrated that the WSP fraction consisted of one type of poiysaccharide, phytoglycogen. Previous workers had reported two types of branched molecules. The basal chain length was determined to be 14 glucose units by periodate oxidation. Phytoglycogen was found to differ from animal glycogen in iodine binding. The iodine uptake was demonstrated to be 3-4 times greater for phytoglycogen than for glycogen. The degree of branching was intermediate between glycogen and amylopectin. However, the molecule more closely resembles glycogen in chain length, enzyme degradation, and sedimentation. On the basis of sedimentation data, this fraction was determined to be practically homogeneous. V. Enzymes i n Starch Synthesis

Recent interest in starch and polysaccharide synthesis has centered around the role of sugar-linked nucleotides. After the discovery of

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glycogen synthetase (Leloir and Cardini, 1957) in liver tissue, Leloir and his colleagues subsequently discovered starch synthetase in bean, potato, and maize starches (DeFekete et al., 1960; Leloir et al., 1961). The starch granules with the bound enzyme were demonstrated to catalyze the transfer of labeled glucose from UDPG to the polysaccharide fraction as indicated by the disappearance of UDPG-14C,formation of UDP, and addition of labeled glucose into the polysaccharide. Recondo and Leloir (1 96 1) have discovered that adenosine diphosphateglucose (ADPG) reacts with the enzyme ten times more rapidly than UDPG. Both UDPG and ADPG have been demonstrated to be in maize kernels (Recondo et al., 1963). Kauss and Kandler (1962) have proposed that ADPG is involved in starch synthesis and UDPG is involved with the synthesis of sucrose. Leloir (1964) and Murata et al. (1963) have suggested that both UDPG and ADPG are involved in starch synthesis. DeFekete and Cardini (1 964) have concluded that the sucrosestarch transformation in maize endosperm involves UDPG with fructose glucosyl transferase. This enzyme transfers glucose from sucrose to either UDP or ADP to give UDPG or ADPG. Frydman and Cardini ( 1964, 1965) have reported a soluble enzyme in sweet corn that transfers glucose from ADPG, but not from UDPG, to phytoglycogen and amylopectin. Maltose and oligosaccharides of the maltose series also served as acceptors. Akazawa et al. (1964) reported a transferase enzyme in rice similar to that in maize described by Leloir and his associates. Other reports (Murata et al., 1963; Murata et a f . , I964b) have indicated that ADPG is present in ripening rice grains and that the amount of ADPG is much higher in rice than in maize (Recondo et al., 1963). Therefore, the sucrose-starch interconversion in rice endosperm also appears to involve nucleoside diphosphate sugars. Recently, Nomura et al. (1967) reported that the major pathway for UDPG utilization in bean and rice chloroplasts appears to be the synthesis of either sucrose or sucrose-phosphate. A specific role of ADPG in the synthesis of chloroplast starch by the ADPG-starch transglucosylase reaction was supported by a coupled enzyme system of ADPGpyrophosphorylase and transglucosylase. Glycogen and the two starch components, amylose and amylopectin, were the first polysaccharides synthesized by the enzyme phosphorylase in v i m from a-D-glucose 1-phosphate (G-1-P). This led to an early, but erroneous, assumption that starch and glycogen were synthesized only from G-1 -P in vivo. Nordin and Kirkwood ( 1 965) are of the opinion that ". . . there now can be very little doubt that the synthesis of amylose,

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amylopectin, and phytoglycogen all proceed from nucleoside diphosphate glucose intermediates, and that the older notion that the primary synthesis involves the action of phosphorylase on G-1-P must be totally abandoned and phosphorylase regarded as being coficerned only with the degradation of starch.” It seems to this author that such a conclusion is not tenable. The complexity of the amyloplast makes it difficult to rule out phosphorylase as a synthetase and categorize it as only a degradative enzyme. Badenhuizen ( 1 963) postulated that phosphorylase may be one of the primary enzymes in starch synthesis. Tsai and Nelson ( 1968) have found two kinds of phosphorylase activity (termed phosphorylase I and 11) in developing maize endosperm. The enzymes differ in their responses to cations and ethylenediaminetetraacetate. It was rather interesting- to note that phosphorylase I1 can synthesize an amyloselike polymer from a “primer free” system while phosphorylase I is not active even with maltose as the primer. Developmental studies indicated that phosphorylase I was present through all the stages of the endosperm examined and during germination, whereas phosphorylase I1 activity was present only during the period when starch synthesis is proceeding rapidly. This rapid synthesis spans 16 to 28 days after pollination (Creech, 1965). This association prompts speculation as to the possible synthetic role of phosphorylase 11. Tsai and Nelson suggested that phosphorylase I and I1 are coded by separate genes because of their unique characteristics and presence at separate stages of endosperm development. Nelson and Tsai ( 1964) reported that ADPG-transferase is confined to the embryo and maternal tissues in the waxy corn kernel and that the endosperm lacks activity for the starch granule-bound enzyme. In this instance a normal amount of starch is synthesized but it consists of all amylopectin. Akatsuka and Nelson (1966) indicated that the ADPGtransferase isolated from the embryo of maize kernels is different from a similar preparation from endosperm. Tsai and Nelson (1966) have also shown the shz lacks ADPG-pyrophosphorylase activity, which appears to be necessary for the synthesis of substantial amounts of starch in maize endosperm. It has not been possible to separate the particulate UDPG- and ADPG-transferase system from the starch granule; however, Frydman and Cardini (1 967) found that mechanical disruption considerably changed the properties of this enzyme. After this treatment, UDPG was no longer a substrate, while the activity with ADPG was enhanced. They suggested that the particulate synthetase and the soluble synthetase could be different forms of the same enzyme.

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Akazawa et al. (1964), studying the transfer of gluco~e-'~C from UDPG-14C, reported that amylose was more highly labeled than amylopectin, and Murata et af. (3964b) noted that ADPG-14C preferentially transfers glucoseJ4C to amylopectin. These results suggested that UDPG is involved in the synthesis of straight-chain amylose; ADPG in the synthesis of amylopectin. The findings of Nelson and Rines (1962) and Nelson and Tsai (1964) would appear to be incompatible with this suggestion. Waxy maize, which produces only amylopectin, lacks glucosyl transferase activity in the endosperm, although maternal tissues are active. Since Nelson and Rines originally studied UDPG-transferase activity, it was thought that the starch granule enzyme of waxy corn was specific for ADPG transferase. It has been shown, however, that activity for both substrates is lacking. Soluble transferase enzymes have been isolated from sweet corn (Frydman and Cardini, 1965), potato, and other plant materials (Frydman and Cardini, 1964; Frydman et al., 1966; Ghosh and Preiss, 1965; Murata and Akazawa, 1966). In potato, two different soluble glucosyl transferases were reported (Frydman and Cardini, 1964). Intact starch granules were used as primer with ADPG. The reaction with UDPG was limited. The other (soluble) acted with phytoglycogen, amylopectin, and oligosaccharides and utilized only ADPG. The lack of activity by waxy granules has been suggested to be due to the lack of complexing between enzyme and amylose, since no amylose is present in the waxy endosperm. Using the glucosyl transferase from rabbit muscle, Brown and Brown ( 1 966), and Brown et al. ( 1 965) showed that almost all of the g l u c o ~ e - ~ ~ C added to limit-glycogen from UDPG-14C was found in the outer main chains, rather than in the side branches which can be removed with a debranching enzyme. Thus, we have the suggestion that ADPG may be the natural or preferred substrate, and that ADPG is involved preferentially in the synthesis of branched molecules. And, if UDPG is a natural substrate, that it may be involved in straight-chain synthesis. The incorporation studies by Leloir, by Murata, and by Akazawa involved the separation of amylose from amylopectin by thymol complexing. This is a standard procedure when dealing with large quantities of starch. It is possible that, on the scale employed in labeled enzyme studies, this separation is subject to error. Or, assuming good separation of amylose and amylopectin, the validity of the incorporation data depends on the accuracy of conversion of the separated fractions to glucose, since specific activities are calculated on the basis of glucose content. Difficulties were experienced in our laboratory (M. L. Tomes, R. G. Creech, and J . D. Loerch, unpublished data) in trying to separate amylose

3 14

ROY G. CREECH

from amylopectin by complexing with thymol when small fractions ( I ml.) were used. The first difficulty involved the solubilization of all types of starch. A method involving the dispersion of starch with dimethylsulfoxide (DMSO), precipitation with alcohol, and solubilization in water was found to be superior. When starch granules from 20-day normal corn were put into solution by this method, and were complexed with thymol for 3 days at room temperature, from 70 to 80% of the total starch was precipitated as amylose. However, on filtering the solution prior to complexing with thymol, only about one-fourth of the starch precipitated. These results suggested that large aggregates were broken up in the filtering process. These results were consistent in repeated trials. Hydrolysis of the total fractions before and after filtering suggested that the loss due to filtering was not sufficient to account for the difference resulting from the removal of large quantities of “aggregates.” Rather, hydrolyses and limited P-amylolyses suggested that an intermediate, lightly-branched fraction might be responsible for this increased precipitate prior to filtering. Limited attempts to demonstrate a lightly-branched intermediate fraction, which would complex with 2-nitropropane after thymol separation, failed to yield any precipitate. To determine whether glucose is transferred from ADPG to amylopectin, or to amylose, another method of checking the results would be desirable. Such a method is suggested by the use of the enzyme “pullulanase.” Pullulanase is a debranching enzyme which acts to cleave the WD-( 1-6) branch points of dextrins and polysaccharides of the glycogen and amylopectin type. This suggests the possibility of incorporating gl~cose-’~C from ADPG-I4C into normal starch granules, using the granules both as enzyme and primer. If then, by the action of pullulanase, the outer branches can be broken at the 1-6 branch points and these branches can be separated from the residual limit-starch, it would be possible to determine whether glucose is transferred from ADPG to the outer branches of the amylopectin molecule, or to amylose. ADPG pyrophosphorylase activity in spinach chloroplasts was reported to be stimulated about 50-fold by 3-phosphoglyceric acid (Ghosh and Preiss, 1965). This may mean that starch synthesis is regulated during photosynthesis. As has been suggested during COz fixation, an increase in 3-phosphoglycerate may cause an increase in ADPG synthesis by stimulating ADPG pyrophosphorylase, which in turn may enhance the rate of starch synthesis. I f this is true, control of starch synthesis may occur at the pyrophosphorylase level rather than at the transglucosylase level as in glycogen synthesis (Rossell-Perez and Lamer, 1964). Studies that indicate that TDPG, GDPG, and CDPG may be of im-

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portance in carbohydrate metabolism are of considerable interest. Much of the recent work has been done with ADPG and UDPG as described above. Milner and Avigad (1965) studied the specificity of a relatively pure preparation of sucrose synthetase from sugar beet roots to various nucleoside diphosphates and showed them to be efficient acceptors (Table XXII). These results indicated that T D P is a very efficient acceptor whereas ADP is much less effective in this case than with sweet corn sucrose synthetase (DeFekete and Cardini, 1964). R. G. Creech and TABLE X X l l Effect of Nucleoside Diphosphates on Sucrose Cleavage by Sucrose Synthetase"

Nucleotide UDP TDP ADP CDP GDP None

Fructose liberated (prnoles/ml/hr.)

Relative activity (% of UDPG activity)

I .29 0.67 0.20 0.15

100 52 16

0.08

12 6

0.02

" From Milner and Avigad ( 1 965).

T. E. Bruszewski (unpublished data) have found that sucrose synthetase from maize endosperm at 20 days post pollination will use TDPG as a substrate to form sucrose. The rate was about 50% of that with UDPG and equal to that with ADPG. GDPG and CDPG were used in trace amounts. Vidra ( 1 967) has discovered the enzyme CDPG-pyrophosphorylase in developing maize endosperm (Table XVIII). The activity was as great or greater than that of ADPG-pyrophosphorylase. These data suggest that CDPG, GDPG and TDPG, especially TDPG, may be as important in starch synthesis as ADPG and additional investigations need to be made. Three possible explanations for the occurrence of phytoglycogen in maize endosperm have been suggested (Hodges et al., 1968). As postulated by Erlander (1958, 1967), the material may be a precursor in the synthesis of starch. Its accumulation in the presence of certain genetic blocks would then be expected. This role would require the presence of a debranching enzyme, and the detection of such an enzyme has been noted (Hodges et al., 1968). Although we have been unable to show the presence of soluble phytoglycogen in the nonmutant (normal) endosperm,

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some preliminary fractional precipitation studies lead us to believe that phytoglycogen-like material may be present in normal starch granules. A second possibility is that phytoglycogen may arise as a shunt product from a material which precedes the formation of amylose or amylopectin and accumulates at their expense in certain mutants. This conforms with the observed presence of smaller starch granules and reduced quantities of starch found in the phytoglycogen containing mutants. The formation of this product would be encouraged by blocking or interfering with normal synthesis or deposition of starch into the granules. A third possibility is that phytoglycogen may be the direct product of the action of an enzyme or enzymes on amylose or loosely branched amylopectin. The reduced quantities of starch and small granules observed in these mutants could, in this case, result from the branching of the substrate prior to its deposition in the starch granule. The structure of the amyloplast may be impaired in these instances, yielding access to the branching enzyme. The correlation between the accumulation of phytoglycogen and the presence of the branching enzyme, in the absence of a 1 :1 genetic correlation, indicates that the enzyme may be inducible. The branching enzyme would appear to have a significant role in each of these possible modes of phytoglycogen formation. An a-amylase was characterized for the first time in developing maize endosperm (Jaynes, 1966; Jaynes and Creech, 1967). Lavintman and Krisman ( 1 964) noted an enzyme in maize with amylase-like properties in their studies of phytoglycogen synthetase. The enzyme degrades amylose, amylopectin, glycogen, and P-limit glycogen to maltose, maltotriose, and other oligosaccharides. Kinetic studies indicated that the enzyme has a pH optimum of about 6.8 in Tris-maleate buffer, which is much higher than the a-amylase from germinating maize kernels. The Michaelis-Menten constant was calculated to be 0.08% amylose, which is a little higher than most but similar to many a-amylases. The enzyme was found to be relatively stable in water for 32 days at 10°C. Copper, iron, lead, mercury, and p-chloromecuribenzoate inhibit enzyme activity. The enzyme was not inhibited by sucrose, maltose, or glucose at the concentrations used in these investigations. A requirement for calcium could not be demonstrated. The enzyme was active after prolonged periods of dialysis against EDTA. An examination of the products with thin-layer chromatography showed that maltose, maltotriose, and maltotetraose were produced in about equal amounts after extended incubation with amylose. Action on glycogen and P-limit glycogen gave less maltose than maltotriose and maltotetraose. The enzyme rate was slower than on amylose. No iso-

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3 17

maltose was detected after extended incubation with branched substrates, and glucose was not detected in any of the experiments; this left open the possibility that the starch degradation was due to amylase plus a debranching enzyme. An argument against such is that only one band was detected in disc electrophoresis that showed activity toward starch. The enzyme was concluded to be an a-amylase because (1) the enzyme cleaved the a- 1,4-glucosidic linkages of amylose; (2) the enzyme cleaved beyond the branch points of p-limit glycogen; (3) the enzyme produced low molecular weight oligosaccharides after exhaustive incubation with starch and glycogen; (4) maltose and maltotriose were not hydrolyzed by the enzyme; ( 5 ) the enzyme was not demonstrated to synthesize higher polymers from oligosaccharides; (6) the general characteristics of the enzyme were similar to those of other a-amylases. The role of this enzyme in starch synthesis is unknown; however, the level of activity corresponds with the rate of starch synthesis during 16 to 28 days after pollination. Possibly, the enzyme degrades the long molecules formed by A D P G - and/or UDPG-transferase to form more acceptors for glucose transfer, thereby causing an increase in the rate and efficiency of the glucose transferases. T h e genotypes normal, ae, bt,, btz, du, PI, 02,shl, shz, sul, suz, and wx were found to possess the enzyme, although in varying quantities, the significance of which is not known at this time. J. Linden, D. French, and P. Peterson (personal communication) have recently detected a-amylase in developing maize endosperm. For a review of other enzymes which degrade starch, the reader is referred to an article by Pazur ( 1 965). VI. General Statement

The fact of genetic control of polysaccharide and starch synthesis in maize endosperm is amply evident from the preceding paragraphs. The advantages of using mutant organisms for the clarification of metabolic pathways have been demonstrated repeatedly since the early pioneering work of Beadle and Tatum. The large collection and quantities of singularly and multiply mutant maize plants and endosperm cultures presently available and the interest of many researchers offer excellent prospects for significant and relatively large contributions to detailed knowledge of the genetic control and biochemistry of starch and carbohydrate synthesis and the differentiation of maize endosperm within the very near future. Initial efforts of long-range programs have been directed toward two

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simultaneous goals. The selective preliminary enzyme assays which have been conducted will be broadened into as complete a study of known carbohydrate-metabolizing enzymes as can be reliably managed. By this means an immediate correlation may be possible between a number of the genetic blocks and specific enzymatic functions. The second immediate goal is to define in detailed physical and chemical terms the nature and quantities of the carbohydrates found in the various mutants. The varied structures and relative quantities of these starches and starch precursors are expected to reflect the specific enzymatic aberrations present in the various strains. Even the carbohydrate separations made thus far have suggested specific pathways of polysaccharide synthesis, as noted above. The detailed chemical analyses need to be continued, and this work must be done before a secure scheme of synthesis can be presented. It is felt that such studies will provide the necessary evidence for evaluating the relative order and importance of specific synthetic steps within the intact scheme. The interactions of multiple mutations on pathways of starch synthesis offer a promising refinement for analyzing the actual pathways involved. Physical studies of the starch granules of the various strains underway provide an additional tool for evaluating the influence of the particular mutations on the synthesis and deposition of starch. These studies have led the research on carbohydrates in maize endosperm into the areas of enzyme characterization, localization of activity, and aspects of enzyme synthesis of polysaccharides in plants, including enzyme repression and activation. ACKNOWLEDGMENT The research reported in this chapter that was conducted at The Pennsylvania State University was partially supported by The National Science Foundations Grants GB- 1876 and (38-4523, and grants from the Corn Industries Research Foundation.

REFERENCES Akatsuka, T., and Nelson, 0. E. 1966.5. Biol. Chem. 241,2280-2286. Akazawa, T. 1965. In “Plant Biochemistry” (J. Bonner and J . E. Varner, eds.), pp. 258297. Academic Press, New York. Akazawa, T., Minamikawa, T., and Murata, T. 1964. Plant Physiol. 39,37 1-378. Anderson, R. A., Uhl, D. E., Deatherage, W. L., and Griffen, E. L. 1962. Cereal Chem. 39, 282-286. Andres,J. M., and Bascialli, P. C. 1941. Univ. BuenosAires Inst. Genet. 2 , l . Andrew, R. H., Brink, R. A., and Neal, N. P. 1944.5. Agr. Res. 69,355-371.

