Advances in Agronomy, Volume 50

VOLUME 50

d:.

Advisory Board Martin Alexander

Eugene J. Kamprath

Cornell University

North Carolina State University

Kenneth J. Frey

Larry P. Wilding

Iowa State University

Texas A&M University

Prepared in cooperation with the American Society of Agronomy Monographs Committee M. A. Tabatabai, Chairman D. M. Kral S. E. Lingle R. J. Luxmoore W. T. Frankenberger, Jr. S. H. Anderson P. S. Baenziger

G. A. Peterson

S. R. Yates

D V A N C E S IN

Agronomy VOLUME 50 Edited by

Donald L. Sparks Department of Plant and Soil Sciences University of Delaware Newark, Delaware

ACADEMIC PRESS, INC. A Division of Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto

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Contents CONTRIBUTORS .......................................... PREFACE ................................................

vii ix

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

1.

I1 .

I11.

IV . V.

R . K . Downey and S. R . Rirnmer Introduction ............................................. Improving Yield .......................................... Improving Resistance to Pests ............................... Future Prospects.......................................... Summary and Conclusions .................................. References ..............................................

1

10 24 39 49

so

POPULATION DIVERSITY GROUPINGS OF SOYBEANBRADYRHIZOBIA Jeffry J . Fuhrrnann

1. Introduction ............................................. I1 . Genotypic Groupings ..................................... Ill . Phenotypic Groupings ..................................... IV . Summary of Phenotypic and Genotypic Relationships ............ V . Taxonomic Srarus of Bradyrhizobium japonirum ................. VI . Concluding Remarks ...................................... References ..............................................

67 68 69 93 93 95 96

CROPRESPONSESTO CHLORIDE Paul E . Fixen I . Introduction ............................................. I1 . Chloride in Plants ........................................ Ill . Yield and Quality Responses to Chloride ...................... IV . Chloride Sources, Losses, and Application ..................... V Predicting Crop Response to Chloride ........................

VI . Summary and Future Research Needs ......................... References .............................................. V

107 108 12.5 133

135 141 143

CONTENTS

vi

REDOXCHEMISTRY OF SOILS Richmond J . Bartlett and Bruce R .James I . Introduction ............................................. I1 . Nature of the Electron ..................................... I11 . Derivation of Thermodynamic Relationships for Electron Activity in Soils ................................................. IV . Kinetic Derivation of Thermodynamic Parameters for Redox ...... V . Uses of pe - pH Thermodynamic Information. . . . . . . . . . . . . . . . . . . VI . Uses of pe - pH Diagrams ................................... Reduction Status of Soils ............ VII . Measurement of Oxidation . Free Radicals in Redox Processes ............................ VIII . IX . Manganeseandlron ....................................... X . Soil Chromium Cycle ...................................... XI . Photochemical Redox Transformations in Soil and Water . . . . . . . . . XI1. Humic Substances ........................................ XI11. Wetland and Paddy Properties and Processes ................... XIV . Empirical Methods for Characterizing Soil Redox . . . . . . . . . . . . . . . References ..............................................

152 153

155 158 160 165 172 176 178 187 188 190 195 198 205

PLANTNUTRIENT SULFURIN THE TROPICS AND SUBTROPICS

N. S. Pasricha and R. L . Fox 1. Introduction .............................................

I1. Extent of Sulfur Deficiency ................................. Ill . Forms of Sulfur in Soil ..................................... IV . Sulfur Cycling in the Tropics ............................... V . Effects of Acid Rain ....................................... VI . Sulfur in Irrigation Waters ................................. VII . Sulfate Retention in Soil ................................... VIII . Diagnosis of Sulfur Needs .................................. IX . Critical soil Solution Concentration .......................... X . Crop Responses .......................................... XI . Sulfur Fertilization and Crop Quality ......................... XI1. Sulfur Interactions with Other Elements ....................... XI11. Summary and Conclusions .................................. References ..............................................

210 211 215 217 223 226 227 237 241 246 252 256 257 260

INDEX

271

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

Contributors Numbers in parentheses indicare the pages on which the authors’ contriburions begin.

R I C H M O N D J. B A R T L E T T (1 5 l ) , Department of Plant and Soil Science, University of Vermont, Burlington, Vermont 05405 R. K . D O W N E Y (l), Agriculture Canada Research Station, Saskatoon, Saskatchewan, Canada S 7 N OX2 P A U L E. FIXEN (107), Potash 6 Phosphate Institute, Brookings, South Dakota 57006 R. L. F O X (209), Department of Agronomy and Soil Science, University of Hawaii at Manoa, Honolulu, Hawaii 96822 JEFFRY J. F U I I R M A N N (67), Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware 1971 7 B R U C E R. JAMES (1 5 l ) , Department OfAgronomy, Univerdy of Maryland, College Park, Maryland 20742 N. S. PASRICHA (209), Department of Soils, Punjab Agricultural University, Ludhiana, India S. R. RIMMER ( l ) , Department ofplant Science, University o f Manitoba, Winnipeg, Manitoba, Canada R 3T 2 N 2

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Preface Volume 50 includes state-of-the-art reviews written by recognized experts on several topics of interest to crop and soil scientists. The first chapter discusses advances in agronomic improvement in oilseed brassicas. These cruciferous crops are cultivated throughout the world as vegetable crops for human consumption, as condiments and spices for improved flavor of human diets, and as fodder crops for livestock feeding. However, the largest cultivation of these crops is for edible vegetable oil production. The first chapter also reviews the world socioeconomic importance of the oilseed brassicas, ways to improve yields and resistance to pests, and future improvement of oilseed brassicas through molecular genetics and other biotechnological means. The second chapter presents a comprehensive overview of population groupings of soybean bradyrhizobia, including discussions on genotypic groupings; phenotypic groupings include serology, intrinsic antibiotic resistance, uptake hydrogenase, dissimilatory nitrate reduction, rhizobitoxine, surface polysaccharides, protein profiles, rhizobiophage typing, plant growth regulating substances, and other phenotypes; a summary of phenotypic and genotypic relationships, and the taxonomic status of Bradyrhizo-

bium japonicum. The third chapter is a comprehensive review of crop responses to chloride. Topics covered are aspects of chloride in crops, including biochemical functions, osmoregulatory functions, disease suppression, crop development, and interaction with other nutrients, plus yield and quality responses of various crops to chloride, chloride sources, losses, and application, and ways to predict crop response to chloride. The fourth chapter presents a thorough treatment of redox chemistry in soils, a topic of immense interest to soil and environmental scientists, and which Advances in Agronomy has not reviewed in many years. Discussions on the nature of the electron, derivation of thermodynamic parameters for redox, use of pe- pH diagrams, measurement of oxidation - reduction status of soils, free radicals in redox processes, manganese and iron, the soil chromium cycle, photochemical redox transformations in soils and waters, humic substances, wetland and paddy properties and processes, and empirical methods for characterizing soil redox are included in this review. The fifth chapter is concerned with plant nutrient sulfur in the tropics and subtropics. Topics reviewed include the extent of sulfur deficiency in these areas, sulfur cycling in the tropics, effects of acid rain, sulfur in ix

X

PREFACE

irrigation waters, sulfate retention in soil, diagnosis of sulfur needs, critical soil solution concentrations, crop responses, sulfur fertilization and crop quality, and interactions of sulfur with other elements. Many thanks to the authors for their excellent chapters. DONALD L. SPARKS

AGRONOMIC IMPROVEMENTIN OILSEED BRASSICAS R. K. Downey' and S. R. Rimmer2 'Agriculture Canada Research Station, Saskatoon, Saskatchewan, Canada S7N OX2 'Depanmenr of Plant Science, Universiry of Manitoba, Winnipeg, Manitoba, Canada R 3 T 2N2

1. Introduction

I!. 111.

IV.

V.

A. World Socioeconomic lmportance of the Oilseed Brassicas B. Brossira Oilseed Species Improving Yield A. Seed Yield B. Oil and Protein Yield Improving Resistance to Pests A. Diseases B. Development of Herbicide-Tolerant Cultivars Future Prospects A. Interspecific Hybridization B. Improvements Based on Biotechnologies C. Uses of DNA Markers Summary and Conclusions References

I. INTRODUCTION Brassica and other closely related cruciferous crops are widely cultivated throughout the world as vegetable crops for human consumption, as condiments and spices for improved flavor of human diets, and as fodder crops for livestock feeding. However, the largest cultivation of these crops is for edible vegetable oil production. In recent years, a number of monographs and reviews (Tsunoda et al., 1980; Downey, 1983; Stefansson, Adwnrtr rn A ~ w n a n y Yo/ , $0 Copyrighi 0 1993 by Academic Press, Inc. All nghts of reproducuon in MY form reserved.

1

2

R. K. D O W N E Y A N D S. R. RIMMER

1983; Scarisbrick and Daniels, 1986; Downey and Robbelen, 1989) have dealt in detail with many aspects of oilseed Brussicu improvement, especially those which relate to improvements in fatty acid composition and the reduction in levels of glucosinolates in the residual meal. Substantive changes in the quality of seed oil and meal composition have resulted in dramatic increases in areas of production in Canada and western Europe (see below). Unfortunately, this has also resulted in a rather narrow germ plasm base of cultivated oilseed brassicas, especially in Brussicu nupus L. Emphasis in plant breeding has consequently shifted from quality improvement toward increasing seed yield, incorporating resistance to diseases and pests, and improving tolerance to stress. This review focuses on recent developments and current and future trends for agronomic improvements in oilseed Brussicu crops.