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Appleman, C. O., and Arthur, J . M. 19 19. J . Agr. Res. 17,137-152. Appleman, C. O., and Eaton, S. V. I92 I. J. Agr. Res. 20,795-805. Axelrod, B. 1965. I n “Plant Biochemistry” (J. Bonner and J . E. Varner, eds.), pp. 23 1-257. Academic Press, New York. Baddiley, J . , Hughes, N. A., and James, A. L. 1961.J . Chem. Soc., pp. 2574-2576. Badenhuizen, N. P. 1939. Protoplasma 33,440-468. Badenhuizen, N. P. I955a. Cereal Chem. 32,286-295. Badenhuizen, N. P. 1955b. Protoplasma 45,3 15-326. Badenhuizen, N. P. 1959. Protoplasmalogia 2,l-74. Badenhuizen, N. P. 1963. Nature 197,464. Badenhuizen, N. P. 1965. 1n“Starch Chemistry and Technology” (R. L. Whistler and E. F. Paschall, eds.), Vol. 1 , pp. 65-103. Academic Press, New York. Badenhuizen, N. P., and Chandorkar, K. R. 1965. Cereal Chem. 42,44-54. Badenhuizen, N. P.,and Dutton, R. W. 1956. Protoplasma 47,156-163. Bakhuyzen, H . L. 1925. Proc. Soc. Exptl. Biol. Med. 23,302-305. Bates, L. L., French, D., and Rundle, R. E. 1943. J . A m . Chem. SOC. 65,142-148. Bear, R. P. 1944. J . Am. Chem. SOC.Agron. 36,89-91. Bear, R. S. 1942. J . Am. Chem. Soc. 64,1388-1392. Bear, R. S., and French, D. 1941.J.A m . Chem. Soc. 63,2298-2305. Bernstein, L. 1943. Am. J . Botany 34,5 17-526. Black, R. C., Loerch, J. D., McArdle, L. J., and Creech, R. G. 1966. Generics 53,661668. Bond, A. B., and Glass, R. L. 1963. Cereal Chem. 40,459-466. Brown, D. H., and Brown, B. 1966. Biochim. Biophys. Acra 130,263-266. Brown, D . H., Illingworth, B., and Kornfeld, R. 1965. Biochemistry 4,486-495. Brown, R. P. 1966. M. S. Thesis, T h e Penn State University, Univ. Park, Pennsylvania. Bunning, E., and Hess, C. 1954. Naturwissenschaften 41,339-340. Burma, D. P., and Mortirner, D. C. 1956. Arch. Biochem. Biophys. 62,16. Burnham, C. R. 1944. Maize Genet. Coop. News Letter 18,15. Buttrose, M. S. 1960. J . Ultrastructure Res. 4,23 1-257. Buttrose, M. S. 1962. J . Cell Biol. 14,159- 167. Cameron, J. W. 1947. Genetics 32,459-485. Cameron, J . W., and Cole, D. 1959.Agron. J . 51,424-427. Cameron, J . W., and Teas, H. J. 1953. Proc. Intern. Congr. Genet. 9,822-823. Caputto, R., Leloir, L. F., Cardini, C. E., and Paladini, A. C. 1950. J . Biol. Chem. 184, 333-350. Caputto, R., Barra, H. S., and Cumar, F. A. 1967.Ann. Rev. Biochem. 36,211-246. Collins, G . N. 1909. U.S. Bur. Plant Inds. 161,3 1 pp. Creech, R. G . 1965. Genetics 52,1175-1 186. Creech, R. G.,and McArdle, L. J. 1966. Crop. Sci. 6,192-194. Creech, R. G . , McArdle, L. J., and Krarner, H. H. 1963. Maize Genet. Coop. News Letter 37,111-120. Culpepper, C. W., and Magoon, C. A. 1924.J . Agr. Res. 28,403-443. Culpepper, C. W., and Magoon, C. A. 1927. J . Agr. Res. 34,413-433. Dankert, M., Passeron, S., Recondo, E., and Leloir, L. F. 1964. Biochem. Biophys. Res. Commun. 14,358-362. Deatherage, W. L., MacMasters, M. M., Vineyard, M. L., and Bear, R. P. 1954. Cereal Chem. 31,50-52. DeFekete, M. A. R., and Cardini, C. E. 1964.Arch. Biochem. Biophys. 104,173.

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DeFekete, M. A. R., Leloir, L. F., and Cardini, C. E. 1960. Nature 187,9 18. Doty, D. M., Smith, G . M., Roach, J . R., and Sullivan, J. T. 1945. Indiana Agr. Expt. Sta. Bull. 503,3 1 pp. Dunn, G. M., Kramer, H . H.,and Whistler, R. L. 1953.Agron. J. 45,101-104. Dvonch, W., and Whistler, R. L. 1949.5. Biol. Chem. 181,889. Dvonch, W., Kramer, H. H., and Whistler, R. L. 195 I . Cereal Chem. 28,270-280. Earle, F. R., Curtis, J. J., and Hubbard, J. E. 1946. Cereal Chem. 23,504-5 I 1. Earley, E. B. 1952. Plant Physiol. 27,184- 190. East, E. M., and Hayes, H. K. 191 1. Conn. Agr. Expt. Stu. Buff.167,142 pp. Elbein, A. D., Barber, G. A., and Hassid, W. Z. 1964. J. Am. Chem. Soc. 86,309-3 10. Erlander, S. R. 1958. Enzyrnologia 19,273-283. Erlander, S. R. 1967. Sturke 19,99- 1 10. Eryster, W. H. 1934.Bibliog. Genet. 11, 187-392. Evans, J. W. 1941. Cereal Chem. 18,468-473. Freudenberg, K., Schaaf, E., Dumpert, G., and Ploetz, T. 1939. Narurwissenschafren 27, 850-853. Frydman, R. B., and Cardini, C. E. 1964. Biochem. Biophys. Res. Commun. 14,353-357; ibid. 17,407-4 1 1 . Frydman, R. B., and Cardini, C. E. 1965. Biochim. Biophys. Acta 96,294-303. Frydman, R. B., and Cardini, C. E. 1967. J. B i d . Chem. 242,3 12-3 17. Frydman, R. B., Cardini, C. E., and DeSouza, B. C. 1966. Biochim. Biophys. Acta 113, 620-623. Ganguli, N . C. 1958. J. B i d . Chem. 232,337. Ghosh, H. P., and Preiss, J. 1965. Biochemistry 4,1354- I36 I. Ginsburg, V. 1958. J. Biol. Chem. 232,55-6 I . Ginsburg, V. 1960. J. Biol. Chem. 235,2196-2201. Ginsburg,.V. 1961. J. Biol. Chem. 236,2389-2393. Greenwood, C. T. 1956.Advan. Carbohydrate Chem. 11,335-393. Greenwood, C. T., and Das Gupta, P. C. 1958. J . Chem. Soc., pp. 703-707. Hageman, R. H., Leng, E. R., and Dudley, J. W. 1967.Advan. Agron. 19,45-86. Hanes, C. S. 1937. New Phytologist 36,189-239. Hassid, W. Z. 1945. Federation Proc. 4,227-234. Hassid, W. Z. 1967.Ann. Rev. Plant Physiol. 18,253-280. Hassid, W. Z.,and McCready, R. M. 1941. J . A m . Chem. Soc. 63,1632-1635. Hizukuri, S., Fujii, M., and Nikuni, 2. 1961. Nature 192,239-240. Hodges, H. F., Creech, R. G., and Loerch, J. D. 1968. Biochim. Biophys. Acrn (in press). Hutchinson, C. B. 1921. J. Hered. 1 2 , 7 6 4 3 . Ingle, J . , Beitz, D., and Hageman, R. H. 1965. Pfantfhysiol.40,835-839. Jaynes, T . A. 1966. M. S. Thesis, The Penn State University, Univ. Park, Pennsylvania. Jaynes, T. A., and Creech, R. G . 1967. Maize Genet. Coop. News Letter 41,163- 164. Jordan, W. S. 1965. Ph.D. Thesis, The Penn State University, Univ. Park, Pennsylvania. Kauss, H., and Kandler, 0. 1962.2. Naturforsch. 17b,858-860. Kirchoff, G. S. C . 18 1 I . Zap. Imperatorskoi Acad. Nauk 4,27 (In Russian). Kramer, H. H., and Whistler, R. L. 1949. Agron. 5. 41,409-4 I 1. Kramer, H. H., Pfahler, P. L., and Whistler, R. L. 1958.Agron.5. 50,207-2 10. Kurahashi, K. 1957. Science 125,114-1 15. Lampe, L., and Meyers, M. T . 1925. Science 61,290-29 1 . Laughnan, J. R. 1953. Genetics 38,485-499. Lavintman, N., and Krisman, C . R. 1964. Biochim. Biophys. Acta 89,193-196.

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TRANSFORMATION A N D AVAILABILITY TO RICE

OF NITROGEN A N D PHOSPHORUS I N WATERLOGGED SOILS Wm. H. Patrick, Jr. and I. C. Mahapatra Louisiana State University, Baton Rouge, Louisiana, and Central Rice Research Institute, Cuttack (Orissa), India

..........

............................................................... 11. Changes Occurring in a Soil as a Result of Waterlogging ... 1. Introduction

A. Establishment of Oxidized and Reduced Soil Zones as a Result of Submergence .................................................................................. B. Oxidation-Reduction Potential .......................................... C. Biological and Chemical Reduction of Various Inorganic Soil ................. Components as a Result of Submergence ..... 111. Nitrogen ............................................................................................. A. Mineralization of Nitrogen ................................................ B. Loss of Nitrogen C. Utilization of Nitrate and Ammonium Nitrogen by Rice ...... D. Evaluating the N I V . Phosphorus ......................... ........................................ A. Distribution of P B. General Reactions and Fixation of Inorganic Phosphate in the Soil .......... C . Effect of Submergence on Phosph sformation and Availability ...... .......................................... D. Phosphorus Nutrition of Rice ..... E. Soil 'rests for Phosphorus in Floo ........................................... V . Summary ...... ................ ..............................................

.................................................................

Page 323 324

3 24 32 6 327 330 330 332 335 337 339 339 34 I 343 35 I 353 354 356

I. Introduction

The soil is submerged for part or all of the growing season in most of the rice-growing areas of the world. Submerging or waterlogging the soil creates conditions markedly different from those of a well drained soil. Land preparation, seeding and transplanting, fertilization, weed control, cultivation, and harvesting are all affected by the presence of excess water. In addition to the physical presence of water in the soil and on the soil surface, waterlogging affects the reactions involving the plant nutri323

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WM. H. PATRICK JR. A N D 1. C. MAHAPATRA

ents in the soil as well as those added as fertilizer. Of paramount importance to the increase in rice yields is the improvement in utilization of applied nutrients by the crop. Rice is noted for its inefficient utilization of applied nutrients, particularly nitrogen. The purpose of this paper is to describe the special soil conditions that exist as a result of waterlogging and to discuss the effect of these conditions on the transformation and availability of nitrogen and phosphorus. II. Changes Occurring in a Soil

CIS

a Result of Waterlogging

Waterlogging causes changes in the properties of the soil because of physical reactions between the soil and water and also because of biological and chemical processes set in motion as a result of the excess water. The structure of the soil, which is considered of great importance in well drained soils, is destroyed by a minimum of manipulation due to the weakening effect of water on the bonds holding the soil particles together as stable aggregates. In areas where there is a shortage of mechanical power, advantage is taken of the ease with which a waterlogged soil can be worked by preparing the seedbed under saturated conditions. The most important change in the soil as a result of waterlogging is the conversion of the root zone of the soil from an aerobic environment to an anaerobic or near-anaerobic environment where oxygen is absent or limiting. Under anaerobic conditions several oxidation-reduction systems that are usually present in the oxidized form are reduced as a result of the activity of facultative and true anaerobes. A. ESTABLISHMENT OF OXIDIZED A N D REDUCEDSOIL AS A RESULT OF SUBMERGENCE

ZONES

A waterlogged soil differs substantially from a well drained soil. In a well drained soil there is usually enough oxygen available from the atmosphere to supply the needs of microorganisms and plants. This condition is changed completely when a soil is flooded. Oxygen movement through the overlying flood water is usually much slower than the rate at which oxygen can be reduced in the soil. Because of the demand for oxygen in the soil and the slow renewal rate through the flood water, oxygen in a warm soil containing an energy source is usually depleted within a day or so after flooding. The greater potential consumption of oxygen as compared to the available supply through the flood water results in two distinctly different layers being formed in a waterlogged soil: an oxidized or aerobic surface layer where oxygen is present, and a reduced or anaerobic layer in which no free oxygen is present. These relationships are illustrated in Figs. 1 and 2.

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FIG. I . A diagram showing the relative rate of oxygen movement through the flood water (small arrow) and potential consumption rate of oxygen in the soil (large arrow). Reduced conditions occur when the oxygen supply through the flood water is not sufficient to meet the requirements for oxygen in the flooded soil.

FIG.2. Differentiation of a waterlogged soil into a surface oxidized layer and an underlying reduced layer as a result o f a limited oxygen supply reaching the soil surface.

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The rate at which oxygen is supplied to the soil surface depends for the most part on the rate of diffusion of oxygen through the flood water and the rate of percolation of water through the soil. These processes depend on depth of flood water, temperature, and soil permeability. The thickness of the oxidized surface layer is determined by the net effect of the oxygen consumption rate in the soil and the oxygen supply rate through the flood water; a high consumption rate results in a thin oxidized surface layer, a millimeter or so in thickness, while a low consumption rate results in a thicker oxidized layer, up to several centimeters in thickness. Because of the higher demand for oxygen, a soil with an appreciable supply of readily decomposable organic matter usually has a thin oxidized layer. The differentiation of a waterlogged soil into two distinct zones based on oxygen penetration was first described by Pearsall and Mortimer (1939), although Shioiri and Mitsui had earlier noted a thin yellowish layer due to oxidized iron when a highly reduced soil was exposed to the atmosphere (Mitsui, 1954). Later Alberda ( I 953) demonstrated the existence of these two layers with oxidation-reduction potential measurements made at 2-mm. intervals from the aerated flood water down into the reduced soil layer. The reduced soil has been shown to consume oxygen at a rate several times that of an oxidized soil (Patrick and Sturgis, 1955; Patrick, 1960). B. OXIDATION-REDUCTION POTENTIAL The most striking and easiest-to-measure change occurring in a soil as a result of submergence is the decrease in oxidation-reduction or redox potential. Aerated soils have characteristic redox potentials in the range +400 to +700 millivolts; waterlogged soils exhibit potentials as low as -250 to -300 millivolts. The narrow range of redox potential values encountered in well drained soils and the poor reproducibility caused primarily by a lack of poising of the oxidation-reduction systems in the oxidized range (Ponnamperuma, 1955) have resulted in the rejection of oxidation-reduction potential measurements as a tool for characterizing aeration in well drained soils. Various methods of measuring oxygen content or oxygen diffusion rate have proved to be more reliable indices of aeration in well drained soils. In waterlogged soils three factors combine to make the oxidationreduction potential the best available measure of the oxidation or reduction status of the soil. First, as mentioned above, the range of potential in waterlogged soils is much wider, approximately 1000 millivolts as compared to a range of approximately 300 millivolts in well drained soils (Fig. 3). Second, in waterlogged soils the higher concentrations of reduced

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Waterlogged Soil

Hlghly Reduced Reduced -300 -200 -100

0

Moderately Reduced

Oxidized

+loo e O 0 woo -00 +500+600 +700

Oxldatlon-Reduction or Redox Potential

- Mllllvolts

FIG. 3. The range of oxidation-reduction o r redox potentials usually encountered in well drained soils and in waterlogged soils.

components that contribute to the potential result in better poising and better reproducibility of the potential reading, although poor reproducibility is still one of the main limitations of redox potential measurements in waterlogged soils. Third, because of the ease with which it is reduced, oxygen is usually absent from waterlogged soils, and methods used for the measurement of oxygen content and oxygen diffusion rate employed in well drained soil cannot be used in waterlogged soil.*

C. BIOLOGICAL AND CHEMICAL REDUCTIONOF VARIOUS INORGANIC SOIL COMPONENTS A S A RESULTOF SUBMERGENCE As long as oxygen is present in the soil, other oxidized components of the soil are relatively safe from biological and chemical reduction. After oxygen has disappeared from a waterlogged soil the need for electron acceptors by facultative anaerobic and true anaerobic organisms results in the reduction of several oxidized components. If an energy source is available to the microorganisms, nitrate, the higher oxides of manganese, hydrated ferric oxide, and sulfate will be reduced. Nitrate and manganese dioxide are reduced at fairly high redox potentials, whereas sulfate is reduced only under the strictly anaerobic conditions associated with extremely low potentials. Ferric oxide reduction is intermediate. Thus, when a soil is depleted of oxygen by submergence, the reduction of the *Note Added in Proof Field measurements of redox potential or measurements in pots in the greenhouse usually have not been successful because of variation encountered among replicate readings. For successful laboratory evaluation of redox potential it has been necessary for the soil to be finely divided and thoroughly mixed. Because of the variation inherent in measurements made where the platinum electrode is in direct contact with the soil solid phase, better reproducibility is obtained with redox potential measurements made in the soil solution which has been separated from the soil without contamination from atmospheric oxygen (IRRI, 1966). Better reproducibility is also obtained if the waterlogged soil is stirred continuously or intermittently without contamination from atmospheric oxygen.

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oxidized inorganic soil components is at least somewhat sequential; nitrate and manganic manganese compounds are reduced first, then ferric compounds are reduced to the ferrous form, and last, sulfate is reduced to sulfide. Takai and Kamura ( 1966) and Turner and Patrick ( 1 968) have presented evidence for the sequential reduction of oxygen, nitrate, manganic manganese, and ferric iron in a waterlogged soil. Usually one component is not completely reduced before the next most easily reduced component begins to be reduced, although in some cases relatively complete reduction of one component is accomplished before the next component begins to undergo reduction (Fig. 4). Ferric Iron

julfaM -300 -200 -100

I///.-. Nitrate

0 +100+200+300+400+5oQ+600+700

-

Oxidation-Reduction or Redox Potential Millivolts (Corrected to pH 7 )

FIG.4. The approximate oxidation-reduction potentials at which oxidized forms of several inorganic redox systems become unstable.