A. WORLD SOCIOECONOMIC IMPORTANCEOF THE OILSEED BRASSICAS Historically, human consumption of vegetable oil obtained from Brussicu spp. was primarily concentrated in asiatic countries, predominantly in the northern Indian subcontinent and in China. The cultivation in these countries of oilseed types of Brussicu rupu L. (syn. Brussicu cumpestris L.) and Brussicu junceu (L.) Czern. dates back to approximately 1500 BC (Prakash, 1980), and these areas today are still major producers and consumers of Brussicu vegetable oils. Since the second world war, a dramatic increase in Brussicu oilseed production has occurred worldwide. In Canada and in Europe this was associated with seed quality improvements through plant breeding involving the modification of the fatty acid composition (elimination of erucic acid) and the reduction of glucosinolate content in the residual meal. The large production increase in Europe was also related to economic support from the Common Agricultural Policy of the European Economic Community (EEC). Thus, in the 1948- 1952 period, 70% of a world total oilseed Brussicu production of 2.8 million tonnes was produced in Asia, but by 1984, Canada (20%) and Europe (35%) produced more than half the total world production of 15.9 million tonnes, with the Indian subcontinent ( 18%) and China (25%) producing the balance (Bunting, 1986). Total world production values of oilseeds, edible vegetable oils, and residual protein meals for the years 1985-1989 are given in Table I. Oilseed brassicas account for approximately 10% of total world oilseed production and 14- 15% of the total edible vegetable oil production. Production by the primary producing regions of oilseed brassicas is shown in Table 11. Total world production is now in excess of 20 million tonnes annually.

3

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS Table I World Production of Oilseeds, Edible Vegetnble Oils, and Derived Protein Meals, 1985 - 1989a

Millions of t o n n e produced by years Commodity

1985- I986

1986- 1987

1975- 19Mb

1988- 1989'

Oilseeds Soybean Cottonseed Peanuts Sunflowerseed Rapeseed Flaxseed Coconut Palm kernel

97.03 30.63 19.94 19.56 18.57 2.36 5.32 2.56

97.92 27.13 20.44 19.25 19.46 2.69 4.72 2.60

103.17 3 1.05 19.72 20.5 1 22.97 2.28 4.24 2.67

93.13 32.24 21.56 20.96 21.72 1.75 4.56 2.89

195.57

194.21

206.61

198.81

13.85 3.47 2.94 6.65 6.19 1.63 3.3 I 1.11 8.17

15.19 3.05 3.10 6.57 6.83 I .56 2.95 I .09 8.09

15.20 3.46 2.85 7.20 7.65 I .90 2.59 8.53

14.85 3.62 3.35 7.57 7.27 1.41 2.76 I .26 9.36

47.32

48.43

50.56

5 1.45

6 1.07

1.89 1.33

67.12 9.83 4.42 7.54 11.09 I .20 1.72 I .32

67.37 11.17 4.01 8.13 12.51 1.14 1S O 1.41

65.54 11.62 4.77 8.59 11.80 0.99 1.60 I .49

98.6 1

104.24

107.30

106.40

Total Edible vegetable oils Soybean Cottonseed Peanuts Sunflowerseed Rapeseed Olive Coconut Palm kernel Palm Total Protein meals Soybean Cottonseed Peanuts Sunflowerseed Rapeseed Flaxseed Coconut Palm kernel Total

11.10

4.22 7.66 10.19 1.15

From United States Department of Agriculture, 1988. Preliminary estimates. 'Forecast estimates. a

1.18

R. K. DOWNEY AND S. R. RIMMER

4

Table 11 Production of Oilseed Brassicas by Main Producing Countries/Regions, 1982- 1989' Millions of tonnes by years Average Country or region

1982/1983- 1986/1987

1987- 1988'

1988- 1989'

India China Canada EEC Europe (excluding EEC) Other

2.64 5.13 3.1 I 3.18 I .70 1.06

3.10 6.61 3.85 5.95 2.16 1.31

3.50 5.04 4.24 5.3 I 2.18 1.45

16.82

22.98

21.72

Total

From United States Department of Agriculture, 1988.

'Preliminary estimates. Forecast estimates.

B. Brcusica OILSEED SPECIES Four species of Brassica have been widely cultivated as oilseed crops, Brassica carinata Braun, B. rapa, B. juncea, and B. napus. Where conditions are appropriate, namely cool temperate climates with good moisture availability, winter forms of B. napus are preferred and are the most productive. Most of the land area cultivated to oilseed brassicas in Europe and China is sown to winter oilseed rape. However, as latitude or altitude increases, the winter form of B. nupus is supplanted by the summer form of B. napus or the winter or summer form of B. rapa. In Canada, cultivation consists of approximately equal amounts of the summer types of these two species. Brassica juncea is well-adapted to drier conditions and is relatively fast maturing. On the Indian subcontinent B. juncea is the dominant species grown, although large areas are also sown to B. rapa types (toria and sarsons) (Prakash, 1980). In these climates, with hot dry summers, nonvernalization types of oilseed brassicas are cultivated in the cool moist winter season. Brassica juncea is also grown in many parts of China outside of the Yangtse/Yellow river flood plains (Stinson et a!., 1982). In western Canada B. juncea is grown as a crop for condiment on some 8 1,000 ha but has strong potential as an oilseed crop for this region (Woods et al., 1991). Brassica carinata may perform well under long season growing conditions.

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

5

Its distribution presently is largely confined to North East Africa, principally Ethiopia. Clearly, these oilseed crops are well adapted to many different parts of the world. 1. Genomic Relationships

The genomic relationships among the four oilseed Brassica species are well known [see Mizushima ( 1980), Olsson and Ellerstrom ( I 980), and Downey and Robbelen (1989)l. Our modem understanding of these relationships was initiated by Morinaga and co-workers (Morinaga, 1934), who provided cytological evidence to show that Brassicu nigra (n = 8; B), Brassica oleracea (n = 9; C), and B. rapa (n = 10; A) are primary species and that 8. carinuta ( n = 17; BC), B. junceu (n = 18; AB), and B. napus (n = 19; AC) are amphidiploids resulting from crosses between corresponding pairs of the primary species. These relationships were later confirmed by U (1935), who succeeded in the artificial synthesis of B. napus from crosses between the diploid species B. rapa and B. oleracea. Synthesis of B. juncea and B. curinata has subsequently been accomplished by the interspecific hybridization between B. nigra and B. rapa or B. oleracea [see Downey et al. (1975) and Olsson and Ellerstrom (198O)J. Understanding the relationship among these Brassicu species has enabled plant breeders to create synthetic amphidiploids and to transfer useful agronomic characteristics from species to species through interspecific hybridization. No cultivars have as yet been released as a direct result of artificial reconstitution of a species through interspecific crosses, although some desirable characteristics have been successfully transferred from one species to another through artificially synthesized amphidiploids that function as a “bridge.” For instance, the first double-low (low erucic acid content in the oil and low glucosinolate content in the meal) strains of turnip rape were developed from interspecific crosses among turnip rape (B. rupa), rape (B. napus), and oriental mustard (B. junceu) (Downey et al., 1975). Similarly, the development of low-glucosinolate B. juncea involved interspecific hybridization of B. rapa and B. junceu (Love et al., 1990).The transfer of resistance to blackleg disease from B. junceu to B. nupus (Roy, 1984) is another example. In China and Japan, interspecific crosses between B. rupa and B. napus have often been used to transfer characteristics such as early maturity, cytoplasmic male sterility, self-incompatibility, and yellow seed coat, from the former to the latter, and to broaden the genetic basis of B. nupus through genome substitution (Liu, 1985).