The critical potentials for several of the important inorganic oxidationreduction system in the soil have recently been studied. These studies have generally been conducted in two ways: the soil is waterlogged and changes in the redox potential, and the concentrations of reducible substances are measured with time; and the redox potential of the waterlogged soil is maintained at a constant potential, and the concentrations of reducible substances are determined. In studies of the first type, Takai et al. (1956) obtained data showing that ferrous iron began appearing in the soil after the redox potential had decreased to approximately +50 millivolts and that sulfide begun to appear after the potential had decreased to -230 millivolts. In a later paper, Takai and Kamura ( 1 966) reported +I50 to -100 millivolts as the critical range for iron reduction in a waterlogged soil. Turner and Patrick (1968) found that the redox potential at which oxygen disappeared was in the range +320 to +340 millivolts. Under controlled oxidation-reduction potential conditions, nitrate in a

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waterlogged soil was found to be unstable at a potential of +340 millivolts at pH 5. I (Patrick, 1960). Assuming a redox potential/pH slope of -60 millivolts per pH unit, this is equivalent to a redox potential of+225 millivolts at pH 7 (E,). Ferric iron was reduced to the ferrous form beginning at about redox potential +200 millivolts at a pH of 5.7. This is equivalent to an E7of+ 120 millivolts (Patrick, 1964). In a study of sulfate reduction in waterlogged soils, Connell and Patrick (1968) found that sulfate began to be reduced when the potential fell to an E, value of -150 millivolts. For the nitrate, iron, and sulfate systems, the greater the decrease in potential below the value at which oxidized components began to be reduced, the greater the concentration of the reduced components. These critical or threshold values for the various inorganic systems are shown in Fig. 4. At times the intensity of reduction is not great enough for sulfide to be produced and sulfate can be found in waterlogged soils coexisting with ferrous iron, manganous manganese, and various reduced organic components. An important consequence of the sequential reduction of the various oxidation-reduction systems is the fact that in soils containing reducible iron there will always be ferrous iron in the soil to precipitate sulfide by the time the reduction processes are intense enough to produce hydrogen sulfide. Even in a soil capable of producing 2000 ppm of hydrogen sulfide, no free hydrogen sulfide was detected because of the presence of a large amount of ferrous iron which precipitated the sulfide (Harter and McLean, 1965). Reduction processes in the soil affect the pH and electrical conductivity of waterlogged soils, as has been shown by Ponnamperuma and his associates at the International Rice Research Institute (IRRI 1964, 1965). Their work has demonstrated the importance of iron and manganese reduction in determining the pH increase that occurs when an acid soil is submerged. They have also called attention to the importance of carbon dioxide in regulating many of the reactions and precipitations that occur in waterlogged soils. Soil reduction per se does not appear to be detrimental to rice except possibly at extremely low redox potentials, potentials low enough for sulfide to be formed. If the maintenance of the redox potential at an intermediate value is necessary for favorable rice growth, then all oxidation-reduction systems that operate between the irreversible oxygen system and the sulfate-sulfide system will function to buffer the potential at an intermediate value. Jefferies ( 1 96 1) has called attention to the importance of maintaining “healthy reducing conditions” in rice soils and has stressed the importance of the iron system in preventing low redox potentials. Because of its high concentration in most rice soils, hydrated ferric oxide is important in preventing the potential from reaching low

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values soon after flooding. A rapid decline in redox potential is characteristic of soils with low contents of reducible iron and manganese and a high organic matter content. It has recently been shown that manganese dioxide is capable of correcting toxic conditions associated with highly reduced soil conditions (Ponnamperuma et al., 1965). Both oxygen and nitrate are important in retarding reduction, but they are usually present in such low amounts (dissolved oxygen at a concentration of approximately 8 ppm. in the water and nitrate at a concentration of 50 ppm. nitrogen or less) that they are soon completely reduced and consequently retard reduction for only a few days after waterlogging. Hydrated ferric oxide and manganese dioxide, on the other hand, are usually present in much higher quantities in paddy soils. The iron system, in particular, generally has a large reserve of solid phase reducible matter. The oxidation and reduction of these compounds is cyclic-that is, they are reduced when the soil is waterlogged and reoxidized when the soil is subsequently drained and exposed to oxygen. Consequently, active iron and manganese compounds serve as buffers against the development of reducing conditions in the soil (Takahashi, 1960). The manganese and iron systems tend to buffer the soil at an intermediate redox potential of +lo0 to +300 millivolts. Both systems must be almost completely reduced before intense reduction can set in. Prolonged waterlogging is usually necessary before all the hydrated ferric oxide is reduced. Manganese compounds were found by Nhung and Ponnamperuma (1966) to be the most effective retardants of highly reducing conditions in waterlogged soils. Takai and Kamura (1966), on the other hand, consider that reducible iron is more important in retarding highly reducing conditions because of the much higher concentration of iron in most soils. I l l . Nitrogen

Throughout the rice-growing areas of the world, nitrogen is the nutrient most limiting for rice production. Nitrogen is usually deficient where rice is grown because the same conditions of climate which favor rice production cause a rapid turnover and loss of nitrogen from the soil. The nitrogen supply for lowland rice comes largely from three sources: ( 1 ) ammonium nitrogen present when the soil is flooded, (2) nitrogen mineralized from soil organic matter and plant residue under waterlogged conditions, and (3) nitrogen added as a fertilizer. Nitrate and nitrite nitrogen present in the soil at the time of submergence are usually lost too rapidly through denitrification to be of value to the crop. A. MINERALIZATION OF NITROGEN Nitrogen mineralization under anaerobic soil conditions cannot pro-

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ceed past the ammonium stage because of the absence of oxygen which is necessary for the microbial conversion of ammonia to nitrate. The breakdown of organic matter which leads to the release of ammonium ions to the soil solution proceeds at a slower rate in a waterlogged soil than in a well drained soil (Tenny and Waksman, 1930). A more restricted, less efficient group of organisms are involved in anaerobic organic matter decomposition. Anaerobic decomposition of organic matter is characterized by (1) the incomplete breakdown of carbohydrates into methane, organic acids, and hydrogen gas with a consequent low energy yield (Alexander, 19611, and (2) a low nitrogen requirement of the anaerobic organisms responsible for organic matter decomposition which results in ammonia being released to the soil solution at a much wider organic matter C : N ratio than would the case with aerobic decomposition. Acharya (1935a) found that rice straw with a wide C : N ratio released approximately 30 percent of its total nitrogen into solution as soluble protein and ammonia soon after the bebinning of anaerobic decomposition. When additional ammonium nitrogen was added to the straw, the immobilization of nitrogen was much more pronounced under aerobic conditions than under anaerobic conditions (Acharya, 1935b). Sircar et al. ( I 940) found that nitrogen was released from decomposing rice straw at a much wider C : N ratio under anaerobic conditions than under aerobic conditions. These studies were done in the absence of soil. Additional information is needed on the effect of aerobic and anaerobic conditions in soils on nitrogen mineralization from native soil organic matter and plant residue. The low nitrogen requirement for anaerobic decomposition results in a more rapid release of ammonia than would be indicated by the wide C :N ratio of much of the organic material returned to rice soils and the generally slow rate of anerobic decomposition. Waring and Bremner (1964a, I964b) recently found for a number of soils that considerably more nitrogen was mineralized under waterlogged conditions than under aerobic conditions. In a study of nitrogen mineralization in an acid rice soil, Patrick and Wyatt ( I 964) found that mineralization of nitrogen under waterlogged conditions was considerable higher than mineralization under well drained conditions. The pH of the waterlogged soil increased to approximately 7 while the pH of the well drained soil decreased to 4.4, probably accounting for part of the difference in mineralization. The ready release of ammonium nitrogen from anaerobically decomposing organic matter accounts for the good response of rice to added organic matter (De Geus, 1954). A fairly good estimate of the amount of nitrogen available to the rice crop can be obtained by measuring the amount of ammonium nitrogen mineralized under anaerobic conditions.

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B. Loss

OF

JR.

A N D I. C. MAHAPATRA

NITROGENFROM WATERLOGGED SOILS

In addition to uptake by the rice crop, nitrogen is removed from a waterlogged soil by (a) denitrification, (b) leaching losses of nitrate and ammonia, (c) ammonia volatilization, and (d) runoff. A small amount of ammonia is fixed in the soil, especially with the cyclic wetting and drying undergone by rice soils, but this is probably of little importance. 1 . Denitracation

Denitrification is one of the major mechanisms by which nitrogen is lost from a flooded soil. Oxidized forms of nitrogen can be used by certain facultative anaerobic microorganisms as electron acceptors and reduced to Nz or N,O. There are a number of organisms capable of this reaction. The rapidity with which nitrate in a waterlogged soil is denitrified depends largely on the availability of an energy source since the denitrifiers occur in large numbers in most soils (Alexander, 196 1). Very little denitrification occurs until all the oxygen is depleted. The reported cases of denitrification under aerobic conditions are apparently the result of anerobic microzones caused by rapid localized oxygen consumption (Jannasch, 1960; Greenland, 1962). It is a common belief that a part of the nitrate present in a waterlogged soil is reduced to ammonia, but several investigators (De and Sarkar, 1936; Broadbent and Stojanovic, 1952; Wijler and Delwiche, 1954; Nommik, 1956) have found that little of the nitrate in a flooded soil reaches the ammonium form. In a series of experiments Broadbent and Stojanovic found 0 to 6 percent of the nitrate reduced to ammonia. Laboratory experiments have shown that nitrate reduction after waterlogging can be very rapid. Yamane (1957) recorded nitrate reduction rates as high as 55 ppm. per day after flooding a soil to which no additional energy source had been added. Wallihan (1938) measured an average loss of 20 ppm. per day. The addition of a readily decomposable energy source increases the rate of denitrification. Bremner and Shaw (1958) found that 1000 ppm. of nitrate nitrogen was lost in 4 days from a submerged soil to which an energy source had been added. The results of one of their laboratory experiments are shown in Fig, 5. The buildup of nitrite is much less pronounced under field conditions, where the nitrate concentration is usually much lower. Patrick (1 960) recorded a nitrate reduction rate of 15 ppm. per day in reduced soil. Except where very high rates of nitrate were added, nitrate reduction rate was a zeroorder reaction with respect to nitrate concentration in most reported experiments. Under rice field conditions denitrification of applied nitrogen fertilizer can be so severe that special precautions must be taken either to

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prevent the formation of nitrate. Figure 6 depicts the processes by

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FIG.6 . A schematic diagram of the processes by which ammonium fertilizer can be lost from a waterlogged soil. Ammonium nitrogen applied to the oxidized soil surface is nitrified and then leaches down into the reduced subsurface layer, where it is denitrified and lost from the soil. (After Mitsui, 1954.)

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WM. H . PATRICK JR. A N D I . C. MAHAPATRA

which ammonium fertilizer applied to the oxidized surface layer of the soil is fmt nitrified and then lost through denitrification after leaching into the underlying reduced layer. Frequent fluctuations in moisture content of a field as a result of flooding and draining create ideal conditions for denitrification. Nitrogen converted to the nitrate form during the period when the soil is drained is lost through denitrification when the soil is flooded. Experiments have shown that rice yields on nitrogen-deficient soils are decreased considerably by draining and reflooding during the growing season while, on soils with excess nitrogen, yields are increased by the same treatment (Yamada, 1965; Patrick et al., 1967a). The Japanese have developed a system of fertilizing lowland rice in which denitrification is minimized. Ammonium fertilizer is placed several inches deep in the soil where it is protected by the reducing conditions of the soil from nitrification and subsequent denitrification (De Geus, 1954; Mitsui, 1954). Deep placement of nitrogen has been shown to be superior to surface placement and has been adopted in many of the rice growing areas of the world. Yield increases of about 10% have been obtained by the use of this method (De Geus, 1954). In the United States, subsurface placement of ammonium nitrogen is recommended for rice in California (Mikkelsen and Finfrock, 1957) and in Louisiana (Patrick et al., 1967b). 2 . A mmonia Volati1iza tion Ammonia volatilization is not considered to be an important mechanism of nitrogen loss from a waterlogged soil except in specialized cases where a high ammonia concentration occurs in conjunction with high pH, high temperature, and low cation-exchange capacity (Willis and Sturgis, 1944). In a waterlogged soil poor incorporation of plant residue can result in an accumulation of organic matter near the surface. The ready release of ammonia from organic matter decomposing in the absence of oxygen and the high pH associated with anaerobic decomposition (Acharya, 193Sa) favor ammonia volatilization from waterlogged soils which have received large additions of organic material. Deep incorporation of the organic matter results in better retention of nitrogen and better utilization of nitrogen by rice (De Geus, 1954). Ammonia formed from applied urea can also be lost from the soil. As shown by Ernst and Massey (1960), losses of ammonia are greatest when ( I ) urea is surface applied instead of being incorporated into the soil, (2) the soil is neutral or alkaline in reaction, (3) the soil is allowed to dry instead of being maintained in a moist condition, and (4) the temperature is high. Delaune ( 1 968) found that ammonia loss from surface applied urea was about the same in waterlogged soils and soils at optimum field moisture at low rates

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of urea application (100 pounds of nitrogen per acre equivalent), but at higher rates of application volatilization loss was more severe at optimum field moisture.

3 . Leaching of Nitrate and Ammonium Nitrogen Nitrate present in the root zone of a soil which is submerged a t the beginning of the season is almost invariably lost by denitrification or by being leached out of the root zone before the plants are large enough to utilize the nitrogen. The biological activity and the rate of percolation of water through the soil will determine which of these mechanisms is most important. Usually there is enough organic matter present so that the nitrogen is lost by denitrification. Nitrate produced in the surface oxidized layer of a waterlogged soil can easily move downward by diffusion and percolation into the underlying reduced layer, where it is rapidly denitrified (Fig. 5). This process is one of the most important pathways of nitrogen loss in waterlogged soil (Pearsall, 1950; Mitsui, 1954). Nitrate nitrogen can also be lost from a flooded field by runoff, but the nitrate content of the flood water seldom exceeds a few parts per million. Ammonium nitrogen is much less subject to leaching from the soil than nitrate because of its adsorption on the cation-exchange complex. Nonetheless, loss of ammonium by leaching is more severe in waterlogged soil than in well drained soil because ( I ) ammonium is not as likely to accumulate in a well drained soil as in a waterlogged soil, (2) reduction reactions in a waterlogged soil produce ferrous and manganous ions which displace ammonium from the exchange complex to the soil solution where it is more subject to removal, and (3) the constant head of water on the soil surface results in greater downward percolation of the soil solution in a waterlogged soil than occurs in a well drained soil. An appreciable loss of ammonium by leaching from a submerged rice soil was measured by Ponnamperuma ( 1 955). He noted that the addition of organic matter increased the amount of ammonium in the soil solution instead of immobilizing the nitrogen as might be expected. An increase in the leaching loss of ammonium due to organic matter addition was also observed by Kiuchi and Omukai ( 1 959).

c. UTILIZATION

OF

NITRATEA N D AMMONIUM NITROGENBY RICE

Rice is noted for its poor utilization of nitrogen. Mitsui (1954) reported that in Japan concentrated nitrogen fertilizers were 30 to 40 percent available to the rice crop under waterlogged conditions as compared to an availability of 50 to 60% when applied to upland crops. Poor

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WM. H. PATRICK JR. AND I. C. MAHAPATRA

utilization of nitrogen occurs in other rice growing areas of the world. Most of the nitrogen obtained by lowland rice is taken up in the ammonium form. Kellner ( I 884) showed that ammonium was superior to nitrate for rice. Nagaoka ( 1 904) found that ammonium sulfate was two and one-half times as effective as sodium nitrate in increasing rice yield. He suggested that the loss of nitrogen by denitrification, the formation of nitrites, and the absence of insufficient sugar to transform nitrate into protein interfered with the utilization of nitrate. Daijuhara ( 1905) confirmed these findings and found the response to ammonium nitrate to be intermediate between sodium nitrate and ammonium sulfate. Later, Daikuhara and Imaseki ( 1 907) noted no difference between ammonium sulfate and sodium nitrate for rice grown under upland conditions. Kelley (191 I ) attributed the chlorotic conditions of young rice seedlings which had received nitrate nitrogen to the inability of the plants to assimilate nitrate as well as to the toxicity of nitrite produced from the nitrate. Later investigations (Janssen and Metzger, 1928; Sturgis, 1936; Reed and Sturgis, 1937; Ponnamperuma, 1955) have indicated that nitrite concentrations high enough to be injurious to the crop are not likely to develop. Although many investigations over the years (Willis and Carrero, 1923; Bartholomew, 1929; Gericke, 1930, for examples) have demonstrated that the rice plant can utilize nitrate nitrogen, nitrate is a relatively unimportant source of nitrogen for lowland rice for several reasons: ( 1 ) nitrate is unstable in a waterlogged soil, as has been pointed out, and is readily denitrified even in a moderately reducing environment; (2) the only nitrate nitrogen available to the crop is that in the soil at the time of flooding or that added as a fertilizer since nitrogen mineralization in a waterlogged soil stops at the ammonium form; and (3) even though in some cases lowland rice can utilize nitrate equally as well as ammonium, the pH and iron status of most waterlogged soils favor the uptake of ammonium. In nutrient solution nitrate is most effective for rice at a low pH while ammonium is favored at near-neutral pH values (IRRI, 1965).

High iron availability is necessary where nitrate nitrogen is used as the source of nitrogen. In nutrient solution high iron availability can be obtained by making the solution more acid. This is not easily done with soil because of the tendency of most soils to approach a neutral reaction after submergence regardless of whether they were originally acid or alkaline (Ponnamperuma, 1965; Redman and Patrick, 1965). In acid soils that are low in free iron oxides, however, submergence may not result in an appreciable pH rise, and nitrate in these soils may be utilized

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by rice as effectively as ammonium, particularly if the organic matter content is low enough that nitrate is not rapidly denitrified upon submergence. In this connection, Go Ban Hong and Schuylenborgh (1 960) found that higher yields were obtained from nitrate than from ammonium on a soil of pH 4.3 that was low in organic matter and iron. On a less acid soil higher in iron and organic matter, ammonium was superior to nitrate. The greater response to nitrate was attributed to secondary conditions of nitrate additions: the increase in pH due to the application of sodium nitrate, the increase in Eh in a reduced soil as a result of the oxidizing capacity of nitrate, and the effect of nitrate in suppressing sulfate formation. Notwithstanding the special conditions under which nitrate is equal to or even slightly superior to ammonium, ammonium nitrogen or ammonium-forming sources of nitrogen (urea) are much more dependable sources of nitrogen for lowland rice. The uncertain responses of rice to nitrate applied as a basal application is discussed in a review by De Geus (1954). Special precautions are usually taken to prevent ammonium nitrogen from being nitrified. This is accomplished by either placing the ammonium fertilizer several inches deep in the reduced zone of a waterlogged soil as discussed above, applying the ammonium fertilizer on the dry soil surface and washing it into the soil with irrigation water (Evatt, 1965), or, less commonly, applying a biological nitrification inhibitor with the ammonium fertilizer (Goring, 1962). Although ammonium nitrogen is usually superior to nitrate as a topdressing for rice, nitrate has often been used successfully as a midseason topdressing (Beacher and Wells, 1960). At least four conditions appear necessary for a good response to be obtained from topdressed nitrate: (1) the plants must be large enough to assimilate the applied nitrogen rapidly; (2) the plants must have an adequate surface root development so that nitrate can come in contact with the roots; ( 3 ) reducing conditions must not extend to the soil surface and into the flood water as is sometimes the case when organic matter is decomposing at the soil surface; and (4) if the nitrate is applied to a soil that is dry enough to have cracks, the soil should not be flooded immediately after application of nitrogen because of the likelihood of washing the nitrate several inches deep into the soil, where it will be denitrified in the flooded soil. D. EVALUATING THE NITROGEN SUPPLYING OF WATERLOGGED SOILS

CAPACITY

Chemical and biological soil tests for available soil nitrogen have generally been less successful than have chemical tests for phosphorus and

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W M . H . PATRICK JR. A N D I. C. MAHAPATFU

potassium. Available phosphorus and potassium analyses are standard procedures in most soil testing laboratories whereas routine chemical or biological tests of available soil nitrogen are seldom employed. Recently, renewed efforts have been made to devise simple tests for available soil nitrogen that will aid in predicting the soil supply of nitrogen to the crop. The chemical method which appeared to be most promising as an index of available nitrogen is based on the liberation of ammonia from organic combination by an alkaline solution of KMn04. This method was first proposed by Truog (1 954). The biological methods used for estimating nitrogen availability generally consist of measuring the amount of organic nitrogen mineralized during incubation of the soil under standard conditions. The most widely used method consists of the determination of the amount of nitrate nitrogen released during incubation under aerobic conditions (Fitts et al., 1953; Stanford and Hanway, 1955). In other methods, ammonium and nitrite are determined in addition to nitrate (Cooke and Cunningham, 1958; Keeney and Bremner, 1965). A biological evaluation method that appears to have special value for lowland rice consists of the determination of the amount of organic nitrogen mineralized to the ammonium form under waterlogged conditions. In the absence of oxygen, which is usually the case under waterlogged conditions, nitrogen mineralization from organic forms does not proceed past the ammonium form, and nitrate and nitrite analyses are consequently unnecessary. Both chemical and biological methods have been used with some suc-

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cess to estimate the availability of soil nitrogen to lowland rice. Using an alkaline permanganate extraction, Tamhane and Subbiah ( 1 960) reported a highly significant correlation bemeen available nitrogen and yield response to nitrogen fertilizer in both pot and field experiments. The same workers reported an even closer association between ammonium produced in the soil under waterlogged conditions and yield response to nitrogen in a pot experiment. Sims ef al. (1 967) found that ammonium production in the soil during a 6-day incubation under waterlogged conditions was the best predictor of grain yield. Little improvement in the correlation was gained by including initial nitrate or ammonium produced after 6 days. The results of one of their experiments are illustrated in Fig. 7, which shows the relationship between grain yield in a greenhouse experiment and the amount of ammonium released after 6 days’ incubation under waterlogged conditions for 19 clay soils in Arkansas. A similar study with silt loam soils failed to show as close a relationship. There was little variation in the amount of ammonium released in the silt loam soils. IV. Phosphorus

The requirement of lowland rice for phosphate is not as widespread or as pronounced as is the requirement for nitrogen. Many experiments show inconsistent responses of lowland rice to phosphate addition even though upland crops on the same soils respond markedly to phosphate. The unique soil conditions created by waterlogging influence the transformation and availability of both native phosphate and applied fertilizer phosphate. This section will consist of a discussion of distribution patterns of phosphate in flooded soil, the reactions and transformation of phosphate as a result of flooding, and the utilization of phosphate by lowland rice. A. DISTRIBUTION OF PHOSPHORUS IN FLOODED SOIL

The phosphorus content of most mineral soils is low, ranging between 0.2 and 0.4% PeOs. As an important soil constituent, phosphorus is present in organic and inorganic forms, of which the inorganic form is more important under waterlogged conditions. According to Chang and Jackson ( 1 957a) inorganic phosphate in the soil can be classified into four main groups: calcium phosphate, aluminum phosphate, iron phosphate, and reductant-soluble phosphate extractable after the removal of the first three forms. Calcium phosphate exists mainly as apatite, but dicalcium, monocalcium and octacalcium phosphates also

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WM. H. PATRICK JR. AND I. C. MAHAPATRA

exist in small amounts as transitional forms. Substantial amounts of phosphorus which resist solution in conventional phosphorus fractionation solvents are found in many soils. The insoluble phosphorus fraction has been termed as “nonextractable” phosphorus (Bauwin and Tyner, 1957) and is equivalent to reductant soluble iron phosphate plus occluded aluminum and iron phosphate fractions of Chang and Jackson (1 957b). This fraction is not believed to contribute toward phosphate fertility in well-drained soils, but its important in waterlogged soils has been realized in recent times. One of the characteristics of many poorly drained mineral soils is the presence of gley horizons resulting from the accumulation of reduced iron and manganese compounds. The gley horizon contains a minimum of total phosphorus. According to Glentworth (1947), a maximum concentration in poorly drained profiles occurs in the A horizon while the maximum concentration in well drained profiles occurs in the C horizon. Soil weathering tends to change inorganic phosphorus from the more soluble calcium forms to the less soluble iron and aluminum forms. Soil organic matter retards this process, bringing about the formation of many alkali and acid soluble complexes of phosphorus (Godfrey and Riecken, 1957). Information on the distribution of soil phosphorus among various fractions in flooded soils is rather limited. Forms of phosphorus and their distribution in swamp mud of Sierra Leone and rice soils of Taiwan and India have, however, been reported. According to Hesse (1962) 87 percent of the total phosphorus in swamp mud was in organic combination and the remainder was almost entirely in association with iron and calcium. In acid red earth or yellow earth and mudstone alluvial paddy soils of Taiwan iron and aluminum phosphates were reported to be the main forms of phosphorus (Tseng, 1960). In slate, schist, and saline alluvial soils calcium phosphate was the predominant form. In sandstone and shale alluvial soils calcium, iron, and aluminum phosphates were present in about equal amounts. The minimum solubility of phosphate in soils of different soils groups was in the range of pH 6 to 8. In the main rice tracts of different soil climatic zones of West Bengal Basak and Bhattacharya ( 1 962) observed that iron and aluminum phosphate represented 47% of the total phosphate. These fractions decreased gradually from planting time to postharvest time and then increased by the planting time of the next season. In general, calcium and aluminum phosphates are considered to be the forms of most probable occurrence in a soil before chemical weathering becomes severe. At an advanced stage of weathering iron phosphate in-

NITROGEN A N D PHOSPHORUS IN WATERLOGGED SOILS

34 1

creases at the expense of aluminum and calcium phosphates with ultimate occlusion of some aluminum and iron phosphate by iron oxide coatings. Because of the resistance of this iron oxide coating to decomposition the “nonextractable” or reductant soluble phosphate is not available to plants in well drained soil while these forms of phosphates are of economic importance for lowland rice because of the instability of hydrated iron oxide under reducing soil conditions.

B. GENERAL REACTIONSA N D FIXATION OF INORGANIC PHOSPHATE IN THE SOIL

The most important factors in determining phosphate fixation and release in the soil have been reported to be the kinds and amounts of clay, the quantities of iron and aluminum compounds and calcium and magnesium compounds, and soil pH (Fried and Shapiro, 1956). Another important factor is the oxidation-reduction state of the soil. There are in general three types of reactions by which phosphorus is fixed in the soil. These are (1) adsorption reactions, (2) double decomposition reactions, and ( 3 ) isomorphoric replacement. One or more ionic forms into which orthophosphoric acid is dissociated may be involved in the abovementioned reactions (Kardos, 1955). Although the adsorption reactions may be chemical or physical, the isomorphorus replacement reaction involves intercrystalline adsorption and replacement of hydroxyl or silicate anions from the crystal lattice by the phosphate ion. In acid soils a substantial quantity of iron and aluminum ions are present in active form. These ions react with soluble phosphates to form both crystalline and amorphorus precipitates. The possibility of the formation of aluminum phosphate minerals (wavellite and variscite) and iron minerals (vivianite and dufrenite) in acid soils has been indicated by Stelly and Pierre (1942), though detection of such minerals in natural soils is difficult because of the extremely small size of the crystals (Haseman et al., 1950). Precipitation and accumulation of aluminum and iron phosphates have been demonstrated in neutral soils (Chang and Jackson, 1957b). In a review on the subject of phosphate fixation Wild ( I 950) cited the importance of iron and aluminum by calling attention to the positive correlation between phosphate fixation and amounts of iron and aluminum in the soil. He noted the reduction in phosphate fixation caused by the removal of iron and aluminum oxides from soil colloids and the increase in phosphate fixation caused by addition of iron and aluminum compounds. Clay minerals play an important role in the mechanism of anion adsorption, which includes mainly the replacement of hydroxyl ions from

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the clay mineral surfaces, especially around the crystal edges. Kaolinite and similar minerals expose many more hydroxyl ions than do montmorillonite and illite. Phosphate has been shown to be fixed by clay minerals by reacting with soluble aluminum originating from the exchange sites and from lattice dissociation of clay minerals to form highly insoluble aluminum phosphate compounds (Hemwall, 1957). The first step in the reaction of soluble phosphate with clay occurs on the surface of the clay (Chang and Chu, 1961). There is evidence of phosphate fixation through a mechanism of replacement of silicate ions (Toth, 1937). It was demonstrated that with increased phosphate fixation the amount of silica in the supernatant liquid increased. Low and Black (1950) studied the reaction between phosphate and kaolinite clay. Digestion of kaolinite in a strong phosphate solution released silica in a quantity proportional to phosphate fixed by the clay and aluminum was changed to a form extractable with aluminum complexing reagents. They postulated two reactions in the above experiment as evidenced by the rate of silica released from kaolinite. The first is an adsorption reaction (replacement of surface silicon-oxygen tetrahedra by phosphorus-oxygen tetrahedra) while the second reaction (phosphate-induced solution of the surface compound and precipitation of aluminum phosphate) is linear with time and dependent on the completion of the first reaction. The phosphate-induced decomposition of clay minerals results in the formation of iron and aluminum phosphates minerals (Haseman et al., 1950). Because the concentration of aluminum in the clay is much higher than that of iron or calcium, phosphates are more likely to be fixed by clay in the initial stages as aluminum phosphate than as iron phosphate or calcium phosphate. As time elapses aluminum phosphate is changed to the less soluble iron phosphate. The mechanism of phosphate fixation in calcareous soils can be accounted for to a great extent by the reaction of phosphates with calcium ions and possible calcium carbonate. These reactions result in the formation of complex calcium phosphate compounds of varying solubilities. The solubility of calcium orthophosphate decreases in the order of monocalcium, dicalcium, and tricalcium phosphates. The formation of insoluble tricalcium phosphate is favored at a high pH. Of the phosphate minerals present in the soil, apatites are by far the most important. Hydroxyapatite is considered to be the stable solid phase in weakly acid, neutral, or alkaline solutions. Some investigators (Moreno et al., 1960) believe that octacalcium phosphate is a very stable form. In alkaline soils that contain free calcium carbonate, phosphate ions, coming

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in contact with the calcium carbonate, are precipitated on the surface of the particles. The quantity of precipitate is dependent on the amount of exposed surface of calcium carbonate. A calcium-saturated clay may also fix phosphate as a linkage of clay-Ca2+-HnPO;. Hemwall ( 1 957) noted that in calcareous soils phosphate fixation was due to the formation of a series of insoluble heterogeneous calcium phosphates. Precipitation of calcium phosphate occurs as a separate phase above pH 6.5 with a calcium or sodium-saturated clay. The initial stage of this reaction is possibly the precipitation of Ca2+and HP042- from solution which proceeds when the solubility product of CaHP04 is exceeded. Subsequent enrichment of the precipitate with more calcium is likely to occur with the increase of OH+ and F+ (Olsen, 1953). The concentration of calcium in the soil solution of calcareous soils is the dominant factor in determining the phosphate concentration in the liquid phase of the soil (Burd, 1948). Phosphorus solubility is influenced greatly by surface adsorption reactions, and the solubility increases regularly with the amounts of phosphate adsorbed in the monolayer region of the surfaces of various materials likely to be present in calcareous soils (Cole and Olsen, 1959a,b).

C. EFFECTOF SUBMERGENCE ON PHOSPHATE TRANSFORMATION AND AVAILABILITY The behavior of phosphate in flooded soils is remarkedly different from phosphate behavior in upland soils. This difference in behavior is of the greatest practical significance in the phosphate nutrition of rice. Relationships between extractable soil phosphate and plant response worked out for upland crops are of little value when the land is used for lowland rice. In general, lowland rice shows considerably less response to phosphate than do upland crops grown on the same soil. The general lack of response of lowland rice to phosphate compared to upland crops is illustrated in Fig. 8, which shows the relative yields of several crops with and without phosphate fertilizer in a large number of experiments carried out in Japan. Clearly, either the rice crop's need for phosphorus is lower than that of other crops or rice has access to sources of soil phosphate that are unavailable to other crops. Soil-water-plant relationships are not identical in the various ricegrowing areas of the world. While rice is grown under continuous flooding in some areas, the practice of alternate flooding and drying is followed in other areas either purposefully or because of natural climatic uncertainties. The transformation of phosphorus is not the same under both conditions. Just as with nitrogen, significant changes in many of the soil

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factors affecting phosphate retention and transformation take place as a result of continuous flooding or alternate wetting and drying.

FIG.8. Relative yields of several crops in Japan with and without added phosphate fertilizer. (After Mitsui, 1954.)

1 . Effect of Continuous Flooding

The occurrence of a marked increase in the availability of native and added phosphates in flooded soils as compared to well drained soils has been well established (Aoki, 1941; Beacher, 1955; Shapiro, 1958a,b; Davide, 1960). These increases in phosphate have usually been attributed to the reduction of ferric phosphate to the more soluble ferrous form and the hydrolysis of phosphate compounds. Different reactions of soil phosphates in flooded and nonflooded soils have been attributed to the combination of phosphate ion with ferrous instead of ferric iron usually associated with well drained soils (Mitsui, 1954). This conclusion is supported by the work of Fujiwara ( 1 950), who found that lowland rice responded much better to iron and aluminum phosphate than did barley or upland rice. More recently Davide (1960) studied the solubility, availability, and changes in the chemical forms of phosphate as a result of flooding and drying. His results showed that with CaH4(P04bas a phos-

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phate source, response to rice was almost the same in flooded and nonflooded soil but iron phosphate showed much better response on flooded soils. He, therefore, concluded that the beneficial effects of flooding on phosphate availability depended on the intensity of reduction and the iron content of the soil. His work further showed that the solubilities of calcium, aluminum, and iron phosphates measured by hydrochloric and sulfuric acid extraction of soils incubated under flooded and nonflooded conditions were greater when soils were flooded; with longer periods of incubation the amount of phosphate extracted depended on the soil type rather than phosphate source. Hesse (1962) observed that when excess soluble phosphate was not continuously present there occurred a loss of aluminum phosphate and a gain in iron phosphate with time. When calcium phosphate was added at a lower and more practical rate of 20 ppm. of phosphorus, aluminum phosphate, after an initial increase, decreased within 30 days to its original value. The absence of aluminum-bound phosphate appeared to be due to its transfer to iron phosphate. This transfer of aluminum phosphate to iron phosphate has been shown to take place in acid sandy soil as a result of the lower solubility product of iron phosphate (Yuan et al., 1960). The availability of native and applied phosphate to rice grown on five waterlogged soils was studied by Valencia ( 1 962). He observed that soon after flooding phosphate increased, apparently through the hydrolysis of AIPO, and the reduction of FeP04. After a long period of waterlogging, however, phosphate became less available, probably due to fixation. This was evident in decreasing A values. Similar results were reported by Chiang (1963). Mandal ( I 964) studied the transformation of inorganic phosphate in waterlogged soils in India. On waterlogging, control samples showed a slight increase in 0.5 N acetic acid-extractable phosphorus and a slight decrease in ferric phosphate while aluminum and calcium phosphates were unchanged. In the presence of starch, acetic acid-extractable phosphate increased and calcium phosphate decreased. The large amounts of carbon dioxide formed by the decomposition of starch may have converted some insoluble tricalcium phosphate to more soluble mono- and dicalcium phosphates. Although much ferrous iron was formed there was no decrease in ferric phosphate. Aluminum phosphate was not affected. In the presence of lime, ferric phosphate decreased considerably and aluminum phosphate decreased slightly. On the other hand, calcium phosphate increased appreciably as a result of liming. Some of the ferric

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phosphate was apparently converted to calcium phosphate. This conversion of ferric phosphate to calcium phosphate would probably not have taken place under well drained conditions. The reversion of ferric iron to ferrous iron under waterlogged conditions and its relation to available phosphate was demonstrated by Islam and Elahi (1954). They found a progressive reduction of Fe3+ to Fez+ and an increase in readily soluble phosphate in lateritic soils incubated under waterlogged conditions in the laboratory. Addition of oxidizable materials, especially green manure, promoted the process of reduction and greatly increased the availability of phosphate. Short periods of waterlogging decreased the availability of both soil phosphate and added phosphate to subterranean clover (Kurtz and Quirk, 1965). Williams et al. (1 958) found that soils containing appreciable amounts of ferric phosphate increased the phosphate supply to plants under anaerobic conditions, but soils without ferric phosphate showed a decrease in available phosphate. A marked increase in the extractable phosphorus with the lowering of redox potential below +200 millivolts (at pH 5.7) was reported by Patrick (1964). Extractable phosphorus increased from 10 ppm. to 35 ppm. between the potentials of +200 and -200 millivolts. The fact that at 200 millivolts ferric iron also began to be reduced to the ferrous form tends to confirm that this increase in phosphate came from the conversion of ferric phosphate to the more soluble ferrous phosphate. Tsubota ( 1959) discussed the process of biological phosphate reduction in volcanic soils and humus-rich alluvial soils of Japan. He found that after 1 week of incubation of reducing medium (containing orthophosphate and incubated with small amounts of soil) considerable amounts of phosphite and hypophosphite were detected. Clostridium butyricum and Escherichia coli were reported to be capable of phosphate reduction.

+

2 . Efect of Alternate Wetting and Drying A good percentage of the world’s rice is still rain fed. Due to uncertainties of natural rainfall and purposeful intermittent drainage of flooded rice fields during the growing season, rice soils are often subjected to alternate wetting and drying. Yuan et al. (1960) observed that prolonged alternate wetting and drying reduced the percentage of phosphate in the aluminum form and increased the percentage in the iron form. Zachariah ( 1964) reported that drying decreased the pH and increased the available phosphate of twenty submerged rice soils in India. Sun-drying or oven-drying produced more

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available nutrients than did air-drying. There was no relationship between available nutrients measured in dried soils and in wet soils. Drying a soil subsequent to flooding generally decreases the solubilities of both native and added phosphates (Paul and DeLong, 1949). Drying leads to the conversion of soluble phosphates to less readily extractable forms and to a decrease in the organic phosphorus fraction. The biological reduction of iron during the flooding phase followed by reoxidation during the drying phase results in the enhanced reactivity of the sesquioxide fraction of the soil, leading to an increase in the phosphatefixing capacity and hence a decrease in the solubility of phosphate. Babayan and Gasparyan ( I 962) reported that air-drying generally increased the easily soluble phosphate content of noncalcareous soils and decreased it in calcareous soils. Moistening air-dry soil to field capacity usually increased the easily soluble phosphorus content. 3. Mechanism of Phosphate Release in Flooded Soils

The distribution patterns and plant availability of different phosphate fractions are alternated by the reducing conditions brought about by flooding. Among the various fractions reductant-soluble phosphate is of special significance in waterlogged soils. This fraction consists of phosphate occluded in hydrated ferric oxide and is unavailable under welldrained conditions. Much of the difference between upland soils and flooded soils in response to added phosphate is apparently due to the release of this phosphate fraction as a result of reduction processes. The changes in inorganic phosphate as a result of waterlogging are illustrated for an acid clay soil from Louisiana in Fig. 9, which shows

E

n

240

I

n 120

0

AI-P

Fa-P

Ca-P

RSFe-P

FIG.9. Transformation of inorganic phosphate in a soil as a result of waterlogging (Mahapatra, 1966).

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WM. H. PATRICK JR. AND I. C. MAHAPATRA

the distribution of the various inorganic fractions before and after 1 month of incubation under waterlogged conditions. The greatest change was the conversion of phosphate from the reductant soluble fraction (RS Fe-P) to ferrous phosphate.

FIG.10. Coprecipitation of phosphate compounds within coating of hydrated ferric oxide on soil particles.