6

R. K. DOWNEY AND S. R. R I M M E R

2. Plant and Seed Description

Brassica rapa (AA, 2n = 20) is one of the primary diploid species and occurs wild in the high plateaus of the Irano-Turanian regon (Hedge, 1976), where it is well adapted to the cool, short season environment of this area. This species has a high relative growth rate under cool temperatures and can produce abundant seed. Both spring and winter forms are cultivated and the most cold-hardy cultivars of the oilseed brassicas occur within this species. This species is considered to be of the seed vernalization type. Full clasping of the upper leaves around the stem, the positioning of the terminal buds below newly opened flowers, and a high ratio of beak to pod length are characteristic of this species. Both dark- and yellow-seeded types occur. Brassica carinata (BBCC, 2n = 34) is the amphidiploid between B. oleracea and B. nigra. It shows a slow steady growth, probably derived from the B. oleracea genome. Leaves, which are generally waxy and light green in color, are attached to the stem with a true petiole. Though seeds are predominantly dark, some types have yellow seed. Cultivation is limited to the Ethiopian plateau and adjacent areas of east Africa. It is currently under evaluation and shows promise agronomically in many other parts of the world. Brassica juncea (AABB, 2n = 36) is the amphidiploid of B. rapa and B. nigra. It has a high leaf area ratio and a high relative growth rate, comparable to B. rapa (Sasahara and Tsunoda, 197 1 ). Asia, especially China, is rich in variations of cultivated forms of this species. It is grown widely for oil in the north Indian subcontinent and in various regions of China (Xinjiang Autonomous Region, Szechuan). This species is also characterized by having leaves with true petioles. Leaves vary considerably in shape but are generally of a dark green coloration. Seeds may be dark or yellow and the “bold” types from India have a large seed size. It has considerable potential as an oilseed crop in many other parts of the world. Brassica napus (AACC, 2n = 38) is the amphidiploid of B. rapa and B. oleracea. The existence of a wild form of B. napus is uncertain; if it does exist it will probably be found in the European-Mediterranean region ( McNaughton, 1976). Olsson (1 960) suggested that the amphidiploid B. napus (genome AACC) might have arisen at different locations by hybridization of various forms of B. oleracea (genome CC) and B. rapa (genome AA). Leaves of this species lack a true petiole as does B. rapa, but only partial clasping of the stem occurs. Seeds are dark, generally larger than those of B. rapa, and no natural yellow-seeded types are known. Development of a yellow seed form that is known to be associated with a thinner seed coat (and thus reduced fiber content in the meal) is one of the current

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

7

objectives in many breeding programs. Production of oil in Europe from B. nupus occurred as early as the thirteenth century, when it was used primarily as lamp oil (Appelqvist, 1972). 3. Mode of Pollination

Brussicu rupu is primarily a self-incompatible species, as are the other diploid brassicas, although some types of B. rupu, e.g., yellow sarson, are self-compatible. The self-incompatibility (SI) in cruciferous species is of the homomorphic sporophytic type determined by a single S locus. About 50-60 alleles are known at the S locus in B. oleruceu (Nasrallah and Nasrallah, 1989). The allelic interactions at the S locus are dominant, codominant, or recessive depending on the alleles involved. This system ensures that B. rupu is normally 100% outbreeding and consequently breeding methodologies for this species are designed to take advantage of this natural heterozygosity. The amphidiploids, B. nupus, B. junceu, and B. curinutu, are normally self-compatible species, though S alleles from B. rupu have been introduced into some genotypes of B. nupus in order to develop SI-based F, hybrids. Such hybrids have recently been registered for commercial production in Canada. Generally, self-pollination occurs readily in the amphidiploid species and selfed seed may easily be obtained by enclosing the flowering racemes in bags. Under field conditions, outcrossing, from pollination due to insects and wind, has been estimated to range from 5 to 15% (Huhn and Rakow, 1979)to about 27 to 35% (Olsson, 1952; Persson, 1956) in winter rape, 22 to 36% in summer rape (Persson, 1956; Rakow and Woods, 1987), and 19% for B. junceu (Rakow and Woods, 1987). 4. Oilseed Quality Improvements

At present, cultivars of two species (B. nupus and B. rupu) have been developed with both low-erucic and low-glucosinolate (double low, or canola) quality, and these are now widely grown commercially. In North America the term “canola” has been coined to describe cultivars that meet specific requirements for erucic acid in the extracted seed oil (less than 2% erucic acid as a percentage of total fatty acids) and aliphatic glucosinolate content in the residual meal (less than 30 pmol g-I). [For a discussion of the development of low-erucic acid cultivars of B. nupus and B. rupu and the genetics of the inheritance of erucic acid in these species, see Stefansson ( 1983).] It is likely that canolaquality cultivars of B. junceu and perhaps B. curinutu will be developed in the near future, and, if this occurs, it will significantly influence the choice of oilseed Brussicu species in some areas.

Table I11 Fatty Acid Composition of Oilseed Brassicu Crops and Other Common Vegetable Oils

Fatty acid composition (%)" CY2

Species, crop, cultivar, and type Brussicu nupus (rape) Victor winter Jet Neuf winter Hero summer Westar summer Stellar summer Brussicu rupu (turnip rape) Duro winter Yellow sarson Echo summer Tobin summer Brussicu junceu (mustard) Indian origin Cutlass

Ref!

14:O

16:O

16:l

18:O

18:l

18:2

18:3

20:O

20:l

22:O

22:l

24:O

0.3 0.4 0.2 0. I tr

0.8 1.4

9.9 56.4 12.9 57.7 59.1

13.5 24.2 12.2 20.8 28.9

9.8 10.5 9.0

0.6 0.7 0.8 0.6 0.5

6.8 1.2 7.5 1.4 1.4

0.7 0.3 0.8 0.3 0.4

53.6 0.0 50.2 0.5 0.1

0.0 0.0 0.3 0.3 0.2

13.4 12.0 18.8 24.0

9.1 8.2 8.9 10.3

0.7 0.9

9.6 6.2 12.0 1.0

0.2 0.0 0.0 0.1

49.8 55.5 23.5 0.3

0.0 0.0

1.2

12.9 13.1 32.5 58.6

1.2 1.2

8.0 17.2

16.4 21.4

11.4 14.1

6.4 11.4

1.2 0.4

46.2 25.8

1

0.0

2 3 4 4

0.0 0.0 0.0 0.0

3.0 4.9 2.8 3.6 4.1

I 2 2 2

0.0 0.0 0.0 0.0

2.0 1.8 2.5 3.8

0.2 0.2 0.2 0.1

5 6

0.0 tr

2.5 3.3

0.3 0.3

1.O

1.6 I .4 I .o

0.9

1.O

11.5

3.3

0.6 0.6 1.2 0.1

0.0 0.0 0.1

0.2

24:l 1 .o

0.0 1.2 0.0 0.0 1.1

1.2 0.0 0.0 I .9 1.7

2km 1

2

tr

3.6

0.4

2.0

45.0

33.9

11.8

0.7

1.5

0.3

0.1

0.2

0.5

6

tr

3.2

0.2

0.9

9.8

16.2

13.9

0.7

7.5

0.7

41.6

0.6

2.0

7

0.0

15.3

0.0

4.2

23.6

48.2

8.7

0.0

0.0

0.0

0.0

0.0

0.0

8

0.1

5.8

0.1

5.2

16.0

71.5

0.2

0.2

0.1

0.7

0.0

0.1

0.0

9 9

9.2 6.7

0.0 0.0

0.0 0.0

3.1 4.3

57.2 71.4

23.4

0.0 0.0

1.4 1.6

1.4 1.0

2.6 2.7

0.0

11.1

0.0

1.8 1.3

0.0 0.0

10

tr

11.5

0.0

2.2

26.6

58.7

0.8

0.2

0.0

0.0

0.0

0.0

0.0

11

0.0 1.0

7.6 23.4

0.0 0.8

2.0 2.5

10.8 17.9

79.6 54.2

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

Brassicu carinafa Ethiopian mustard Glycine may (soybean) Group 1 variety Helianrhus annuus (sunflower) Peredovik Arachis hypoguea (peanut) Virginia Bunch Cook Jumbo Zea mays (corn) United States sources Curlhamus finctorius (safflower)

us10 Gossypium hirsutum (conon)

12

Fatty acids represented by carbon chain length and number of double bonds; tr, trace amounts. References: (1) Appelqvist (1969), (2) Downey (1983), (3) R.Scarth (unpublished), (4) Scarth ef al. (l988), (5) Appelqvist (1970). (6) R. K. Downey (unpublished), (7) Hymowitz ef al. (1972), (8) Earle ef ul.(1968). (9) Worthington and Hammons (1971), (10) Beadle ef al. (1965), ( 1 1) Knowles (1968). and ( 1 2) Anderson and Worthington (197 1). a

10

R. K. DOWNEY AND S. R. RIMMER

In developed countries, the production of edible oil from oilseed brassicas is now obtained exclusively from low-erucic acid cultivars and this trend is expected to continue for production in developing countries. Low-erucic acid strains of B. juncea have been recently obtained. These strains were obtained by crossing plants from an accession with intermediate levels of erucic acid content and screening for low erucic acid in the F, progeny using the half-seed technique (Kirk and Oram, 1981). The development of low-glucosinolate B. junceu required interspecific hybridization of B. rupu and B. juncea (Love el ul., 1990) in order to transfer the Bronowski block for aliphatic glucosinolate synthesis from a B. rupu line producing low glucosinolate to a strain of B. juncea that produced 3-butenyl glucosinolate but no 2-propenyl (allyl) glucosinolate. Continued improvement for oilseed quality includes development of strains with modified fatty acid composition. These include development of strains with lower levels of linolenic acid, higher levels of linoleic acid, high oleic acid levels, and other modifications (see Table 111 for a comparison of the fatty acid compositions of oilseed brassicas and other vegetable oilseed crops). A cultivar with low levels of linolenic acid (<4%) has been developed and registered in Canada (Scarth el ul., 1988). The oil with reduced levels of linolenic acid from this cultivar has been shown to have a prolonged cooking and shelf life (Kay, 1988). Oilseed brassicas with high (>60%) levels of erucic acid would also be desirable for industrial purposes. Breeders have found it extremely difficult to achieve levels of erucic acid higher than 55% of the total oil content. Because of the inability of acyl transferases to insert erucoyl moieties in the 2-position of the triglyceride there may be a natural upper limit of 66% erucic acid obtainable in Brussicu spp. (Taylor el ul., 1992).