The coating of hydrated ferric oxide which is present on silt and clay particles has occluded in it several forms of phosphate, some of which would be available to the crop if they were not separated from the soil solution by the insoluble ferric oxide layer (Fig. 10). Reduction of this hydrated ferric oxide to the more soluble ferrous hydroxide as a result of waterlogging releases the occluded phosphates. Calcium phosphate released in this manner is available to the crop whereas the occluded ferric phosphate is probably not available to the rice crop until it has been reduced to the more soluble ferrous phosphate. It has been demonstrated by various workers that fertilizers added to well-drained soils change gradually into ferric phosphate which upon reduction forms ferrous phosphate. Precipitation of two ferrous phosphates ( 1 ) vivianite, Fe3(P0&.8 H 2 0 , and (2) hydrated calcium ferrous H 2 0 , have been observed in the fertilizer phosphate, Ca2Fe(PO4)~.4 residue under waterlogged conditions when monocalcium phosphate monohydrate was added to soil samples and incubated at 86°F. for 6 to 7 months (Lehr er af., 1959). Vivianite rarely occurs, however, in submerged soils (Eriksson, 1952). Ferric phosphate has also been reported to be reduced by Bacillus circufans(Bromfield, 1954). The phosphate solubility-pH curves for flooded and nonflooded soils were found by Aoki (1941) to resemble those of precipitated ferrous and

NlTROGEN A N D PHOSPHORUS IN WATERLOGGED SOILS

349

ferric phosphate, respectively. Based on this work, Mitsui (1954) postulated that phosphate availability under submerged conditions was governed mainly by the solubility of iron phosphate. The high solubility of phosphate in the alkaline pH range was presumed to be due to the hydrolysis of this compound. Fractionation studies on the inorganic phosphorus in paddy soils have since revealed the dominance of iron phosphate (Davide, 1960; Chang and Chu, 1961; Tyner and Davide, 1962; Mahapatra, 1966). The mechanism of phosphate release in a flooded soil may be explained by (1) reduction of insoluble ferric phosphate to more soluble ferrous phosphate (Islam and Elahi, 1954), (2) release of occluded phosphate by reduction of hydrated ferric oxide coating (Chang and Jackson, 1958; Mahapatra, 1966), (3) displacement of phosphate from ferric and aluminum phosphates by organic anions (Bradley and Sieling, 1953), (4) hydrolysis of ferric and aluminum phosphates (Ponnamperuma, 1955), and ( 5 ) anion (phosphate) exchange between clay and organic anions (Russell, 1962). Another possible process of increase in the availability of phosphorus has been explained by the following reaction, which occurs under highly reduced conditions (Sperber, 1958; Kawaguchi, 1965): Fe,(P0J2 3 Has+ 3 FeS 2 H3P04.This process probably prevails in soils poor in free iron. The presence of large amounts of aluminum enhances the refixation of phosphate that becomes soluble during the reduction of iron phosphate. Large quantities of iron compounds, when incompletely reduced as a result of flooding, also encourage refixation of phosphates. After the soil is dried, oxidation and dehydration cause precipitation of phosphate. Bromfield ( 1960) pointed out that the decrease in phosphate availability may be caused by resorption of phosphate by clay or aluminum hydroxide. Phosphate availability may be considerably reduced as a result of the precipitation of F e P 0 4 in the oxidized rhizosphere of the rice roots (Ponnamperuma, 1965). Fe2+ is oxidized to Fe3+and combines with the phosphate ion. The reactions cited here emphasize the fact that submergence does not invariably lead to an increase in phosphate solubility and availability. Several factors obviously affect the release and refixation of phosphate in a flooded soil.

+

+

4 . E f e c t of Organic Matter

Organic matter affects phosphate transformation in waterlogged soils through the mechanism of reduction and chelation. Both processes lead to increases in the solubility and availability of soil phosphate (Shapiro,

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W M . H. PATRICK JR. A N D 1. C. MAHAPATRA

1958b). The transformation of inorganic phosphate to the organic form, observed in flooded soils (Bartholomew, 193 1; Paul and DeLong, 1949), result in decreased phosphate availability. Evidence of the formation of insoluble aluminum organic complexes of phosphorus due to anaerobic fermentation of rice straw has been provided by Gasser (1956). The role of organic matter in reducing the intensity of phosphate fixation by sesquioxides was considered statistically with data from 15 Indian soils (Datta and Srivastava, 1963). The results indicated a strong interaction of organic matter and pH with the sesquioxide content; when correction was made for these factors the sesquioxide level and phosphate bonding were highly correlated. Organic phosphorus can serve as a plant nutrient only after its mineralization into inorganic forms, Since apparently little mineralization takes place under anaerobic condition, organic phosphate appears to be of little practical value in flooded soil, though organic matter is very important in the mechanisms of reduction and chelation under waterlogged conditions.

5 . Efect of Added Phosphate Fertilizers The manurial value of various phosphate compounds is, to a great extent, determined by the strength with which their Ca2+ ions are bound, for this determines the solubility of phosphate ions in the soil solution. In addition to the solubility of the added phosphate, the relative efficiency of such sources is affected to some extent by soil reactions, native phosphate level, rate, method, and time of application (Davide, 1965). Studies with lateritic rice soils of India indicate that superphosphate, monocalcium phosphate, dicalcium phosphate, ammoniated superphosphate, bone meal, and rock phosphate were almost equally effective in increasing rice yields (Datta and Mistry, 1958; Mahapatra and Sahu, 1961). The percentage of plant phosphorus derived from fertilizer was greatest from monocalcium and dicalcium phosphate. Nelson (1 957), working in the Philippines, got no response to various rates of phosphate applications even on soils with very low levels of soil test (Truog) phosphorus. This lack of response to phosphate application was attributed to fixation of added phosphate, transformation of native phosphate to available forms under flooded conditions, and inadequacy of the analytical method used to evaluate the available phosphate. In a study of phosphate distribution in a soil which had received applications of superphosphate for thirty-one years, Chang and Chu ( 1 96 1) found that the phosphate was retained mostly as iron phosphate with aluminum phosphate next, and calcium phosphate least. These

NITROGEN A N D PHOSPHORUS IN WATERLOGGED SOILS

35 1

authors postulated that the first stage of fixation of phosphate in the soil probably occurs on the surface of the solid phases with which phosphate comes in contact and that the relative amounts and kinds of phosphates formed depend on the specific area of the solid phases associated with aluminum, iron, and calcium. In time aluminum and calcium phosphates gradually change to less soluble iron phosphates, the rate of transformation increasing with the moisture content of the soil. Valencia ( 1962) also noted that all soluble phosphate fertilizers added to the soil undergo similar reactions in that they are converted to aluminum and iron phosphates.

6 . Movement of Phosphate in Flooded Soils The movement of phosphate through the profile is very slow in well drained soils. In flooded soils phosphate moves much more readily through the profile, possibly in association with ferrous iron (Glentworth and Dion, 1949; McGregor, 1953; Cooke and Gasser, 1955). It has been observed that phosphorus movement does not involve aluminum. Ferrous and possibly calcium phosphates are the principal forms involved in movement in waterlogged soils. Describing the mechanism of degradation of paddy soils in Japan, Mitsui (1 954) reported that the iron of the plow layer is first of all reduced as a result of waterlogging. The ferrous iron produced combines with carbonic acid to form bicarbonate which is leached down in the percolating water. Soluble organic matter and especially excessive H2S enhance the process of leaching. The leached ferrous and manganous compounds reprecipitate in the subsoil where oxidation predominates even after waterlogging. In this way, part of the subsoil becomes an accumulation layer, equivalent to the B horizon of a true podzol. It is likely that iron phosphate compounds undergo similar oxidation and reprecipitation in waterlogged soils as evidenced from the data cited by Mitsui (1954). This is borne out by the low total phosphorus content of gley horizons of poorly drained soils (Glentworth, 1947). The maximum concentration of total phosphorus in poorly drained soils occurs in the A horizon. D. PHOSPHORUS NUTRITION OF RICE Although the total phosphorus requirement of rice is similar to that of other small grain crops rice differs markedly from other crops in its response to phosphate fertilizer. As has been demonstrated in a large number of field experiments, rice usually makes little or no response to phos-

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WM. H. PATRICK JR. AND 1. C. MAHAPATRA

phate fertilizer on soils where marked response to phosphate is made by small grain crops grown in rotation with rice (Mitsui, 1954). Where additional phosphate is needed for rice, the crop appears to respond as well to phosphate added to the preceding crop grown in rotation with rice as it does to phosphate added directly to the rice crop (Takihashi, 1965). The residual effect of phosphate added to a previous crop is much more apparent with rice than with other crops. Insofar as phosphorus nutrition is concerned, lowland rice appears to differ from upland crops in at least two important respects. First, the rice plant can absorb an adequate amount of phosphorus from a soil solution with a low concentration of phosphate (Okajima, 1965). Tanaka ( 1 962) found that rice plants grew normally when supplied with a nutrient solution in which the phosphorus content was maintained at 0.1 ppm. At this low concentration a large volume of solution was required. Second, waterlogging releases phosphate from fixed forms to the soil solution as a result of reduction reactions, which probably ensures an adequate supply of phosphorus throughout most of the season in soils that are otherwise defficient in phosphate but contain appreciable amounts of iron phosphate and reductant soluble phosphate. High concentrations of phosphate in the nutrient solution or in the soil solution produce chlorosis. The iron utilization mechanism of lowland rice is weak compared to that of barley and other plants, and high concentrations of phosphate hamper the intake and translocation of iron in the plant (Kimura, 1932; Kimura and Okajima, 1950). A high phosphate content may also create a zinc deficiency as a result of precipitation of zinc phosphate in the soil and in the plant tissue. The absorbing power of rice roots is high during the initial growth stages and decreases as the plant ages (Takahashi et al., 1955). Rao and Rao (1961) found that the absorption curve for phosphorus was sigmoidal with peaks occurring during the vegetative and reproductive stages. Mitsui (1954) reported that the application of phosphorus during the initial growth stages was most effective for good yield. This conclusion was corroborated by Fried and Broeshart ( 1963) from their studies with 32P. Split application of phosphate fertilizer was, however, suggested by Gama and De ( 1 960) from their study of phosphorus uptake during the growth of the rice plant. Patnaik et al. ( 1 965), working with indica rice varieties of different maturation periods, observed that phosphorus absorbed during the tillering stage was efficiently utilized for grain production and was adequate to give an optimal grain yield. Phosphorus taken up beyond this period tended to accumulate in the grain, straw and roots with no advantage to the grain yield.

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E. SOILTESTSFOR PHOSPHORUSI N FLOODEDSOILS As pointed out above, many of the soil test methods commonly used for phosphate generally have proved inadequate as indicators of the soil phosphorus supplying capacity for lowland rice. The poor correlation between extractable phosphate and response of rice to phosphate fertilizer is apparently the result of the rice plant being able to utilize fractions of the soil phosphate other than those attacked by the chemical extractant. Acid extractants that dissolve only calcium and aluminum phosphates will give a poor indication of the available phosphate in soils that contain appreciable amounts of iron phosphate and reductant-soluble phosphate which become available to the crop after waterlogging. It is a well established fact that an increase in the availability of phosphate to lowland rice is caused by waterlogging (Fujiwara, 1950 Shapiro, 1958a; Davide, 1960). Improvement in soil phosphate fractionation methods, especially the scheme developed by Chang and Jackson (1957a) and modified by several other investigators, has permitted an assessment of the dominant fractions of inorganic phosphate in soils and has provided a basis for the development of soil test extractants that dissolve the same form of soil phosphate that are available to rice. The necessity of considering the iron phosphate fraction when selecting a chemical soil test for available soil phosphorus for lowland rice has been emphasized by Tyner and Davide (1 962) and Chang and Juo (1 963). Iron phosphate appears to be the main source of soil phosphate for lowland rice (Rodrigo, 1966) and may be an important source of phosphate for other crops (A1 Abbas and Barber, 1964). In Taiwan Wang and Tseng (1 962) and Tseng and Wang ( 1 963) found that several extracting solutions gave satisfactory correlations with growth response in the greenhouse but failed under field conditions. Both alkaline extractants and acid extractants containing fluoride were used in this study. Vajragupta et al. ( 1 963) reported satisfactory correlation between soil test values for available phosphorus using Bray’s P-2 extractant (0.1 N HCI 0.03 N NH4F) and yield of rice in Thailand. In Louisiana Peterson et al. ( 1 963) also found a significant correlation between phosphorus extracted with Bray P-2 extractant and response to added phosphate fertilizer. In Ceylon, Rodrigo ( 1966) used Olsen’s method (extraction with 0.5 M NaHC03) to correlate soil test values of available phosphorus with response of rice to field applications of phosphate fertilizer. The use of this extractant, which releases at least part of the iron phosphate, enabled Rodrigo to make accurate predictions of response to phosphate fertilizer. The relationship between the amount of

+

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WM. H. PATRICK JR. A N D I. C. MAHAPATRA

phosphate fertilizer required for optimum yields and the amount of extracted phosphate is shown in Fig. 1 1 . Waterlogging the soil for several days before extraction usually increases the extractability of phosphate, especially in soils containing

I 0

I

10

I

1

I

I

I

30 40 50 60 Avallable P20J Ibs per acre 20

-

I

70

FIG. 1 1 . The amount of phosphate fertilizer required for optimum yield of rice as a function of the amount of phosphate extracted from the soil with 0.5 M NaHC03.

iron phosphate and reductant soluble phosphate. Mahapatra ( 1966) found that iron phosphate and reductant soluble phosphate made a significant contribution to the phosphorus extracted with Bray’s P-2 extractant in waterlogged soils. This extractant did not dissolve significant amounts of these forms of phosphate when air-dried samples were used. V. Summary

Waterlogging causes changes in the properties of soils which profoundly affect the nutrition of lowland rice. The root zone is changed from an aerobic to an anaerobic or near-anaerobic environment as a result of the drastic decrease in the oxygen supply in the soil. Oxidationreduction systems in the soil which are generally stable as long as the soil is bathed in oxygen become unstable when the oxygen supply is restricted. Nitrate, manganic compounds, ferric compounds, and sulfate

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are stable in well aerated soils but become unstable when the oxygen supply of the soil is cut off. Nitrate nitrogen is subject to loss through denitrification as a result of the ability of facultative anaerobes to substitute nitrate for oxygen. Manganous manganese and ferrous iron are produced from oxidized compounds of these elements under waterlogged conditions. If reduction is intense enough, sulfate is reduced to sulfide by anaerobic bacteria. Nitrogen reactions in the soil are affected greatly by waterlogging. The decomposition of organic matter is slower under restricted aeration, but the low nitrogen requirement of the organisms involved results in an early release of nitrogen to the soil solution and to the rice crop. Denitrification is probably the major mechanism by which nitrogen is lost from waterlogged soils, although volatilization losses of ammonia can occur under special conditions. Leaching losses of nitrogen are usually not severe. Both nitrate and ammonium ions can be assimilated by the rice plant, but better stability of the ammonium form in waterlogged soils make it the superior form of nitrogen for lowland rice. Advantage can be taken of the reducing conditions in waterlogged soils to increase the utilization of nitrogen fertilizer. Ammonium sources of nitrogen fertilizer are applied several inches deep in the soil before flooding so that there will be little danger of the nitrogen being oxidized to nitrate, in which form it is easily lost from the soil. Phosphate reactions in the soil and phosphorus uptake by rice are affected by waterlogging. The reducing conditions caused by waterlogging activate forms of phosphate that are normally insoluble in well drained soils. Ferric phosphate is reduced to the more soluble ferrous phosphate. Several forms of phosphate which are coprecipitated with ferric oxide are released as a result of the reduction of ferric oxide in the soil. These reactions usually result in a larger amount of phosphate becoming available to a flooded rice crop than would be the case with an upland crop. Fixation of phosphate is also greater in seasonally waterlogged soils as a result of the activation of iron during the submerged period and its oxidation and precipitation when oxygen reenters the soil. Lowland rice is not as likely to respond to additions of phosphate fertilizer as are upland crops. This is due not so much to a lower nutritional requirement for phosphate as to the release of soil phosphate under waterlogged conditions. On soils where phosphorus is required for adequate rice yields the phosphate can either be applied to the rice crop or to crops grown in rotation with rice with about equal results.

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Fitts, J . W., Bartholomew, W. V., and Heidel, H. 1953. Soil Sci. SOC.A m . Proc. 17, 119122. Fried, M., and Broeshart, H. 1963. 5th Japan Conf. Radioisotopes, Tokyo, M a y 1963. Fried, M., and Shapiro, R. E. 1956. Soil Sci. SOC.A m . Proc. 20, 471-475. Fujiwara, A. 1950. Tokoku J . Agr. Res. 1. 223-228. Gama, M. V., and De, L. M . 1960. Agron. Lusit. 22,193-203. Gasser, J. K. R. 1956. Rappt. 5th Congr. Intern. Soil Sci. C 479-49 1 . Gericke, W. F. 1930. Soil Sci. 29, 207-225. Glentworth, R. 1947. Nature 159, 441-442. Glentworth, R.,and Dion, H . G . 1949. J . Soil Sci. 1, 35. Godtrey, C. L., and Riecken, F. F. 1957. Soil Sci. SOC. A m . Proc. 21, 232-235. Goring, C. A. I. 1962. Soil Sci. 93, 21 1-218. Greenland, D. J . 1962. J . Agr. Sci. 58, 227-233. Harter, R. D., and McLean, E. 0. 1965. Agron. J . 57, 583-588. Haseman, J . F., Brown, E. H., and Whitt, C. D. 1950. Soil Sci. 70, 257-271. Hemwall, J. B. 1957. Soil Sci. 83, 101-108. Hesse, P. R. 1962. Nature 193, 295-296. Hong, G o Ban, and J. V. Schuylenborgh, 1960. Netherlands J . Agr. Sci. 8,305-3 16. IRRI. 1964. Intern. Rice Res. Inst. Ann. Rept., Los Banos, Philippines. IRRI. 1965. /ntern. Rice Re,s. In.y/. Aun, R r p t . . Los Banos. Philippines. IRRI, 1966. Intrrn. Rice Res. Inst. Ann. Rept., Los Banos. Philippines. Islam, M. A., and Elahi, M. A. 1954. J . Agr. Sci. 45, 1-2. Jannasch, H. W. 1960. Arch. Hydrobiol. 56, 355-369. Janssen, G., and Metzger, W. H . 1928. J . A m . SOC. Agron. 20, 459-476. Jeffenes, J . W. 0. 1961. J . Soil Sci. 12, 172-179. Kardos, L. T. 1955. I n “Chemistry of the Soil” (F. E. Bear, ed.), ACS Monogr. No. 126. Reinhold, New York. Kawaguchi. K. 1965. “The Mineral Nutrition of the Rice Plant,” p. 266. John Hopkins Univ. Press, Baltimore, Maryland. Keeney, D. R., and Brernner, J. M. 1965. Soil Sci. SOC.A m . Proc. 31,34-39. Kelley, W . P. 191 1. Hawaii Agr. Expr. Sta. Bull. 24. Kellner, 0. 1884. Landwirrsch. Vers. Sta. 30, 18-32. Kimura, J . 1932. J . Imp. Agr. Expr. Sta. Tokyo 2, 1-32. Kimura, J . , and Okajima, H. 1950. Sci. Rept. R I T U , D1, 1-13. Kiuchi, T., and Ornukai, S. 1959. Soil Plant Food (Tokyo) 5, 108-1 13. Kurtz, L. T., and Quirk, J. P. 1965. Australian J . Agr. Res. 16, 403-412. Lehr, J. R., Brown, W. E., and Brown, E. H. 1959. Soil Sci. SOC. Am. Proc. 23, 3-7. Low, P. F., and Black, C. A. 1950. Soil Sci. 70, 273-290. McGregor, A. J . 1953. J . Soil Sci. 4, 86. Mahapatra, 1. C. 1966. Ph.D. dissertation, Louisiana State Univ., Baton Rouge, Louisiana. Mahapatra, I. C., and Sahu, B. N. 1961. Indian J . Agr. 4, 219-224. Mandal, L. N. 1964. Soil Sci. 97, 127-132. Mikkelsen, D. S., and Finfrock, D. C. 1957. Agron. J . 49, 296-300. Mitsui, S. 1954. “Inorganic Nutrition, Fertilization, and Soil Amelioration for Lowland Rice.” Yokendo Ltd, Tokoyo. Moreno, E. C., Lindsay, W. L., and Osborn, G . 1960. Soil Sci. 90,58-68. Nagaoka, M. 1904. Bull. Coll. Agr. Imp. Univ. Tokyo 6, 285-334. Nelson, L. E. 1957. Soil Sci. 1, 63-73. Nhung, M. T. M., and Ponnamperurna, F. N . 1966. Soil Sci. 102, 29-41.

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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed. A

B

Abbot, R. C., 300,301,322 Abifarin, A. O., 77,87 Ableiter, J. K., 18.46 Acharya, C. N., 33 I , 334,356 Adda, Y., 168,196 Adler, I., 154, 157, 166, 173, 185,192 Afanasiev,J. N., 17.44 Agrell, S. O., 154,192 Ahluwalia, M., 69,7 1,74,76,87,88

Babayan, G. B., 347,356 Baddiley, J., 307,319 Badenhuizen, N . P., 275, 286, 288, 297, 308,309,310,312,319

Akatsuka,T.,276,307,311,312,313,318, 321 Al-Abbas, A. H., 353,356 Albee, A. L., 176,193 Alberda, T., 326,356 Alexander, L. T., 257,274 Alexander, M., 33 1,332,356 Al-Fakhry, A. K., 59,87 Allaway, W. H., 244, 245, 246. 248, 259. 261,263,264,265,271 Andersen, C. A,, 154, 173, 176, 178, 180, 192 Anderson, R. A,, 285,318 Anderson, R. J., 244,259. 260,262,271 Anderson, W. B., 133,149 Anderssen, R. S., 148,149 Andres, J. M., 276,318 Andrew, R. H., 277,303,318 Aoki, M., 344,348,356 Appleman, C. O., 302,303,319 Archard, G. D., 170,192 Arisz, W. H., 142, 143, 150 Arnold, R. W., 38,44 Arnon, D. I., 267,271 Arthur, J. M., 302,319 Asher, C. J., 144, 146,150 Athwal, D. S., 55, 56, 66,67,68,69,70,7 I , 72,74,80,8 I , 82,87,88 Atkins, I. M., 21 I, 222,223,232,233 Avigad, G., 3 15,321 Avdulov, N. P., 59,87 Axelrod, B., 304,306,307,319 Aykroyd, W. R., 50,87 Ayyangar, G. N. R., 63,65,87,88

361

Baird, A. K., 184,192 Bais, D., 253,272 Bakhuyzen, H. L., 309,319 Balassa, J. J., 249, 251, 252, 253, 267, 274 Balasubramanian, S. C., 50,87 Baldridge, D. E., 212, 214, 217, 218, 219: 233

Baldwin, M., 28,47 Banejee, B. R., 188,193 Barber, B., 23,44 Barber, G. A., 307,320 Barber, S. A., 92, 138, 140, 141, 144, 145, 147,150,15I, 353,356

Barley, K. P. J., 138, 147,150 Barman, M. L., 165,193 Barnard, C., 202,232 Barnes, D. K., 54,78,87 Barra, H. S., 276,319 Bartholomew, R. P., 336,350,356 Bartholomew, W. V., 8 , 4 4 , 338,357 Basak, M. N., 340,356 Bascialli, P. C., 276,318 Bass, S. T., 300,322 Bates, L. L., 276,319 Bates, L. S., 292,321 Batt,A.P., 161,193 Bauman, L. F., 7 1,87 Baun, W. L., 163,194 Bauwin, G. R., 340,356 Bayard, M., 188, 189, 190,193 Beacher, R. L., 337,344,356 Beaman, D. R., 173,193 Bear, F. E., 260,271 Bear, R. P., 276, 277, 286, 292, 293, 300, 301,309,319 Bear,R.S.,310,319 Bearden,J. A., 166,193 Beardsley, D. W.,74,87 Beath, 0. A,, 244, 246, 248, 262, 266, 273

362

AUTHOR INDEX

Beck,T. R., 11,12,23,45 Beeson, K. C., 244,247,248,262,271,272, 2 73 Beitz, D., 303,320 Bence, A. E., 176,193 Bennett, H. H., 15,28,46 Berg, M. A., 212, 214, 217, 218, 219, 222, 232,233 Berger, K. C., 244,272 Bernfield, P., 308,3 10,321 Bernstein, L., 304,319 Bethe, H. (Von), I7 I, 1 9 3 Bhadrachalam, A., 352,358 Bhatnagar, M. P., 66,87 Bhattacharya, R., 340,356 Bhowmick, H. D., 331,358 Biever,K. 5.,213,219,232 Biggar,J. W., 132,133, 145,151 Bilquez, A. F., 6 8 , 8 7 Bingle, W. D., 188,193 Birks, L. S., 154, 157, 161, 165, 166, 170, 173, 183,193,194 Bitzer, M. J., 211, 212, 213, 214, 224,232, 233 Black, C. A,, 9 , 4 4 3 4 2 , 3 5 7 Black, R. C.,288,289,290,291,310,319 Bollard, E. G., 246,272 Bolt, G. H., 117,150 Bond, A. B., 304,319 Bone, J. F., 262,272,274 Bonner, J., 149, 150 Bonsteel, J. A., 28,44 Borlung, N. E., 202, 207, 21 1, 213, 216, 217, 218, 219, 220, 221, 222, 225, 226, 229,230,232,233 Borom, M. P., 185,193 Bouldin, D. R., 97, 98, 104, 145, 146, 147, 150 Bowen, G. D., 146,150 Bowen, H. J. M., 236,238,243,246,272 Bowles, J. M., 257,272 Bradley. D. B., 349,356 Braidwood, R. J., 3,4,44 Bray, R. H., 92, 114,150, I51 Brehaut, E., 5 , 4 4 Bremner, J. M., 331, 332, 338, 356, 357, 359 Brentano, W., 308,321 Brewer, R. F.. 255,258,272 Briggle,L. W.,211,212,219,232

Brimhall, B., 276,322 Brink, R. A., 277,303,318 Bnx, K., 63,64,88 Broadbent, F. E., 332,356 Broeshart, H., 352,357 Bromfield, S. M., 346,349,358,356,359 Brouwer, R., 142, 143, 144,150 Brown, B., 3 13,319 Brown, D. H., 313,319 Brown, E. H., 341,342,348,357 Brown, J., 247,272 Brown,J. D., 154, 164, 173. / 9 ~ ’ Brown, P. E., 1 5 , 4 4 Brown, R. P., 292,294, 295, 296, 297, 298, 301,302,319 Brown, W. E., 348,357 Broyer,T. C., 145,150 Bruno,G. W., 164,196 Buckman, J., 253,274 Biinning, E., 309,319 Burd, J . S., 343,356 Burhop, E. M. S., 168,193 Burk, R. F., 263,272 Burleson, C. A,, 252,271 Burma, D. P., 307,319 Burnham,C. R., 277,319 Burton, G. W., 52,54,55,56,57,58,59,60, 61, 62, 63, 65, 66, 67, 68, 69, 70,71, 72, 74,78,19,80,82,83,84,85,87,88,89 Butler, B. E., 11,45 Butler, G. W., 246,247,272 Buttrose, M. S., 308,309,319 Byers, H. G., 253,273 Byrd, J. D.. 309,322 C

Cady,J.G., 182,193 Cadwell, D. E., 183,193 Cahn, E., 223,232 Cain, A. J., 2 , 4 5 Calais, D., 168,196 Cameron, F. K., 8 , 4 7 Cameron, J. W., 277,278,319 Campbell, A. S., 186,197 Campbell, W. J., 154, 163,193,196 Cannon, H. L., 257,267,272 Capitant, M., 154, I95 Caputto, R., 276, 307,319 Cardini,C. E.,307,311,312,313,315,319, 320,321

AUTHOR INDEX

Carnahan, H . L., 59,87 Carrero, J. O., 336,359 Carroll, R. L., 151,272 Carslaw, H.S., 96,97,98,99, 100, 101,

112,135,150 Carter, D. L., 244,264,273 Cary, E. E., 244,248,264,265,272,273 Castaing, R., 154,157, I61, 162,164,165,

166,168,169,177,193 Cescas, M. P., 154,181, 185,186,193,194,

I98 Chandola, R. P., 66.87 Chandorkar, K. R., 286,288,319 Chang, S. C., 339,340,341,342,349,350,

353,356 Chapman, H . G., 238,243,244,245,246,

269,272 Chase, Agnes, 5 1,88 Chaudhari, B. B., 80,88 Chavan, V. M., 80.88 Chiang, C. T., 345,356 Chu, W. K., 342,349,350,356 Church, C. G., 302,322 Clark, H., 171,198 Clark, J. G. D., 3,45 Clarke,A. L., 138,139,146,150 Clayton, J. S., I , 46 Cline, M. G., 33,34,35,45 Coe, M.D., 3,45 Coffey,G. N., 1 I , 12,16,21,22,24,29,45 Colby, J. W., 163,165. 166,168,169,171,

173,178,I Y 4 Cole, C. V., 343,356 Cole, D., 277,319 Collins, G. N.,276,319 Columella, 4,5,45 Conant, J. B., 20,45 Connell, W. E., 329,356 Conty,C., 191,194 Cook, M . J., 253,274 Cooke, C. J ., I 9I , 194 Cooke,G. W.,338,351,356 Cooper, R. B.,54,58,88 Cordier, S., 106,15I Cosslett, V. E., 161,169,194 Cox, E. L., 2 I2,219,232 Crank,J., 112,135,I50 Creech, R. G., 278, 284, 285,286,288,

289, 290, 291, 292, 294, 297, 300, 304,312,315,3 16,319

363

Criss,J. W., 173,194 Crowther, E. M., 35,45 Culpepper, C. W., 286,302,303,3 10,319 Cumar, F. A., 276,319 Cunningham, R. K., 338,356 Curtis, B. C., 206,233 Curtis, D. L., 67,88 Curtis, J. J., 308,320 Cutolo, E., 307,321

D daCasa,C., 170,171,194 Daijuhara, G., 336,356 Dainty,J., 143,151 Daniel, L., 259,272 Danielson, R. E., 139,151 Dankert, M., 307,31 1,319,321 Das Gupta, P. C., 290,310,320 Datta, N . P., 350,356 Daum, R. J., 21 1,212,232 Davide, J. G., 344,349,350,353,356 Davies, E. B., 264,265,272,274 Davy, H., 7,45 De, L. M . , 352,357 De, P. K., 332,356 De,S.C.,331,358 Dean, L. A., 149,I50 Deatherage, W. L., 277,285,286,318,319, 322 DeFekete, M. A. R., 311, 315,319,320, 321 DeGeus, J. G., 33 1,334,337,356 Delaune, R. D., 334,356 DeLong, W. H., 347,350,358 Delwiche, C. C., 332,359 Denny, P.J., 183,184,188,190,198 Derouin, D. H., 195 Descamps,J., 154,168,169,193 DeSouza, B.C . , 3 13,320 DeVane, E. H., 78,87 Devasahayan, P., 63,88 Dewey, J., 23,43,45 Deziel, C., 253,272 Dirnler, R. J., 294,322 Dion, H. G., 35 1,357 Dittmer, H . J., 149,150 Dokuchaiev, V. V . , 17,20,43,45 Dolby, R . M . , 161,162,194 Dowker, B. D., 75,88 Doty, D. M . , 302,320

364

AUTHOR INDEX

Dudley, J. W., 276,320 Duggan, R. E., 250,25 1,272 Dumpert, G . ,310,320 Duncan, D. L., 244, 245, 248, 249, 250, 2 73 Duncurnb,P., 165, 170, 171,172, 191,194 Dunn, G . M., 277,320 Durant, Y .,7 1,88 Dutton, R. W., 309,319 Dvonch, W., 277,297,210,320 Dye, W. B., 259,272

E Earle, F. R., 308,320 Earley, E. B., 303,320 East, E. M., 276,277,320 Eaton, A., 11, 12,23,45 Eaton, S. V., 303,319 Eddy, H., 6,46 Edgington, G.. 253,273 Ehlig,C. F., 132,150,248,264,272 Ehwald, E., 4,5,6,45 Elahi, M. A., 346,349,357 Elbein, A. D., 307,320 Elgin, R. L., 166, 168,195 Elion,H. A., 154, 155, 157,194 Elrick, D. E., 154, 182,195 Engstrom, A., 154,194 Eriksson, E., 348,356 Erlander, S. R., 29 I , 3 15,320 Ernst, J. W., 334,356 Eryster, W. H., 276,320 Etasse, C., 77,88 Evans, E. E., 3,45 Evans, J. W., 303,320 Evans, S. D., 141,150 Evatt, N . S., 337,356 F Fallou, F. A., 10, 14, 15,45 Fanshier, D., 307,321 Faulkner, M. D., 334,358 Feldman, F. J., 253,272 Fergason,L. A., 161,194 Finfroch, D. C., 334,357 Fink, R. W., 168,194 Finley, P., I9 1, I94 Fippin, E. O., 15.45 Fireman, P., I7,20,45

Fischer, D. W., 163,194 Fisher, R. M., 157,194 Fitts, J . W., 338,357 Fitzgerald, R. W., 173,195 Flannery, K. V., 3,45 Fodorova, T. N., 20 I , 233 Fonseca Martinez, S., 2 1 I , 2 12,232 Foote, W. H.,214,232 Fortson, J. C., 70,80,85,87 Frazer,J. Z., 166, 168, 173,194,195 Fredricksson, K., 173,196 French,D.,276,309,310,319,322 Freudenberg, K., 3 10,320 Fried, M., 143,150,341,352,357 Frost, D. V., 249,250,272 Frydman, R.B., 31 1,312,313,320 Fujii, M., 298,309,320,322 Fujita, A., 186,196 Fujiwara, A., 344, 353,357 Fukasawa, H., 200,204,205,209,232 G

Gabbiani, G., 253,272 Galle, P..154,195 Garna, M. V., 352,357 Ganguli, N . C., 307,320 Gardner, D. R., 35,47 Gardner, W. R., 92, 112, 131, 132, 148, I50 Gasparyn, 0. B., 347,356 Gasser, J. K., 35 1,356 Gasser, J. K. R., 349,357 Gates, C. T., 149,150 Geering, H. R., 135, 138,150 Gericke, W. F., 336,357 Gerloff, G. C., 246,272 Ghosh,H. P..313,314,320 Gibbs,G. V., 163,198 Gildenhuys, P., 63,64,88 Gill, B. S., 59,60,61,66,67,87,88 Gill, G . S., 66,67,87 Gillespie,J. E., 154, 182,195 Gilmour, J. S. L., 2,45 Ginsburg, V., 307,320,322 Glass, R. L., 304,319 Gledhill, V. H., 149, I50 Glentwood, R.,340,35 I , 357 Glinka, K. D., 2 I , 22,29,45 Godfrey, C. L., 340,357

AUTHOR INDEX Goldschmidt, J . R., 253,272 Goldsmith, J. R., 257,272 Goldstein,J., 166, 192 Gopalan, C., SO, 8 7 Goring, C. A,, 337,357 Graham-Bryce, 1. J . , 92, 103, 108, 109,150, 15 I Gray, L. F.. 2S9,272 Gray,L. J., 154, 166, 173, 185,197, 198 Green, M., 165, 169,195 Greenland, D. J., 332,357 Greenwood, C. T., 276,290.3 10,320 Griffen, E. L., 285,318 Grogan, C. O., 59,87,277,286,322 Guillemin, C., 154,195 Guinier, A,, 154,193 Gupta, A. K., 59,60,88 Gupta, V. P., 69,70,7 I , 72,74,88 H

Hagedoorn, H. L., 168,195 Hagernan, R. H., 276,303,320 Haley, L. E., 353,359 Hallerman, G., 179,197 Handreck, K. A., 145,150 Hanes, C. S., 3 10,320 Hanneman,R. E., 173, 185,193,196 Hansen, R. G., 300,322 Hansen, W. W., 171,198 Hanway, J., 338,358 Hardas, M. W., 63,88 Hariharan, P. V., 65,87 Harley,N.,257,274 Ham, J. R., 262,272,274 Hart, R. H., 74,87 Hart, R. K., 163,195 Harter, R. D., 329,357 Hartley, W. J., 264,272 Hartmans, J., 248,272 Haseman, J. F., 341,342,357 Hasler,M.F., 154, 173, 178, 180,192 Hassid, W. Z., 276, 303, 307, 3 10,320,321 Hathcock,J. N.,267,272 Haunold, A., 2 I 3 , 2 19,232 Hayes, H . K., 276,277,320 Hayes,R.M.,212,219,232 Healy, W. B., 259,272,273 Hegsted, D. M., 255,272

3 65

Heidel, H., 338,357 Heinrich, K. F. J., 154, 164, 165, 166, 168, 176,195, 198 Hemwall, J . B., 342, 343,357 Hendricks, S. B., 142, 143, I50 Henke, B. L., 166, 168,195 Henoc, J., 169,195 Hermsen, J. G., 202,232 Hess, C., 309,319 Hesse, P. R., 340,345,357 Hexter, A. C., 257,272 Heyne, E. G., 2 1 I , 233 Heywood, B. J., 25 I , 272 Hilgard, E. W., 9, 13, 14,22,29,45,46 Hill, C. R., 267,272 Hill, W. W., 244,273 Hill, Helen D., 59,87 Hillier, J.. 154, 195 Hitchcock, A. S., 5 I , 88 Hitchcock, E. W., 13,46 Hixon, R. H., 276,322 Hizukuri, S., 298,320 Hoagland, D. R., 145,150 Hobby, M. G., 173,195 Hodges,H.F.,315,320 Hodgson, J. F., 118, 119,150, 245,272 Holland, R. F., 205,233 Hong, Go Ban, 337,357 Hopkins, L. L., 263,272 Hough, B. K., IS, 46 Howe, B., 3 , 4 4 Howie, R. A., 173,195 Hrishl, N. J., 63,88 Hubbard, J. E., 286,308,320,322 Hudson, H., 7 , 4 6 Hudson, T. G. F., 267,273 Hughes,N.A.,307,319 Hunt, C. B., 15.46 Hutchinson, C. B., 277,320 I Illingworth, B., 3 13,319 Imaseki, T., 336,356 Imrie, B. C., 222,224,232 Ingersoll, R. M., 195 Ingle, J., 303,320 Ingold, M.,201,233 Irving, S. M., 183, 184, 188, 190,198 Islam, M. A., 346,349,357

3 66

AUTHOR INDEX

J Jackson, M. L., 339, 340, 341, 349, 353, 356 Jackson, R. D., 92, 93, 100, 102, 104, 105, 106, 107, 108, 114, 129, 130, 133, 144, 147,148,l49,151 Jaeger, J. C., 96, 97, 98, 99, 100, 101, 112, 135,150 Jain, S. K., 69,87,88 James, A. L., 307,319 Jannasch, H. W., 332,357 Janssen, G., 336,357 Jarilow, A., 4,46 Jaynes, T. A., 290,292,3 16,320 Jefferies, J. W. O., 329,357 Jenny, H.,92, 147,150 Jenny, Hans., 35,46 Jensen, N. F., 22 1,226,232 Johns, A. T., 247,272 Johnson, C. M., 260,26 I , 273 Johnson,Jr.,G. G., 163,198 Johnson, V. A., 202, 204, 205, 206, 107, 21 1, 213, 215, 216, 217, 219, 220, 221, 222,224,232,233 Johnson, W. M., 38,46 Jones, L. H. P., 145,150 Jones, S., 'I 64,197 Joppa, L. R., 202,222,232 Jopson, R. C., 168,194 Jordan, W. S., 291,292,304,320 Joshi, A. B., 63,69,87,88 Jost, W., 109, 110, 122,150 Juo, S. R., 353,356