11. IMPROVING YIELD

A. SEEDYIELD 1. Yield Components and Breeding Methods

Although improved nutritional quality of the oil and meal has been a major breeding objective of Brussicu oilseed breeders, yield of seed, oil, and protein must all be maintained and improved if these crops are to remain competitive. Because seed yield is probably the most difficult and costly trait to measure accurately, numerous attempts have been made to identify the most important yield component(s). Positive relationships have fre-

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

11

quently been cited between the seed yield and the numbers of pods per plant and per main raceme, as well as the numbers of seeds per pod and seed weight per pod (Thompson, 1983; Shabana et al., 1990). In examining yield and yield components of 10 European winter rape cultivars over a 3-year period, Grosse et al. (1992) concluded that high yields could be attained from different combinations of three yield components- seeds per pod, number of pods, and individual seed weight. However, as noted by Thurling (1974b) and others, compensation among the various yield components in response to environment occurs to such an extent in oilseed brassicas that few breeders practice selection for one or even a few yield components. Observations on the contribution of various yield components to the observed heterosis in hand-crossed hybrids have confirmed earlier findings. Heterosis effects varied for each yield component depending on the environmental and/or genotypic effect when number of pods per plant, number of seeds per pod, single seed weight, and plant density were considered (Lefort-Buson and Dattke, 1982; Schuster et al., 1985; Uon, 1989; Schuler et al., 1992). Given the importance of the oilseed brassicas, very few studies have been undertaken to determine the physiological basis for increased yield. Thurling ( 1974a), who studied three Australian cultivars, found that correlations of total dry matter and yield were positive and highly significant (r = 0.70). Allen and Morgan (1975) reported that leaf area index at first flower was correlated to yield and concluded that a greater photosynthetic source at flowering and after first flower would result in higher yield. Campbell and Kondra (1978), studying single plants of three B. napus cultivars, found that seed yield was significantly correlated with total dry matter production (r = 0.2 1 to 0.52) per plant. Thurling (199 1) concluded from a series of experiments with cultivars and breeding lines that early flowering and maximum light penetration of the crop canopy are required to maximize seed yield. The importance of light penetration of the crop canopy is supported by the findings of Mendham et al. ( 1991). Comparing the seed yield of an apetalous strain to a closely related petalous variety, they attributed the higher yield of the apetalous strain to the 30% greater solar radiation transmitted through the apetalous canopy. Although these studies provide the breeder with some insight into the plant type that may be highly productive, the measurement of such parameters is normally not as efficient or effective in oilseed brassicas as the total measurement of yield. In conventional B. napus and B. juncea breeding programs for yield, various forms of the pedigree system are employed [see Thompson (1983), Downey and Rakow ( 1987),and Downey and Robbelen ( I989)]. However,

R. K. DOWNEY AND S. R. RIMMER

12

Table IV Average Relative Yield of Winter Rape Parental Lines and Cultivars Compared to Performanceof Syn-1 Synthetics and Seed Mixtures of P a r e d seed yield as percentage of parents Average of parents

Syn- I synthetic

seed mixtures

Reference

100 100 100 100

I04 I14 I06 108

97 I05 106 104

Grabiec and Krzymanski ( 1 984) Schuster and Friedt ( 1985) E o n ( 1987) Lkon and Diepenbock (1987)

'After Becker (1988). the parameters of these systems differ from pedigree cereal programs in two important respects. First, the oilseed brassica crops have a high multiplication rate per generation (- 1000: I), and second, the plant-to-plant outcrossing rate is much higher, ranging from 5 to 36% (see Section I,B,3). Thus replicated progeny testing can begin as early as the F, and a certain level of heterosis from the initial cross can be captured and retained in subsequent generations. In a comparison of winter rape selection techniques, Sauermann (1989) found that in winter B. napus visual selection in the F, for yield was superior to a random line selection, but the highest yielding lines were identified by measuring yields of single-row F, progenies in a three-replicate test at one location or by testing sublines in the F4 with one replicate at each of three locations. Because of the potential for significant levels of heterosis for yield in the oilseed brassica species, the degree of natural interplant crossing, and the absence of a highly efficient system of pollen control, synthetics have been suggested as a means of capturing part of the available heterosis. Becker ( 1988) compared the performance of experimental synthetics of winter B. napus to their parent lines or cultivars and noted that the synthetics yielded some 4 to 14% more seed (Table IV). He postulated that even higher levels of heterosis could be captured if the parents were selected on the basis of their combining ability. On the other hand, in three of the four experiments, sowing mixed seed of the parents in the same drill run also resulted in yield increases, which in some instances approached or equaled the yield of the synthetics (Table IV). U o n (199 I ) found that cultivar mixtures and Syn- 1s displayed greater yield stability than their corresponding F, hybrids or any of the individual cultivars, suggesting that heterozygosity and het-

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

13

erogeneity of both cultivar mixtures and synthetics have a positive effect on yield stability. In B. rupu, where self-incompatibility ensures a high degree of heterogeneity, recurrent selection has been the most effective method for increasing seed yield as well as oil content (Downey and Rakow, 1987). However, with the numerous agronomic and quality traits that must now be incorporated into any new B. rupa canola cultivar, more than one specialized recurrent selection program, to be run in parallel with the main recurrent selection program, may be required. After sufficient progress is made within each specialized composite, two or more may be combined to create a new composite cultivar source. The presence of the natural self-incompatibility (SI) system in most B. r a p cultivars suggests that synthetic cultivars would be attractive alternatives to F, populations derived by recurrent selection. Falk (1991), using four parental cultivars of B. rupu, compared the seed yield of the parent cultivars and all possible F,s with their various two-, three- and four-component Syn- Is and Syn-2s. It was found that the agronomic performance of the synthetics could rival the single-cross F,s. On average, hybrids yielded 15 to 30% more seed than their parent cultivars over 2 years of testing. However, the Syn-1 populations averaged only 1% less seed than the F,s in each testing year, while the average of the Syn-2s in the last year of testing yielded only 3 and 2 percentage points less than the F,s and Syn-ls, respectively. 2. Heterosis and F, Hybrids

Although B. nupus is usually classified as a largely self-pollinating species, significant levels of heterosis for yield have been obtained in F, hybrids of both the spring and the winter forms. Based on the results from reciprocal top crosses of seven summer rape cultivars with the Canadian cultivar Regent, Sernyk and Stefansson ( 1983) concluded that it should be possible to develop hybrid cultivars of summer rape with a commercial heterosis for yield of about 40%. Also, in spring rape Grant and Beversdorf ( 1985)found high-parent heterosis for seed yield of up to 72%, with specific combining ability being more important than general combining ability. In winter rape, several researchers have documented the potential for hybrids, reporting heterosis for seed yield up to 60-70% (Schuster and Michael, 1976; Lefort-Buson and Dattke, 1982, 1985). Lefort-Buson et al. (1987) related heterosis to genetic distance in crosses between and within groups of European and Asiatic B. nupus cultivars and strains. As might be expected, hybrids between distant groups showed greater heterosis than

14

R. K. DOWNEY AND S. R. RIMMER

within-group hybrids. Additive and dominant genetic variance was more important for the within-group than the between-group hybrids. In the self-incompatible B. rapa species, Arunachalam and Bandyopadhyay ( 1984)were also able to relate the magnitude of heterosis exhibited in the F, to the genetic divergence of the parental phenotype. Singh and Gupta (1985) outlined a procedure to identify diverse genotypes using 3 1 B. rapa strains tested in 12 different environments. Indian researchers have reported varying degrees of heterosis for yield within and among Indian oilseed types of B. rapa (i.e., Toria and brown- and yellow-seeded sarson). In general, such hybrids have shown positive high parent heterosis for seed yield with a high proportion showing commercial heterosis. Unfortunately, many of these observations are based on single plant yields (Prasad and Singh, 1985; Yadav and Yadava, 1985; Singh and Gupta, 1985) or have been taken from space-planted, single-row plots (Devarathinam ef al., 1976; Labana et al.. 1978), and the results reported are often based on only I year and a single location. In most of the studies, combining-ability analysis has indicated yield to be primarily under the control of nonadditive gene action. High parent heterosis reported from Indian studies has been as great as 63% on single plant yields in yellow sarson (Labana et al., 1978). In Canada, a natural top cross of a canola breeding line onto the yellow sarson rapeseed cultivar, R500, yielded 46% more seed than the commercial B. rapa canola cultivar Candle (Hutcheson ef al., 1981). Further studies over a 2-year period, using R500 as the female parent in crosses with three Canadian oilseed cultivars and strains, showed high parent heterosis for seed yield between I6 and 37% (Hutcheson, 1984). Schuler (1989) tested hand-crossed hybrids between the B. rapa canola cultivar Tobin and 19 European and Canadian parental strains and cultivars at four locations over a 2-year period. He reported the highest average commercial heterosis for seed yield to be 64%, the same range as that found by Labana et al. ( 1978). However, the best canola-quality hybrid yielded only 22% over the commercial cultivar Tobin. Falk ( I99 1) reported that 4 of 12 hybrids from diallel crosses between four B. rapa Canadian and European cultivars exhibited high parent heterosis for seed yield. The maximum high parent heterosis found in the 2-year multilocation study was 26%, whereas the best commercial heterosis for seed yield, between canolaquality parents, was 22%. Studies of heterosis in B. junceu have for the most part been camed out on the Indian subcontinent and, as in the B. rapa studies, are largely based on single plant yields in space-planted plots. Singh (1 973) reported that six B. juncea hybrids showed an average high parent heterosis of 49% over 2 years of testing. Banga and Labana (1 984) found the range of seed yield