Kellogg, C. E., 18,46 Kemper, W. D., 92, 93, 100, 102, 103, 104, 105, 106, 107, 108, 109, 114, 116, 117, 118, 119, 121, 127, 128, 129, 130, 133, 140,144,147,l48,149,150,151 Khan, M-U D-D., 64,88 Kherde, M. K., 222,224,232 Kihara, H., 200,20 I , 205,224,232 Kihlman-Falk, E., 146,150 Kimura, J., 352,357 Kirchoff, G . S. C., 308,320 Kirianenko, A., 168, 169,196 Kirkham, D., 8,44 Kirkwood, S., 275,3 11,321 Kiuchi, T., 335,357 Knoblock, E. C., 253,272 Knox, E. G., 35,46,87 Knox, F. E., 75,87 Koffman, D. M., 163,196 Kornfield,R.,313,319 Koyama, T., 328,359 Kramer, H. H., 277, 278, 286, 287, 293, 294,297,301,304,319,320,321 Kramer, P. J., 142, 148,151 Krishnaswamy, N., 52, 58, 59, 61, 62, 63, 64,65,66,67,68,77,88,89 Krisman, C. R., 290,3 16,320 Kronstad, W. E., 214,232 Krull, C. F., 22 I , 232 Kubota, J., 244, 245, 246, 247, 248, 259, 26 1,263,.264,271,273 Kulkarni, R. K., 65,88 Kurahashi, K., 307,320 Kurtz, L. T., 346,357

K Kadam, B. S., 65,88 Kajjari, N. B., 55,88 Kalckal, H. M., 307,321 Kamura, T., 328,330,359 Kandler, H. H., 3 1 I , 320 Kardos, L. T., 341,357 Kauss, H., 3 I I , 320 Kawaguchi, K., 349,357 Keener, H. A., 244,273 Keeney, D. R., 338,357 KeiLK., 154, 157,163, 173,196 Kelley, W. P., 336,357 Kellner, O., 336,357

L Labanauskas,C. K., 261,273 Lacadena, J. R., 206,224,232 Lahr, K. A., 224,233 Lai,T. M., 140,150 Lakin, H. W., 246,262,271,273 Lampe, L., 303,320 Langan, L. N., 244,247,273 Lapham, J. E., 15,28,46 Lapham, M. H.,15, 28,46 Larner,J., 314,321 Larrea, P. E., 212,213, 214, 216,217, 218, 2 19,232

AUTHOR INDEX

3 67

Laties, G. G., 143,150, 151 MacDonald. J. R., 143,150 Laughnan, J. R . , 277,285,292.320 McGregor,A.J.,3SI,357 Lavintman, N., 290,316,320 McIllrath,W. 0.,213,214,233 Lavy, T.L., 140,145,150 McLean, E. O., 329,357 Lazar, V . A., 244,247,264,273 MacMasters, M. M., 277,286,319 Leach, R. M., Jr., 25 I , 273 McNeal, F. H., 202,212,214,217,218, Lebsock, K. L., 21 I , 233 2 19,222,232,233 Ledingham, R. B., 166,168,195 MacNeish, R . S., 3.46 Lee, F. S., 163,196 MacRobbie, E. A., 143,151 Leggett, R. F., 15,46 Magdalena,T., 183,196 Lehr, J. R., 348,357 Magoon, C. A,, 286,302,303,310,319 Leloir, L. F., 307,311,319,320,321 Mahapatra, I. C., 347,349,350,354,357 Lemon, E. R., 246,261,273 Mains, E. B., 277,321 Leng, E. R., 276,320 Majaj, A. S., 263,272 Lent, R.L., 166,168,195 Mamuro,T., 186,196 Leroux,J., 166,196 Mandal, L. N.,345,357 Leuck, D. B., 57.88 Mander, A. E., 1 I , 46 Lewis, D.G., 103,147,150,151 Mangelsdorf, P. C . , 3,46,276,321 Liebig, G. J., 250,273 Manners, D. J., 275,321 Liebig, J., 7 , 8 , 4 6 Marais, P. G., 144,151 Liebhafsky, H. A,, 161,178,196 Marbut, C. F., 6, 15, 2 I, 28,29,30,3 I , 33, Lifshin, E., 173,196 35,46 Lindsay, W. L., 118, 119,150,342,357 Margolin, M., 265,273 Lindstrom, B., 168,196 Marignan, R., 106,151 Livers, R. W., 206,2I I , 224,232,233 Mark, H., 168,194 Loerch, J . D., 288,289,290,291, 307,310, Martin, J. P., 25 1,273 315,319 Maruo, B., 309,322 Loneragan, J. F., 144,146,I50 Mason, K. E., 253,273 Long, J. V. P., 154,192 Mason, P. K., 172,197 Loper, G . M., 246,248,273 Massey, H.F., 334,356 Losee, F . L., 259,263,27 1,272,273 Matrone, G., 267,272 Low, P.F., 92,109,151,342,357 Matsunami, T., 186,196 Lowery, R.S., 74,87 Mattern, P.J., 202,204,206,207,21 I , 2 15, Lucken, K. A,, 20 I , 206,233 216,217,220,222,224,232 Lucretius,., 10,46 Maurice, F., 168,169,196 Ludwig,T. G., 259,272,273 Mayhugh, M. S., 112, I50 Luehrs, F. U., 154,198 Maywald, E. C . , 309,322 Luthra, R. C., 74,87 Melsted, S. W., 1 14,151, 353,359 Lyford, W. H., 29,46 Menon, P.M., 6I , 62,63,68,80,88,89 Meredith, D., 5 I , 88 M Merkle, 0.G., 21 I , 222,224,232,233 Maan, S. S., 201,205,206,233 Mertz, E. T., 292,321 Maasland, D. E. L., 93,108, 116,150 Mertz, W., 253,273 McArdle, L. J., 278,285,286,288,289, Metzger, W. H., 336,357 290,291,304,310,319 Meyer,K.H.,308,309,310,321 McCready, R.M., 303,310,320 Meyers, M. T., 303,320 McCuistion, W. L., 206,207,233 M e n , C . ,22I , 232 MacDonald, H . A., 248,272 Miears, R. J., 334,353,358

368

AUTHOR INDEX

Mikkelsen, D. S., 334,357 Miles, J. W., 148,151 Milner,Y.,315,321 Minamikawa, T., 307.3 1 I , 313.318,321 Minocha, J. L., 6 1,88 Misra, C. S., 352,358 Mistry, K. B., 350,356 Mitchell, J., 1,46 Mitchell, R. L., 245,257,258,273 Mitsui, S., 326, 332, 334, 335, 344, 348, 349,35 I , 352,357 Mo1l.S. H., 161, 163, 164, 188, 191,196 Monteagudo, A,, 206,232 Moore, D. P., 245,264,271 Moreno, E. C., 106,151, 342,357 Morns, C. T., 3 10,321 Morris, D. L., 3 10,321 Morrison, G. H., 270,273 Morrow, K. S., 244,273 Mortimer, C. H., 326,358 Mortimer, D. C., 307,319 Mortland, M. M., 140,150 Moss, H. C., 1,46 Miiller, P. E., 19,46 Muhrer, M. E., 269,273 Muir, A., 17, I 9 , 2 1,46 Mulvey, J,, 170,192 Munch-Petersen, A,, 307,321 Muntzing, A., 52,88 Murakami, M., 186,196 Murata, T., 307,3 I I , 3 13,318,321 Murty, B. R., 76,88 Muth, 0. H., 245,262,264,271,273 N

Nabos, J., 77,88 Nageli, C., 308,321 Nagaoka, M., 336,357 Naidu, N. A., 138,150 Nair,N. H.,61,88,89 Nakayama, N., 3 I 1,321 Nambiar, A. K., 8 0 , 8 9 Narvaez, I., 202, 207, 211, 213, 216, 217, 218, 219, 220, 221, 222, 225, 229, 230, 232,233 Nason, A. P., 25 I , 252,274 Neal, N. P., 277,303,318 Nek Alam, 2 12,233 Neller, J. R., 345,346,359

Nelms,A.T., 171,196 Nelson, L. E., 350,357 Nelson, 0. E., 286,287,292,299,300.3 12, 313,318,321,322 Nesheim, M. C., 25 I , 273 Nettevich, E. D., 201,233 Newfeld, E. F., 307,321 News, O., 10,415 Nhung, M. T. M., 330,357,358 Nicholas, D . J. D., 246,273 Nicholson,J. 8.. 161,196 Nielsen,D. R., 132, 133, 145,151 Nikuni, Z., 298,309,320,322 Nomura, T., 3 1 1,321 Nommik, H., 332,358 Nordin, J. H., 275,3 11,321 Norrish, K., 154, 183, 184,197 Northcote, D. H., 275,321 Nye, P. H.,96,98, 105, 109, 147,151 0

Oberleas, D., 269,273 Odell, B. L., 269,273 Odhiambo, T. R., 22,46 Oehler, E., 20 1,233 Ogilvie, R. E., 157, 161, 174, 175, 188, 191, 196 O’Harra, J. L., 259,272 Okajima, H., 352,358 Okuda, S., 307,321 Oldfield, J. E., 245,264,271 Oliver,S., 140, 141, 145,151 Olsen, S. R.,92,94,96, 100, 102, 104, 105, 106, 108, 114, 121, 129, 130, 139, 140, 144, 146, 147, 148, 149, 151, 343, 356, 358 Olson, L., 6,46 Olson, R. J., 220,223,224,233 Omukai, S., 335,357 Ong, P. S., 164,196 Ortega, J., 22 I , 232 Osborne, G., 342,357 Overstreet, R., 92,150 Ozanne, P. G . , 144,146,150

P Page, N. R., 252,271 Pal, B. P., 2 12,233 Paladini, A. C., 307,319

369

AUTHOR INDEX

Pantulu, J. V., 52,59,60,62,63,88 Parker, M. W., 303,3 10,321 Parnell, F. R., 276,321 Parsons, R., 109,151 Passeron,S., 307,319 Passioura, J. B., 147, 148,151 Patel, S. M., 65,88 Patil, B. D., 62,63,84,88 Patil, J. A., 5 5 , 80,88 Patnaik,M.C.,71,87 Patnaik, S., 352,358 Patrick, W. H., Jr., 326, 327, 328, 329,331, 332,334,336,346,356,358,359 Paul, H., 347,350,358 Paul,J. L., 103, 139, 149,151 Patterson, F. L., 21 I , 212, 213, 214, 224, 232,233

Pazur,J.H.,276,307,317,321 Pearsall, W . H., 326,335,358 Pearson, W. N., 263,272 Peat, S., 288, 304,3 10,321 Percival, G . P., 244,273 Petersen, H. D., 212,219,232 Peterson, F. J., 334,353,358 Pfahler, P. L., 277, 286, 287,293,294,301, 320,321 Pfeiffer,H.G., 161, 178, 196 Pfeifer, R. P., 2 12,2 13,226,233 Philibert,J., 154, 165, 170,196, 1 9 7 Picklesimer, M. L., 179,197 Pierre, W. H., 34 I , 358 Pilney, D. G., 163,195 Ping-Hua Lee, M., 4,46 P1ace.G.A.. 140, 147,151 Ploetz, T., 3 10,320 Polanyi, M., 23,46 Ponnaiya, B. W. X., 63,89 Ponnamperuma, F. W., 326, 330, 335, 336, 349,357,358 Poole, D. M., 170, 172,197 Porter, H. K . , 275,321 Porter, K. B., 2 I 1,222,224,232,233 Porter, L. K., 93, 106, 107, 108, 116, 129, 133,150,151 Potter, A . L., 3 10,321 Powell, J. B., 52, 59, 60, 61, 63, 65, 66, 84, 87,88,89 Prasad, A. D., 268,273 Pratt, P. F., 244,254,267,272,273

Preiss,J.,313,314,320 Proctor, B. E., 309,321 Purves, D., 257,273 Putnam, E. W., 307,321

Q Quackenbush, G . A,, 29,46 Quinones, M. A., 202, 207, 21 1, 213, 216, 217, 218, 219, 220, 221, 222, 225, 229, 230,233 Quirk,J. P., 103, 147,150,151,346,357 Quirk, W. A., 111,334,358

R Rachie, K. 0 . , 7 1 , 7 6 , 8 1 , 8 2 , 8 7 , 8 9 Radok, J. R. M., 148,149 Rahman, H-UR., 64,88 Rajki, E., 207,2 I I , 223,224,233 Rajki, S., 207,2 I I , 223,224,233 Raman, V. S., 6 I , 6 2 , 6 3 , 6 4 , 8 8 , 8 9 Rangasamy, S. R. S., 6 1 , 6 3 , 8 9 Rao, D. V. K., 352,358 Rao, P. K., 80,89 Rao, V. S., 352,358 Rausell-Colom,J.A., 154, 183, 184,197 Ravikovitch, S., 265,273 Raychaudhuri, S. P., 4,46 Reading, R. E., 261,274 Recondo, E., 307,3 11,319,321 Redman, F. H., 336,358 Reed, J. F., 336,358 Reed, S.J. B., 166, 172,197 Reid,A. M., 173,195 Reith, J. W. S., 257,258,273 Reitz, L. P., 226,229,230,23 1,233 Reuther, W., 261,273 Ribbe, P. H., 173,197 Riecken, F. F., 340,357 Rines,H.W.,286,313,321 Roach, J. R., 302,320 Roberts, E. A., 309,321 Roberts, T. H., 205,233 Robertson, L. D., 206,233 Robertson, W. K., 345,346,359 Robinson, W. O., 253,273 Rodrigo, D. M., 353,358 Rodriquez, R., 202,207,2 I 1 , 2 13,2 I6,2 17, 218, 219, 220, 221, 222, 225, 229, 230, 232,233

370

AUTHOR INDEX

Rollins,J.B., 121,150 Rosenfeld, I., 244,246,248,262,266,273 Rosenquist, C. E., 2 12,233 Ross, W. M., 201,205,224,233 Rossell-Perez, M., 3 14,321 Roth, M., 259,263,271 Rovira, A. D., 146, I50 Ruffin, E., 12,46 Rundle, R. E., 276,3 10,319,322 Russell, E. W., 5 , 6, 9, 46, 144, 151, 349, 358 Russell, F. C., 244,245,248,249,250,273 Russell, R. S., 144, 145,151

s Sahu, B. N., 350,357 Salvinien, J., 106,151 Sanchez-Monge, E., 206,232 Sanstedt, R. M., 300,301,322 Sarkar, S. N., 332,356 Sarvella, P., 59,87 Sawatzky, A., 164,197 Schaaf, E., 3 10.320 Schippert,M.A., 161,188,191,197 Schmidt, J. W., 202, 204, 205, 206, 207, 211, 213, 215, 216, 217, 219, 220, 221, 222,224,232,233 Schoch, T. E., 309,322 Schofield, R. K., 92, I51 Schroeder, H. A., 249,251,252, 253, 254, 267,273,274 Schulze, W. E., 277,286,322 Schuylenborgh, J. V., 337,357 Scott,M.L.,263,274 Seaholm, J. E., 334,358 Seckinger, H. L., 294,322 Semple, E. C., 5,46 Shafer, S., 221,232 Shainberg, I., 117,150, 151 Shaler, N. S., 15, 22,29, 4 7 Shankar, K., 69,75,76,87,811 Shapiro, R. E., 143,150, 341,347,349,353, 357,358 Shaw, C. F., 28,47 Shaw, K., 332,356 Shebeski, L. H., 211, 214, 216, 217, 218, 233 Shields, P. K., 165, 170, 172,194 Shirai, S., 186,196

Shuey, E. W., 307,321 Sibertzev, N. M., 18,47 Sieling, D. H., 349,356 Simonsen, G . H., 244,273 Simonson, R. W., I1,26, 27, 29, 33, 35, 38, 39,43,47 Sims, J. L., 339,358 Sing, A., 63,88 Sircar, S. S. G ., 33 1,358 Smith, D., 246,248,273 Smith, E., 307,321 Smith, F. F., 262,271 Smith, G. M., 302,320 Smith, J. V., 154, 169, 173, 174, 175, 183, 197 Smith, P. E. L., 3,47 Soremark, R., 267,274 Somers, G. F., 303,3 10,322 Spanswick, R. M., 143,151 Spencer, V. E., 261,274 Sperber, J. I., 349,358 Sprague, G. F., 276,322 Sraon, H. S.,61,88 Srivastava, S. C., 350,356 Stanford, G., 338,358 Stelly, M. B., 341,358 Stenstrom,R.C., 173, 174,175,197 Stevens, H., 21 1,232 Stewart, B. C.,93, 106, 107, 108, 129, 133, 151 Stojanovic, B. F., 332,356 Strahan, D.,300,301,322 Straughn, M. N., 302,322 Sturgis, M. B., 326, 334, 336, 353, 358, 359 Su, J. C., 307,322 Subbiah, B. V., 339,359 Sugiyama, T., 307,3 11,3 13,321 Sullivan, J. T., 302,320 Summer, J. B., 303,3 10,322 Surft, C. D., 168,194 Sutcliffe, J. F., 146, I51 Suzuki, N., 307,321 Suzuki, S., 307,321 Sweatman,T. R., 154, 183, 184,197 Syers, J. K., 186,197

T Tackett, D. L., 339,358

37 1

AUTHOR INDEX

Takahashi, J., 330,352,358,359 Takai, Y., 328,330,359 Tamhane, R. V., 339,359 Tanaka, A., 352,359 Taylor, J. E., 195 Taylor, T. A., 166,197 Taylorson, R. B., 6 I , 8 9 Teas, H. J., 278,319 Tenny, F. G., 33 I , 359 Teubner, F. G., 244,274 Thacker, E., 262,271 Thatcher, J. W., 154,193 Theisen, R., 165,197 Thomas, P. M., 170, 171, 172,197 Thompson, J. N., 263,274 Thornburg, W. L., 309,322 Thran, L. W., 261,274 Tinsley, I. J., 262,272,274 Tipton, I. H., 25 I , 252,253,267 Toth, S. J., 342,359 Tatuka, I., 6 6 , 8 7 Tousirnis, A. J., 154,198 Truog, E., 338,359 Tsai, C. Y., 286, 287, 299, 300, 312, 313, 321,322 Tscherkes, L. A,, 262,274 Tseng, H. D., 340,353,359 Tso, T. C., 257,244 Tsubota, G., 346,359 Tsunewaki, K., 200,20 I , 224,232 Tulaikoff, N . M., 20,47 Tull,J.,6,47 Turner, D. H., 307,322 Turner, F. T., 328,359 Turner, J . F., 307,322 Turvey, J. R., 288,304,3 10,321 Tyner, E. H., 154, 185, 193, 194, 198, 340, 349,353,356, 359 U

Ueda, S., 300,30 I , 322 Uhl, D. E., 285,318 Underwood, E. J., 238, 243, 250, 251, 2 5 5 , 259,26 I , 274 Usher, A. P., 7 , 4 7 V Vajragupta,Y., 353,359 Valencia, I. G., 345,351,359

Vanderford, C. F., 20,47 Van Der Grift, J., 248,272 Van Schaik, J. C., 93, 103, 105, 106, 107, 108, 109, 121, 127, 128, 129, 140, 150, 151 van Warnbeke, A., 34,47 Vasey, E. H., 140, 145,150 Varro, 5 , 4 7 Vavilov, N . I., 52,89 Vazquez, G., 22 I , 232 Verachtert, H., 300,322 Vidra, J. D., 299,300,301,307,3 15,322 Vieth, D., 164,195 Viets, F. G., 245, 269,274 Villegas, E., 218,233 Vineyard, M. L., 277,286,300,319,322 Vinton, W. H., 251,252,274 Viten, F., 263,272 Vohra, S. K., 62.88 Volgarev, M. W., 262,274 Vose, P. B., 246,274

W Waksman, S. A., 33 I , 359 Walker, J. M., 140, 145, I50 Walker, T. W., 186,197 Wallace, A,, 244,274 Wallerius, J. G., 7 , 4 7 Wallihan, E. F., 332,359 Walters, S. M., 2 , 4 5 , 4 7 Wang, C. H., 353,359 Wapstra,A. H., 168, 195 Wanng, S. A., 33 1,359 Watanabe, F. S.,94,96, 104, 139, 144,151 Watkinson, J. H., 264,265,272 Watson,C. A., 212, 214,217,218,219,233 Weatherwax, J. R., 250,25 1,272 Weatherwax, P., 276,322 Webster, D. L., 171,198 Webster, 0.J., 67,88 Weiblen, P. W., 183,193 Weijer, J., 302,322 Wells,C. B., 154, 183, 184,197 Wells, J. P., 337,339,356,358 Welsh, J . R., 202,232 Wert, C. A., 166, 195 Weswig, P. H., 262,272

372

AUTHOR INDEX

Whelan, W. J., 275,276,288,304,3 10,321, Wolf, M. J ., 294,322 322 Wood, G. C., 173,195 Whistler, R. L., 277,286,287,293,294, Wood, R. F., 263,272 297,30I , 309,310,320,321,322 Wyatt, R.,331,358 White,E. W., 162,183,184,188, 190,198 Whitney, M., 8,9,24,25,26,47 Y Whitt, C. D., 34I, 342,357 Yakowitz, H., 164,176,195,198 Widner,J.N.,211,233 Yamada, N.,334,359 Wiebe, H. H., 142,148,151 Yamamoto, R. S., 262,272 Wiezorek, G. A., 265,272 Yamane, I., 332,359 Wijler, J., 332,359 Yarilov, A. A., 18,47 Wild, A., 341,359 Yoshida, M., 309,322 Williams, B. A,, 298,322 Young,J.0.,253,273 Williams, C. H.,346,359 Young, T.C., Jr., 3,47 Williams, D. E., 144,151 Yuan, T. L., 345,346,359 Williams, E. J . , 143,151 Ywan, W. L., 330,358 Williams,J. D. H., 186,197 Williams, R.F., 346,359 Z Willis, L. G., 336,359 Zachariah, P. K., 346,359 Willis, W. H., 334,359 Wilson, J. A., 201,202,205,207,208,209, Zaslow, B., 309,322 211,212,214,216,217,218,222,223, Zechman,G. R.,Jr., 163,198 Zemany, P. D., 161,178,196 224,225,226,228,233 Zenger, D. H., 184,192 Winslow, E. H., 161,196 Wittry, D. B., 157,161,163,166,178,196, Ziebold,T. O., 174,175,176, 177,178,198 198 Zobel, H. F., 296,297,298,309,322 Wittwer, S. H., 244,274 Zuber, M.S., 277,286,322

SUBJECT INDEX E

A A q i l o p s rctudara, 200

Aegilops ovuta, 200,204,209 Aegilops ventricosa, 201 Alfalfa, 248 Alfisols, 28 Alluvion, 12 Aluminum, 141-142, 245, 340, 342, 345, 346,349,350 Ammonia, 33 I , 332,334-337 Amylopectin, 276, 303 Amylose, 276, 303 Anion absorption, 341 Anions, interdiffusion, 92-1 10 Arsenic, 239,249-250 Avena sativa, I49

B

Electron microprobe analyzer, 153- 198 Environmental cycles, 236-239 Ergot, 226-227 F Fluorine, 239, 255-257 Forage yield, pearl millet, 70-71, 72-74, 77, 85-86 G

Geest, 12 Genetics corn endosperm, 276-302 cytoplasmic sterility, 200-2 10 fertility restoration, 204-2 10 heterosis, 2 10-2 14 H

Barley, 144 Beans, 269 Beryllium, 239 Bluegrass, 149 Boron, 141-142.244 Bromine, 25 I

Holcus, 5 I Humus, 7

I Iodine, 238,239,247,25 I Iron, 244, 247, 327, 328, 329, 330, 336, 340, 345,346,349

C Cadmium, 25 1-253 Calcium, 140-141, 255, 256, 340, 341, 342, 350 Cations,diffusion in soil, 110-120 Chaetochloa, 5 1 Chamaeraphis, 5 1

K Kaolinite, 342 L Lead, 238,257-258 Lime, 250,345 Loose smut, 226

Chelating agents, 118-120 Chernozem, 17, 18 Chloride, 138-139, 147, 148, 149,251 Chromium, 238,239,253-254 Claviceps purpurea, 226 Clay, 1 17 Cobalt, 238,239,246,248 Copper, 141-142, 238, 239, 244, 248, 258262 Corn, 139, 140, 141, 144, 147 carbohydrate synthesis, 275-322 Cytoplasmic sterility, 55-56, 67-68, 80-83, 200-2 10

M Magnesium, 140-141 Maize, carbohydrate synthesis, 275-322 Male sterility, cytoplasmic, 55-56, 67-68, 80-83,200-2 I0 Manganese, 141-142, 238, 244, 245, 327, 329,330,340 Medicago sativa, 248 Mollisols, 8 , 2 8 Molybdenum, 139-140, 244, 246, 247, 249, 258-262,267

373

374

SUBJECT INDEX

N Napier grass, 84 Nickel, 239 Nitrate, 138-139, 147 Nitrogen, waterlogged soil, 330-339 Nutrient, movement to plant roots, 9 1- 15 1 0

Oats, 149 Ortstein, 19 P Panicum, 5 I Pearl millet, 49-89 breeding, 72-86 cytogenetics, 52,58-65 forage, 70-7 1,72-74,77,85-86 genetics, 65-72 male sterility, 55-56,67-68,80-83 reproduction, 53-58 Pedon, 35-38,40-42 Penicillaria, 5 1 Pennisetum albicauda, 5 1 Pennisetum americanum, 5 I Pennisetum dubium, 63 Pennisetum echinurus, 5 1 Pennisetum glaucum, 5 I , 83 Pennisetum nigritarum, 5 1 Pennisetum orientale, 63 Pennisetumpurpureum, 63,64,83,84 Pennisetum spicatum, 5 1 Pennisetum squamulatum, 63 Pennisetum iyphoides, 50,63,64,83 Phosphorus, 139-140, 144, 147, 148-149, 245,252,255 waterlogged soil, 338,339-354 Photoperiodism, pearl millet, 54-55.68 Phytoglycogen, 288-291, 303-304, 3 10, 3 15 Plant root, ion diffusion, 146- 148 nutrient sink, 143-145 Poa pratensis, 149 Podzol, 7, 19,38 Potassium, 140-141,338 R

Rhizosphere, 147 Rice, availability of nitrogen and phosphorus, 323-399

Rye, 144 Ryegrass, 247

S Secale cereale, I49 Selenium, 238,244,246,248,250,262-266 Setaria, 5 1 Soil basic entities, 33-42 classification, 4-6, 13, 15, 18,39-42 concept of, 1-47 electron, microprobe analyzer, 153- 198 ioncapacity, 104-106, Ill-115, 129-130 ion diffusion coefficient, 106- 1 10, 1 15- 1 18, 129 mapping, 24,39-42 plant growth medium, 2-10 redox potentials, 326-330 waterlogged, 323-359 zones in submerged, 324-326 Soil series, 26 Soil type, 26-27 Soybean, 141,247 Spodosol. 19,38 Starch formation, corn, 292-299,308-3 17 Sugar beet, 247 Sugar cane, 247 Sulfate, 327,329 Sulfur, 139-140

T

Tessera, 36 Tomato, 149 Trace element, cycling, 235-274 Triticum aestivum, 20 1, 202,205,209 Triticum boeoticum, 20 I Triticum dicoccoides, 200,204,205,209 Triticum dicoccum, 20 I , 205 Triticum durum, 200,209 Triticum macha, 209 Triticum timonovum, 20 1 Triticum timopheevi, 201, 202, 205, 206207,209,2 10,2 I7 Triticum vavilm'i, 209 Triticum vulgare, 200 Triticum zhukovskyi, 20 1

375

SUBJECT INDEX

U Ultisols, 7, 8 Uranium, 257 ustilago tritici, 226

Waterlogged soil, nitrogen and phosphorus, 323-359 Wheat, 138,148,264 hybrid, 199-233 Winter rye, 149

V Z

Vanadium, 266-268 W

Water, ion transport, 145, I 49

Zinc, 118, 141-142, 2 3 6 , 2 3 7 , 2 3 8 , 2 3 9 , 2 4 5 , 246,250,252,268-270

K E Y W O R D TITLE I N D E X Volumes 1-20 A

Agricultural engineering, progress in, 5, 185 Agricultural lands, abundance of earthworms, 13,249 Agricultural machinery, recent developments in, 11, 171 Agricultural trends, in Old Cotton Belt, 9, I Agriculture, in England and Wales, 3, 165 international collaboration in, 6,95 Agronomy, in Canada, changing pattern, 5,3 19 in Southwest United States, 12,197 statistics contributions to, 9, 177 Alfalfas, creeping, 15, 3 I7 Alfalfa improvement, 1, 205 Alfalfa meadows, management after seeding, 3, 93 Alkali soil, plant growth on, 1, 1 Ammonia, anhydrous, 8,61 reactions in soils, 10, 325 Amorphous inorganic materials, in soils, 16,327 Animals, nitrate poisoning, 16, I97 Atomic energy and plant sciences, 4, 279 Australia, grassland agronomy in, 4, I Australian soils, responses to fertilizers, 10,167 B Barley yellow dwarf virus disease, 13, 2 I7 Boron, in soils and crops, 1,32 1 Braunerde, concept of, in Europe and United States, 9,217 Breeding grasses, for southeastern states, 3, 197 C Carbohydrate synthesis in maize, 20, 275 Castorbeans, 10, 257 Chemical weathering, of soil minerals, 5, 219

Chemical weed control, impact on farm management, 15, 161 Clay fraction of soils, 12, 1 Clay minerals, in soils, 1, 159 Clay-water interaction, physical chemistry of, 13,269 Climate, and crop adaptation, 6, 199 Clover, ladino, 2,207 subterranean, 13,57 Coal mine spoils, reclamation, 2, 3 17 Colorado mountain soils, 17,233 Conservation, and productivity, 5, 383 new grasses and legumes for, 10, 349 Copper, in nutrition, 4,147 Corn, mineral nutrition related to growth, 8,32 1 Corn belt soils, new concepts of management, 6, 12 1 Corn breeding, biochemical, 19,45 Corn improvement, objectives, 16, 101 Corn plant population, relation to soil productivity, 10,435 Cotton, 2, 1 Crops, nitrate accumulation, 16, 197 Crop adaptation, and climate, 6, 199 Crop production, fertilizeruse in, 11,133 Cytogenetics, and pearl millet breeding, 20,49 D Defoliation, 9, 67 Desiccation, 9,67

E Earthworms, and significance in agriculture, 13,249 Electron microprobe X-ray analyzer, and soil, 20, 153 Energy environment, of plants, 18, 17 1 Erosion, rainfall, 14, 109 wind, physics of, 15,2 I I Europe, Braunerde concept, 9,217

376

KEYWORD TITLE INDEX, VOLUMES

F Fertilizer(s), advances in, 17, 1 Australian soil responses to, 10, 167 crop response in relation to “available” phosphorus, 16,59 eficient water use, 14, 223 evaluation by biological methods, 14, 265 in forestry, 12, 127 molybdenum, 8, 163 new, 1, 39 residual effect of, 9, 205 Fertilizer use, changing concepts, 10,67 economics in crop production, 11, 133 Fertilizer production and technology, 11, 233 Field crop production and soil management, 8, 1 Field plant physiology, 16, I Forage crops, preservation and storage, 2,273 Forage seed mixtures, factors in compounding, 4, 179 Forests, soil and growth, 4,329 Forestry, fertilizers in, 12, I27 Fruit and nut production, progress in, 17, 283 G Grain disease, barley yellow dwarf, 13,217 Grasses, for conservation, 10,349 Grassland agronomy, in Australia, 4, 1 Grass breeding, progress, 8, I27 Grass seed production, technological advances in, 12,41 Great Plains, agronomic trends and problems, 10, 1 Green manuring, 7, I4 I H Horticulture in Canada, changing pattern, 5,319 Hybrid wheat, 20, 199

1-20

377

Irrigation agriculture, in the United States, 3,323 Irrigation-salinity relation, 16, 139 Isotopes, in soil physics research, 14, 321

L Ladino clover, 2,207 Laterite, 14, I Legumes, for conservation, 10,349 Legume seed production, technological advances in, 12,4 1 Lespedezas, 9, 113 Liming, 10,475 M Maize, carbohydrate synthesis in, 20, 275 Mechanical resistance, soil factor influencing growth, 19, 1 Mediterranean pasture, ecology of, 18, 1 Microbial activities in soils, 7, 75 Microbiology in soil science, 7, 399 Micronutrient elements, chemistry in soils, 15, 119 Mineral nutrition, of corn, 8, 321 of plants, 7, 75 of soybeans, 12, 229 Mineralization of nitrogen in soil, 7, 299 Mississippi Valley prairie soils, 2,157 Molybdenum as fertilizer, 8, 163 N Nematode control, chemical treatment of soil, 3, 243 Nitrate, in crops and animals, 16, 197 Nitrogen, 2, 8 1 applied to soils, 18,2 19 availability to rice in waterlogged soils, 20, 323 effect on phosphorus availability to plants, 11, 369 mineralization of, 7,299 Nitrogenous fertilizer, anhydrous ammonia, 8,61 Nutrients, movement to plant roots, 20, 91

0

I Iron chlorosis in plants, 13, 329 Irrigated pastures, 2, 351

Old Cotton Belt, agricultural trends in, 9, I Organic soils, 8, 377 classification, 17, 115

378

KEYWORD TITLE INDEX, VOLUMES

P

Pastures, irrigated, 2,35 1 Pearl millet breeding, and cytogenetics, 20, 49 Petroleum hydrocarbons, soil contamination, 13, 197 Phosphorus, availability to rice in waterlogged soils, 20,323 nitrogen effect on availability to plants, 11,369 soil, 19, 15 1 Phosphorus fixation, by soils, 9, 95 Pinus radiata forests, soil and fertilizer requirements, 19, 307 Plants, crop, evaluation for winter hardiness, 8,203 crop and pasture, competition, 15, 1 energy environment of, 18,17 1 iron chlorosis in, 13,329 response to soil compaction, 16, 181 soil colloid and mineral nutrition, 4,67 Plant breeding, population variability in, 16,28 1 Plant growth, and soil aeration, 18,57 soil manganese in, 4,22 1 Plant introduction, federal service to agriculture, 7, 189 Plant nutrient behavior, changing concepts, 10,67 Plant nutrient losses, by water erosion, 15, 303 Plant nutrition, potassium in, 6,253 Plant roots, movement of nutrients, 20, 91 Plant sciences, and atomic energy, 4, 279 Podzolic soils, 13, 1 Potassium, in plant nutrition, 6,253 soil, 3, 113 Potato production. 1, 353 Prairie soils, Mississippi Valley, 2,157 R

Rainfall erosion, 14, 109 Range improvement, progress in, 6, 1

1-20

Reclamation, of coal mine spoils, 2, 317 Red clover, 12,365 Rice culture in United States, 14,61 Root and shoot growth, mechanical resistance influencing, 19,l S

Safflower, 10,289 Saline soil, plant growth, 1,l Salinity, relation to irrigation, 16,139 Seed-flax improvement, 6,143 Seed stock preservation, 19,87 Silica, in soils, 15,339 in plants and animals, 19, 107 chemical treatment, nematode control, 3,243 clay fraction of, 12, 1 clay minerals in, I, 159 fate of nitrogen, 18,2 19 Soil@), and forest growth, 4,329 land use, in the Netherlands, 11, 333 micronutrient elements in, 15,119 reactions of ammonia in, 10,325 water infiltration, 12,3 1 1 Soil aeration, and plant growth, 18,57 Soil aggregation, 7, 1 dynamics, 18, 107 Soil bulk density, measurement of, 9, 159 Soil colloid, mineral nutrition of plants, 4, 67 Soil compaction, plant response to, 16,181 Soil concept, 20, 1 Soil conservation, 3,265 Soil contamination, by petroleum hydrocarbons, 13, 197 Soil fertility, role of sulfur, 10,407 Soil humic substances, structural chemistry, 17, 327 Soil investigations, and electron X-ray analyzer, 20, 153 Soil management, for conservation and productivity, 5,383 Soil manganese, in plant growth, 4,22 1 Soil microorganisms, and plant roots, 1,241 Soil minerals, chemical weathering of, 5,2 19 Soil nitrogen, 2 , 8 1

KEYWORD TITLE INDEX, VOLUMES

Soil nitrogen balance sheets, 7 , 2 13 Soil organic fraction, 5, 1 5 3 Soil oxygen, characterizing with platinum microelectrode, 16,249 Soil penetrability, measurement of, 9, I59 Soil phosphorus, 19, I5 1 fixation, 1, 391 Soil physics, isotopes in, 14,321 Soil potassium, 3, 113 Soil tests, determination of lime and fertilizer requirements, 8,24 I Soil water, control of, 2, 233 requirement and availability, 6,67 Soil-water behavior, transport coefficients, 18,327 Sorghum, maturity genes of, 19,267 Sorghum improvement, 6,305 Southwest United States, agronomy in, 12, I97 Soybean(s), 1,77 management, 14,359 mineral nutrition, 12, 229 physiology of, 12, 265 Soybean genetics and breeding, 14, 149 Statistics, contributions to agronomy, 9, 177 Stubble mulch farming, 13,125 Subsoil, 3 , l Subterranean clover, 13,57 Sugar beet improvement in United States, 7, 89 Sulfur, in soil fertility, 10,407 Swedish crop production, recent changes, 7,39 Sweetclover improvement, 17,163

T Tall fescue, 8,283 Tobacco, growth and mineral nutrition, 19, 21 1

1-20

379

Trace elements, agronomic control over environmental cycling, 20,235 Trace element usage, technological aspects, 6,183 Turfgrass in the United States, 18,259 U

United States Braunerde concept in, 9,2 I7 turfgrass management in, 18,259

V Vegetable production, 2, I 13 Vegetation control, on industrial lands, 4, 305

W Water, relation to soils and crops, 11,l Water erosion, plant nutrient losses, 15, 303 Water inflltration, into soils, 12,3 1 I Water use and fertilizers, 14,223 Weed control, 1,289 in southern United States, 7,25 1 Wheat, hybrid, 20,199 Wheat breeding, for quality, 17,85 Wheat improvement, half century of, in United States, 5, I Wheat stem rust, race 15B, 10,143 Wind erosion, and control, 15, 2 1 1 Winter hardiness, crop plant evaluation, 8,203 Y Yield variation, physiological basis of, 4, 101

Z Zinc deficiency, its control, 9,3 I

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