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

IS

heterosis to extend from -76 to 263% over the high parent whereas commercial heterosis ranged from -77 to 172%. However, yields were measured using only 20 selected single plants per entry. Singh and Singh ( 1989, also using single plant yields, observed a heterotic yield response ranging from 9 to 9 1% over the high parent. It would appear that the greatest opportunity for commercial exploitation of the heterosis phenomenon within the oilseed brassicas occurs in B. nupus and B. junceu. In general, higher levels of heterosis have been found within these species than in B. rupu. In addition, these self-compatible amphidiploid species do not exhibit the same degree of inbreeding depression as found in the self-incompatible B. rupu during the development of high-combining parents. It is also important to keep in mind that heterosis is measured as a percentage of actual yield. As a result, the commercial advantage of a hybrid cultivar is highly dependent on the average yield of the crop being grown. Given that the average yield of winter B. nupus in some countries is over 3000 kgjha, while average yields of summer B. nupus and B. rupu are, respectively, closer to 1500 and 1000 kgjha, the level of heterosis in the latter two crops would have to be substantially higher to return to the growers the same number of extra kilos. Given the extra cost of producing hybrid seed and the moderate yield and level of heterosis found in B. rapu studies, the development of synthetic varieties of this species appears to be a more attractive alternative to hybrids. To date, no investigations have been published on the potential level of heterosis that might be obtained in B. curinutu. 3. Pollination Control Systems

The potential for increased yield from hybrid cultivars in oilseed brassica crops is substantial and has generated considerable commercial interest. However, to capture the heterotic potential exhibited in hand-crossed hybrids, some form of pollination control is required. The pollen control systems investigated include chemical gametocides, various cytoplasmic male sterility (CMS)- nuclear male fertility restorer systems, genic male sterility (GMS), and sporophytic incompatibility (SI). a. Gametocides Although gametocides have been extensively investigated in cereal crops ( McRae, 1985), only limited work on Brussicu species has been reported. Van der Meer and Van Dam (1979) were able to maintain some plants of several B. oleruceu cultivars in a completely male sterile condition for up to 24 days by repeatedly spraying with varying concentrations of GA 417 in isopropyl alcohol. Reversion to complete male fertility occurred a few days

16

R. K. DOWNEY AND S. R. RIMMER

after treatment. In B. junceu, Banga and h b a n a (1984) induced male sterility with 2-chlorethylphosphonic acid (etherel). Although they reported 54% hybridity in seed harvested from the treated plants, only shriveled seed was obtained. In general, the indeterminate flowering habit of the oilseed brassicas suggests that gametocides will be of questionable value for the commercial production of hybrid seed. b. Self-Incompatibility In the Cruciferae, SI systems, where present, are of the strong sporophytic type, characterized by an interaction between the papilla cells of the stigma and the pollen or pollen tube (Hinata and Nishio, 1980). Genetic analysis of self-compatibility(SC) and SI revealed that SI expression is not only controlled by a multiple series of alleles at a single locus but also by alleles at a complementary modifying locus (Hinata el ul., 1983; Hinata and Okazaki, 1986). Self-incompatibility has been used since the 1940s to produce hybrid cruciferous vegetables (Tsugimoto and Minato, 198 1) and since the 1960s to produce hybrid marrow - stem kale (Thompson, 1967). Thompson ( 1983) proposed the development of a three-way B. nupus hybrid, using a dominant self-compatible line as the third parent. Recently, B. nupus SI hybrids of spring canola have been registered in Canada. The first SI hybrids were produced using a system patented in Canada by Kingroup Inc. (Scott-Pearse, 199 1 ) that involves using microspore culture to produce doubled haploid plants that are homozygous for either SC or dominant SI alleles. When pollen from SC plants is transferred to SI plants, a heterozygous, self-incompatible parent is produced. This SI parent is used as the female in hybrid seed production fields, with an SC line serving as the pollen parent. Although one-half of the resulting hybrid plants in commercial fields will be self-incompatible, the surrounding non-SI plants can provide an effective pollen source. Banks (1989) provided the SI genes for these hybrids by introgressing S alleles from B. rupu and B. oleruceu into oilseed B. nupus, demonstrating their effectiveness as a practical means of pollination control. Unlike Schweiger and Eicke (1981), he found that the stability of the SI system in B. nupus was sufficient for hybrid seed production. Banks (1989) reported a good correlation between pollen tube growth and degree of seed set and concluded that this test was effective in S allele identification. One of the major constraints to the use of the SI system is the need to suppress the SI bamer so that sufficient parental seed can be produced for hybrid seed production. Various methods have been used to overcome SI [see review by Ito (198 I)], but the most efficient method for the large-scale maintenance of B. nupus SI parental lines appears to be the use of high CO,

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

17

concentration in an enclosed environment when plants are flowering (Nakasishi and Hinata, 1973, 1975; Thompson, 1978). An alternative method is to apply a series of salt (NaCl) sprays to the flowering plants (Guan and Wang, 1987; Monteiro et uf., 1988). The use of the SI system for hybrid development could be extended to B. junceu as well as B. rupu, which already has an SI system. However, the use of the SI system in B. rupu has two limitations. First, the level of heterozygosity within the crop is already high, which tends to limit the expected level of heterosis, and second, unlike B. nupus and B. junceu, inbreeding to maintain the SI parent results in a significant loss in vigor and seed set in B. rupu, thus increasing production costs for such hybrids. c. Cytoplasmic Male Sterility The cytoplasmic male sterile- nuclear restorer system in brassica oilseeds parallels systems in maize and sunflower and has been reviewed by Shiga (1980) and Rousselle et ul. ( 1983). Several cytoplasms have been reported to induce male sterility in B. nupus. The nap cytoplasm was identified independently by Shiga (1980) and Thompson (1983), in progeny of crosses between winter and spring cultivars, where the male parent was the Polish cultivar Bronowski (Shiga et ul., 1983). Bronowski camed the recessive male sterility rfallele together with a male fertility (F)-conditioning cytoplasm. Nearly all B. nupus cultivars carry the dominant restorer, R/;and a sterility-inducing cytoplasm. Unfortunately, the male sterile lines with an (S) rf $genotype become male fertile at moderate temperatures (26/20°C) (Fan and Stefansson, 1986) and two to five genes are involved in maintenance and restoration (Grant, 1984). Because of these complications little research is now being done on this system. The ogu cytoplasmic male sterility system, found in Japanese radish (Ruphunus sutivus L.) by Ogura (1968), was transferred to B. oleruceu and then to B. nupus (Bannerot et ul., 1977). The male sterility of the recovered CMS B. nupus plants was highly stable under a wide range of environments ( Bartkowiak-Broda et ul., 1979). However, the leaves of these plants were chlorotic at low temperatures (<12°C) and the flowers were unattractive to pollinators due to the absence of even rudimentary nectaries (Mesquida and Renard, 1978; Rousselle, 1982). These deficiencies were overcome through protoplast fusion resulting in the production of male sterile, nonchlorotic plants with well-developed nectaries (Pelletier, 1990). However, the development of restorer lines with B. nupus proved to be difficult, although it was known that restorer genes were present in the Ruphunobrussicu (R. sutivus X B. nupus) strains used by Heyn (1976), as well as R. sutivus X B. oleruceu progeny used by Rousselle and Dosba (1985) in crosses with ogu CMS B. nupus plants. Introgression of restorer genes by

18

R. K. DOWNEY AND S. R. RIMMER

Heyn (1976, 1979) resulted in restored white- or yellow-flowered B. nupus plants with a significantly decreased seed set. Pellan-Delourme and Renard (1988) crossed restorer lines derived from Heyn’s (1979) experiments with a reconstituted cultivar Brutor containing ogu cytoplasm. Completely male sterile white- and yellow-flowered plants were recovered and, as in Heyn’s (1979) experiment, the white flower trait appeared to be linked to male fertility restoration. Although all restored plants examined had the normal complement of 38 chromosomes, up to three multivalents per cell were observed and the fertility of the restored material was always significantly less than that of the male sterile or non-CMS material. High ovule abortion was tightly linked to male fertility restoration. Restored lines with normal seed set were obtained through continued backcrossing and selection for improved pod development. The recovery of high female fertility was linked to the loss of a radish isozyme marker closely linked to the restorer gene (Delourme ef ul., 1991). Thus the ogura-INRA system is now completely functional in B. nupus and is being transferred into B. junceu and B. cumpestris (Renard ef ul., 1992). Prior to solving the fertility restoration problem, Renard and Mesquida (1987) suggested that ogu CMS plants could be used to produce “mixed B. nupus hybrids” containing about 10% male fertile plants that would act as a pollen source in commercial production fields. The pol cytoplasm, or the Polima system, originated in China where cytoplasmic male sterile plants were found in a B. nupus introduction from Poland (Fu, 1981). Most B. nupus cultivars maintain this cytoplasm to some degree but good maintainers and restorers have been difficult to find. Fan ef al. (1986) found that low-erucic acid (Zem) lines of B. junceu were effective restorers. However, when the restorer gene(s) were transferred into B. nupus, fully restored plants always carried an additional pair of chromosomes, presumably from B. junceu (Tai and McVetty, 1988). In China, a single dominant restorer gene was identified in a B. nupus introduction from Europe called “Italy” (Fang and McVetty, 1987). It is the Italy restorer that is now used in most Polima-based hybrid breeding programs. Although the male sterility of pol CMS plants is good at moderate temperatures, at high temperatures (24-30°C) some pollen is produced (Fan and Stefansson, 1986). Initial experience with the pol cytoplasm indicated that the level of heterosis expressed in such CMS hybrids was substantially below what would be expected from the same cross when parents with normal cytoplasm were used. McVetty ef ul. (1990) compared the performance of male fertility-restored intercultivar F, hybrids in the pol as well as in the nap cytoplasms. They concluded that there was indeed a biological cost to the

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

19

hybrid plants attributable to the pol cytoplasm, relative to those with the nap cytoplasm. Although the relative cost of pol compared to nap cytoplasm was consistent over a range of environments, the yield depression was not uniform among the various parental cultivars evaluated. This latter observation suggests that selection for more compatible nuclear restorers could bring about a reduction of the cytoplasm’s biological cost and produce pol CMS-based hybrids with the same level of heterosis obtained from hand-crossed hybrids with normal cytoplasm. Despite the present reduced heterosis levels, two pol-based spring B. napus hybrids have been registered for sale in Canada by ICI Seeds and others are expected to follow from this and other breeding programs in the near future. In B. rapa, Sovero ( 1987) successfully selected for pol maintainer alleles in a population derived from crosses with three widely diverse B. rapa cultivars. However, restorer genes for the pol CMS have not yet been reported in B. rapa. The rnur or Diplolaxis CMS was obtained by Hinata and Konno (1 979), from the interspecific cross of Diplotaxis muralis X B. rapa cv. Yukina. Restorer genes for the mur CMS source were readily available in B. rapa. Indeed, all Canadian and European spring B. rapa cultivars fully restore male fertility. Thus, maintainer alleles from the vegetable cultivar Yukina had to be transferred to oilseed B. rapa before hybrids could be produced. A mur CMS female oilseed B. rapa strain derived from cultivar Tobin is now available for distribution from the Agriculture Canada Research Station, Saskatoon (D. S. Hutcheson, personal communication). Attempts to utilize rnur CMS in B. napus have not yet been successful. Fan et af. ( 1985),following six backcrosses of B. napus into mur cytoplasm and selecting for male sterility, found that all male sterile plants camed an extra chromosome, making the system unreliable. However, Pellan-Delourme and Renard ( 1987), in test crosses involving 147 cultivars and lines from 14 countries, found two lines that produced some male sterile progeny in mur cytoplasm. No euploid male sterile plants were found. Sovero (1 987) examined F, progenies of rnur CMS B. napus crossed with seven strains of B. rapa. The F,s were mostly fertile, but a few male sterile plants were observed. Thus, the Diplolaxis system provides a complete hybrid production mechanism for B. rapa but requires additional development for use in B. napus. In B. juncea, Rawat and Anand (1979) identified the nig CMS and believed this CMS cytoplasm probably originated in Brassica nigra. Unfortunately, although male sterility in this cytoplasm is stable, fertility restoration, with restorer genes isolated from B. rapa and B. nigra, appears to be incomplete. Recently, scientists at the TATA Institute, using cDNA

20

R. K. D O W N E Y AND S. R. RIMMER

genome probes, reported that they have identified the male sterile cytoplasm in this CMS B. junceu as being derived from Brussicu tourneforfii, rather than from B. nigru as originally believed (Pradhan ef al., 1991). Should this be the case, then the search for restorer genes for this cytoplasm may soon be successful. Prakash and Chopra (1988) obtained oxy CMS plants through backcrossing the nucleus of B. rupa spp. oleiferu var. brown sarson into Brussicu oxyrrhinu cytoplasm. Male sterility in this system is very stable and the flowers show no abnormalities except that the anthers are small, slender, nondehiscent, and contain only nonfunctional pollen. Some chlorosis of the first leaves of male sterile plants was noted but this has now been overcome through protoplast fusion (S. Prakash, personal communication). Gene(s) for fertility restoration have been transferred recently from B. oxyrrhinu (S. Prakash, personal communication). If the restorer proves to be effective, this system appears to have a high potential for commercial development. The Mok-po CMS system in B. nupus was initially described by Lee ef ul. (1976) as a CMS system with maintainer and restorer lines. However, in Canada and Australia this system has performed as a GMS rather than a CMS (D. S. Hutcheson and G. Buzza, personal communications). d. Genic Male Sterility A number of monogenic recessive male sterile mutants have been identified in B. rupu. GMS plants in brown sarson (Chowdhury and Das, 1967; Das and Pandey, 1961), yellow sarson (Chowdhury and Das, 1966), and toria (Zuberi and Zuberi, 1983) were normal with respect to their general appearance except for smaller flowers and pointed, sterile anthers. In contrast, the male sterile B. nupus mutant reported by Takagi (1970) was characterized by narrow leaves as well as small flowers and degenerative anthers. When inherited as a monogenic recessive, GMS appears to have little commercial value. However, a diallelic, epistatic system developed by Chinese researchers is being commercially utilized in Shanghai province (Lee and Yan, 1983; Li ef ul., 1988). Pollen production in this system is controlled by two loci with epistatic effects. At one locus the allele for male sterility (MSl) is completely dominant over the alternative (msl) allele, while at a second locus the allele for male fertility (MS2) is completely dominant over the recessive male sterility allele (ms2). The male sterile phenotype can only occur if the MS2 locus is homozygous recessive and the MSI locus is either heterozygous or homozygous dominant. Male sterile seed is produced by crossing genotypes MSlMSlms2ms2 X

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

21

rnslrnslrns2rns2. F, hybrids are produced by crossing the resulting male sterile MSlrnslrns2rns2 parent with a male fertile rnslmslMS2MS2 parent. Although this system alleviates the problem of hand roguing in the hybrid seed production field, considerable labor is required to maintain and produce the specific parental genotypes. The larger bud size of male fertile plants makes possible the visual identification and roguing of such contaminants prior to flowering. Commercial hybrid production might also be economic if a monogenic recessive male sterile genotype could be induced to shed pollen by hormone application. V. K. Sawhney (personal communication), has shown that the application of cytokinin to GMS B. napus genotypes results in a temporary restoration of male fertility. e. Transgenic Pollen Control Systems Paladin Hybrids has proposed and patented a number of systems that result in male sterility through precise disruption of pollen development through the actions of inserted genes in B. napus and other crop plants (Fabijanski and Amison, 1989). Normal pollen development is inhibited by linking pollen-specific promoters to genes expressing a toxic molecule or to antisense genes or to genes that have enzymatic activities that inhibit cell growth. Plant Genetic Systems has applied the toxic molecule technique to prevent pollen production in B. napus plants and restoring male fertility through the use of a second gene that inhibits the expression of the pollen toxic gene. Two nuclear dominant genes that interfere with the vital functioning of male fertility, RNase TI from Aspergillus oryzae and Barnase from Bacillus arnyloliquefaciens, were combined with a promoter from tobacco that expresses only in the tapetum cells of immature anthers (Mariani ef al., 1990). The transfer of these genes to B. napus plants resulted in completely male sterile plants but with otherwise normal flowers and growth habit. The Barstar gene, also isolated from B. amyloliquefaciens, was found to completely inhibit the activity of Barnase and RNase TI. When B. napus plants transformed with Barstar were crossed with the male sterile Barnase plants, the progeny were all male fertile. However, maintenance of the female parent requires backcrossing to a nontransformed maintainer, resulting in a 1 : 1 ratio of male sterile to male fertile plants. To overcome this difficulty, the bar gene, which confers tolerance to the broad-spectrum herbicide phosphinotricin (Ignite or Basta) (see Section III,C), was engineered into the chimeric constructs with both the Barnase and Barstar genes. Thus the female parent is maintained by backcrossing to the nontransformed maintainer parent and the male fertile,

22

R. K. DOWNEY AND S. R. RIMMER A line maintenance MS HT #4# -me& #4# --

*M#M

meM#M

Commercial F1 hybrid production me& RFHT MSHT#4# Xw# f w & R F H T

--

Herbicide roguing Male fertile, Herbicide tolerant Commercial hybrid

MS HT #4# --

m6# # #

MS = Male sterility (Barnase) HT = Herbicide tolerance (bar) RF = Restoration of fertility me, M,# = the absence of (Barstar) these genes Figure 1. Method of producing A lines and commercial B. napus hybrids, using a transgenic pollen control system and herbicide tolerance, proposed by Plant Genetic Systems.

herbicide-susceptible segregates are rogued out in the seedling stage through the application of the herbicide Ignite or Basta. Commercial F, seed is produced from fields containing the male fertile parent, homozygous for the Barstar and herbicide-tolerance genes, and the male sterile female heterozygous for Barnase and herbicide tolerance (Fig. 1). The resulting F, progeny are, therefore, male fertile and herbicide tolerant. This system is now under extensive evaluation in western Canada. f. Commercial Hybrids The most successful hybrid to date is the Chinese winter B. napus rapeseed variety Qinyou No. 2. This cultivar is reported to yield 43% above local cultivars and in 1990 was sown on some 400,000 ha (Li et al., 1990). This hybrid results from a cross between European and Chinese winter B. napus strains. The CMS pollen control system used has not been described except to claim that it is different from the Polima and other known CMS systems (D. Li, personal communication). Unfortunately, the hybrid does not produce canolaquality seed.

B. OILAND PROTEIN YIELD Oilseed brassicas are produced and marketed for the oil and highquality protein they contain. Thus any increase in these two seed components will

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

23

increase the value of the seed to the end user. Oil contents within and among Brussicu oilseed species range from 35 to 44% of the air-dried seed. The oil is normally worth two to three times that of the residual meal. In the majority of European countries the seed is purchased on an oil content basis, but in most other regions the grower is paid for the weight of the seed delivered (Downey and Rakow, 1987). Because oil content has a relatively high heritability (Grami ef al., 1977) and can be quickly and accurately measured by wide-line nuclear magnetic resonance (NMR) (Conway and Earle, 1963) or near infrared (NIR) (Starr et al., 1985), a more rapid genetic advance can be made for oil percentage than for seed yield or protein content. However, intensive selection for oil content usually results in lower protein values. Grami et ul. (1977) showed that oil and protein contents are significantly and negatively correlated both phenotypically (-0.81) and genotypically (-0.71). To avoid sacrificing protein for oil, Grami et ul. (1977) found simultaneous selection for the sum of oil and protein to be effective, with a heritability of oil plus protein in B. nupus of 0.33. Because of the cost and limited precision of Kjeldahl protein analysis for meals and seed of the oilseed brassicas, most breeders have concentrated on increasing oil content while attempting to maintain protein levels (Downey and Rakow, 1987). However, with the availability of automated and accurate elemental nitrogen analyzers, greater selection pressure will undoubtedly be placed on protein in the future. Higher oil and protein levels coupled with a desired lower fiber content can also be obtained through the development of yellow-seeded cultivars. Yellow seeds exhibit these advantages over the normal brown- or blackseeded forms from which they are derived because yellow seed coats are thinner. Thus the proportion of oil and protein-rich embryo within the seed is increased in relation to the high-fiber hull (Jonsson and Bengtsson, 1970; Stringam et ul., 1974). Hutcheson ( 1984) showed that similar modifications in oil, protein, and fiber levels could be obtained through increasing seed size. However, there are other advantages to yellow seed in that the yellow hulls have higher digestibility values (Downey and Bell, 1990) and more closely match the color of other feedstuffs in livestock and poultry rations, thus allowing feed formulas to be modified without changing the appearance of the feed. Almost completely yellow-seeded cultivars of canola-quality B. rupu are now commercially available, whereas complete yellow-seeded forms of B. junceu (condiment mustard) and B. curinutu have been developed. Unfortunately, the development of a stable, pure yellow-seeded form of B. nupus with an acceptable agronomic performance has yet to be achieved (Shirzadegan and Robbelen, 1985; Chen and Heneen, 1989).

24

R. K. D O W E Y AND S. R. RIMMER

111. IMPROVING RESISTANCE TO PESTS A. DISEASES A number of diseases are important in the culture of the various oilseed brassicas. This section focuses on the development of cultivars resistant to some of the most important diseases, primarily blackleg, white rust, Sclerotinia stem rot, and Alternaria black spot diseases. A review of the resistance of brassicas to blackleg has recently been published (Rimmer and van den Berg, 1992). Some consideration will be made of other diseases, of which grey leaf spot, club root, Verticillium wilt, and turnip mosaic virus (TuMV) disease are probably the most important on a worldwide basis. For the main diseases the following topics will be discussed: disease importance and distribution, variation for pathogenicity, sources of resistance, the genetics of resistance, and screening methodologies. 1. Blackleg

Blackleg, or stem canker, caused by Leptosphaeria maculans (Desrn.) Ces. et de Not., is a serious disease of crucifers throughout the world. Although it has been known to cause yield loss in Cole crops for many years, the importance of the disease as a threat to oilseed rape production has been more recent and, at least in Europe and Canada, is associated with the increase in area of production. Rapid expansion of cultivation of B. napus was followed by epidemics in France and Great Britain (Alabouvette and Brunin, 1970; Cook and Evans, 1979). In Australia, damage was so severe that the crop was virtually eliminated until resistant cultivars could be developed (Bokor et al., 1975). It has been estimated that blackleg caused an overall yield reduction of 7.2% in Saskatchewan, Canada, in 1985 (Petrie, 1986). In 1987, a blackleg disease survey conducted in Manitoba indicated an overall yield reduction of 9.6% (Platford, 1988). The disease is not important in India or China, probably due to the widespread practice of harvesting the stems for fuel, thus limiting the survival of the pathogen on host residues. Leptosphaeria maculans may be categorized into two distinct populations, which differ in pathogenicity, cultural characteristics, and physiological characters. One population of isolates usually described as nonaggressive or avirulent produces a redbrown or yellow/brown pigment in Czapek - Dox medium, whereas the second group of aggressive or virulent isolates generally produces no pigment. Nonaggressive isolates grow more rapidly on V8 agar and prune lactose yeast agar and produce few pycnidia. In contrast, aggressive or virulent isolates grow slowly and produce abun-

A G R O N O M I C I M P R O V E M E N T I N OILSEED BRASSICAS

25

dant pycnidia. Aerial mycelium is white or green in aggressive types and mycelium in nonaggressive strains varies between white, grey, yellow, or orange/brown (Delwiche, 1980; Humpherson-Jones, 1983; Hanacziwskyj and Drysdale, 1984). Only aggressive isolates produce sirodesmins, which are phytotoxic secondary metabolites (Koch ef al., 1989). Although a number of workers have successfully crossed isolates of the aggressive types, no success in mating either aggressive with nonaggressive or nonaggressive with other nonaggressive strains has been obtained to date (Petrie and Lewis, 1985; Mengistu et al.. 1990). Evidence for pathogenic specialization within the aggressive types of L. maculans has been reported by Koch et ul. (1991). They distinguished three pathotypes of aggressive isolates by the reaction of isolates on the cotyledons of the B. napus cultivars, Westar, Glacier, and Quinta. These were designated pathogenicity group (PG)2, PG3, and PG4, respectively. Nonaggressive isolates were designated as PG 1. A large variation for resistance has been reported among oilseed rape (B. nupus) cultivars and breeding lines (Alabouvette ef al., 1974; Cargeeg and Thurling, 1980a; Delwiche, 1980; Jonsson, 1974; Kriiger, 1978; Lammerink, 1979; McGee and Petrie, 1978; Rimmer and van den Berg, 1992; Roy, 1978a; Roy and Reeves, 1975; Thurling and Venn, 1977; Wratten, 1977). Resistance in some lines may be traced back to the French cultivars Major and Ramses (Roy, 1978a; Wratten, 1977),which possibly have a common origin in Nain de Hambourg (Rollier, 1978). Resistance in several winter cultivars developed in Europe was obtained from the variety Jet Neuf (Renard d ul., 1983). Resistance to blackleg in spring types of B. napus has been observed in the French cultivar Cresor and in Australian cultivars, e.g., Maluka. Taparoo, and others. Many Australian cultivars derive their resistance from Japanese cultivars, e.g., Chisaya, Chikuzen, and Mutu natane. Little resistance to blackleg disease has been observed in B. rapa accessions. The rather weak resistance that has been observed seems to be largely restricted to winter forms of the oilseed types (Kutcher, 1990). Species with the B genome (B. nigru. B. juncea, and B. carinufu) generally appear to be highly resistant to blackleg disease (Roy, 1978a; Sacristan and Gerdemann, 1986; Sjodin and Glimelius, 1988), although some susceptibility has been reported in a few accessions. However, the apparent lack of symptoms in cotyledons and leaves is often associated with subsequent extensive colonization of root and basal stem tissues (Gugel ef al., 1990; Ken, 199 I ). Keri observed that over 80% of 250 accessions of B. juncea inoculated with Canadian isolates of L. maculuns on the cotyledons were subsequently found to have root infection. Success in the development of winter rape cultivars with effective resist-

26

R. K. DOWNEY AND S. R. RIMMER

ance to blackleg, e.g., Jet Neuf, indicates that resistance is a heritable character. Roy and Reeves (1975) found that 25% of the F, population was resistant in some crosses and that blackleg resistance was readily incorporated from the cultivar Major into the Australian cultivar Wesreo (Roy, 1978b). The incorporation of blackleg resistance into rapeseed lines with desirable agronomic qualities is a major objective of breeding programs in countries where the disease has seriously reduced yields. The inadequacy of control by cultural practices and chemical means has further emphasized the necessity of breeding for resistance. Considering the importance of the disease and the standard practice of breeding for resistance, the paucity of published data concerning the inheritance of resistance is surprising. Seedling resistance to blackleg has been investigated by Delwiche ( 1980), who found that cotyledon resistance of a French winter breeding line was controlled by a single dominant gene, Lml. A second dominant gene (Lm2) conferred resistance in another cultivar. Tests for independent inheritance of these two genes were highly significant, indicating linkage. Inheritance of cotyledon resistance in spring oilseed rape, however, was controlled by a single recessive gene (Sawatsky, 1989). After inoculation of seedlings of many genotypes with Australian isolates of the pathogen, Cargeeg and Thurling (1980a) observed continuous variation in disease reaction, which they considered to be indicative of polygenic control of resistance. A significant cultivar by isolate interaction occurred. Comparative studies between resistance observed in the glasshouse and in the field indicated variation in resistance both among and within cultivars of B. napus (Cargeeg and Thurling, 1980b). Sawatsky ( 1989) found resistance in the French spring rape lines (R8314 and R83- 17) to be governed by two genes with dominant alleles, designated Bl-1 and Bl-2. The presence of both dominant alleles (Bl-I, BI-2) conferred a high level of resistance whereas a single dominant allele (either (BI-1 bl-1, bl-2 bl-2) or (bl-1 bl-1, 81-2 bl-2) provided intermediate levels of resistance. Seedling resistance observed in both B. juncea and B. carinata lines is thought to be controlled by genes located on the B genome and considered to be more effective than seedling resistance in B. napus. Recent work by Ken ( 1991 ) indicates that resistance in B. juncea is controlled by two genes. These genes exhibit dominant recessive epistasis. The presence of wild-type alleles or homozygous recessive alleles at both loci confers resistance. Interaction between the wild-type resistance gene and the homozygous recessive at the second locus results in a compatible or susceptible phenotype. Numerous techniques have been employed to screen breeding materials of oilseed brassicas for resistance to blackleg in the field, greenhouse, or

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

27

growth chamber. Although selection for resistance can be conducted in blackleg-infested nurseries in the field, only one generation can be evaluated each year. This is probably the best that can be achieved with winter forms of B. napus (Wittern and Kriiger, 1989, but with summer forms it is more efficient to use screening procedures in growth chamber tests to obtain selections from two generations a year. Various workers have screened plants at different growth stages using different modes of inoculation. Cotyledon testing has involved application of drop or spray suspensions of pycnidiospores or ascospores (Williams, 1985; Alabouvette ef al., 1974; Thurling and Venn, 1977; Wittern and Kriiger, 1985; Cargeeg and Thurling, 1980b). Seedlings have been inoculated by placing fungus-infested oat kernels at the plant base or by incorporating infected kernels in the soil (McGee and Petrie, 1978; Wittern and Kriiger, 1985). A method devised by Helms and Cruickshank (1979) involved covering seeds with perlite mixed with a pycnidiospore suspension. Other tests have screened for blackleg resistance at later growth stages. Older seedlings have been infected by injecting the base of the petiole with a pycnidiospore suspension (Newman, 1984). The true leaves can also be inoculated with a pycnidiospore suspension to test for disease response (Alabouvette ef al., 1974; McGee and Petne, 1978). A method of inoculating stems at the bolting stage with a pycnidiospore suspension has been used in blackleg resistance screening (Newman and Bailey, 1987). In order to evaluate which of these varied techniques are useful it is important that blackleg resistance testing in the greenhouse accurately represents the disease reaction obtained in field trials. A technique developed by Delwiche ( 1980) involved inoculating cotyledons with a droplet of pycnidiospore suspension and rating plants 10 days postinoculation. This test distinguished differences in disease response between four varieties. However, disease reactions under field conditions were not determined. Wittern and Kriiger (1985) found only slight differences between five varieties in various experiments in which cotyledons were inoculated with droplet or spray suspensions. A poor correlation between field and greenhouse results were observed. Alabouvette et al. (1974) tested the susceptibility of three rapeseed varieties by applying a spray suspension at the cotyledon stage. Their results indicated a difference between greenhouse and field classification of varietal susceptibility. Cargeeg and Thurling (1980b) compared responses to inoculation in the glasshouse and field. Seedlings were inoculated with an ascospore suspension and tested in four different glasshouse environments. A significant positive correlation was found between disease scores in two of the glasshouse environments and the field trial for six field selected lines. However, the correlation between field and glasshouse results of the glasshouse-selected lines was nonsignificant.

28

R. K. DOWNEY AND S. R. RIMMER

Wittern and Kriiger (1985) used ascospore inoculum as droplet or spray suspensions to infect cotyledons or hypocotyls. The technique in which a spray suspension of ascospores was applied to cotyledons resulted in the greatest differences between varieties, whereas the other tests resulted in only small differences in disease reactions between the different varieties. Greenhouse and field results did not correspond for this method. McGee and Petrie (1978) tested disease responses of 18 B. napus and B. rupu lines in the greenhouse by inoculating plants at the soil level with oat kernels infested with the blackleg fungus. These lines were also tested against a population of L. mucufuns in field trials. The correlation between disease rankings in each test was highly significant. Wittern and Kniger (1985) obtained severe infection of rapeseed by mixing L. rnucufuns-infected oat kernels in the soil. However, differences in disease between varieties were slight and greenhouse and field results did not agree. Helms and Cruickshank (1979) tested cultivars of B. nupus, B. rupa, and B. rapa ssp. pekinensis growing in inoculated perlite but could not detect any differences in susceptibility between cultivars based on symptoms found on cotyledons and hypocotyls. Newman and Bailey (1987) reported a high correlation between glasshouse seedling tests and field results using a technique in which petioles were inoculated. However, this method did not detect all resistant types; field-resistant selections were uniformly susceptible for two sets of selections. Results obtained from mature plants tested at the bolting stage were inconsistent with field scores in most experiments. W. McNabb and S. R. Rimmer (unpublished) found a slight modification of this technique to correlate best with field observations compared to four other methods. 2. White Rust

White rust (also called white blister) is an important disease of cruciferous species in India and Canada. The disease, caused by Albugo cundida (Pers. ex Hook.) Kuntze, affects primarily B. rupa and B. junceu. It caused considerable yield losses of B. rapa in western Canada during the 1970s (Berkenkamp, 1972; Bernier, 1972; Petrie, 1973). The disease on B. rupa has been controlled with resistance since the registration of the cultivar Tobin (B. rupu) in 1981. However, a new pathotype of A. cundida, virulent on Tobin, has developed recently and may have been responsible for heavy local infections in Alberta (Conn and Tewari, I99 1). Canadian cultivars of B. nupus are highly resistant to western Canadian isolates of A. cundida in both field and laboratory studies (Petrie, 1975, 1988; Verma el uf., 1975). Albugo candida occurs as a number of specific pathotypes that differ in their pathogenicity to species and to genotypes within species of crucifer-

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

29

ous hosts. A number of pathotypes are specific to Brussicu species and closely related crops. A numerical classification of races or pathotypes based on pathogenicity and species specificity to a range of cruciferous hosts (Pound and Williams, 1963) has been used widely for genetic studies on host resistance. In North America, at least eight pathotypes of A. cundidu have been identified and classified based on their compatibility with different cruciferous host species or genera. These include race 1 on R. sutivus, race 2 on B. junceu (Pound and Williams, 1963), race 7 on B. rupu (Verma et ul., 1975; Pidskalny and Rimmer, 1985), and race 8 on B. nigru (L.) Koch (Delwiche and Williams, 1977). It should be noted that although A. cundidu pathotypes are virulent on many genotypes of their homologous host species, they are also capable of inciting disease on some genotypes of closely related species (heterologous hosts). Other as yet uncharacterized isolates have been obtained with pathogenicity on other Brussicu species (see Table V). Pathotypes occur that are more or less specific to B. rupu (race 7) and to B. junceu (race 2). No pathotypes are known whose homologous host is B. nupus, although certain genotypes of this species are susceptible to race 7 (Fan et ul., 1983), to an Indian isolate from B. junceu (Verma and Bhowmik, 1989), and to isolates from B. oleruceu and B. curinutu (S. R. Rimmer, unpublished data). Recently, Ethiopian isolates of A. cundidu from B. curinutu have been obtained and preliminary examination of the pathogenicity of these isolates indicates that they are virulent on some genotypes of species with the CC genome. a. Resistance to Race 2 Alhugo cundidu race 2 mainly infects B. junceu (Pound and Williams, 1963; Pidskalny and Rimmer, 1985; Petrie, 1988), but some genotypes of other Brussicu spp. are susceptible. Sources of resistance appear to be very limited. Parui and Bandyopadhyay (1973) found that a strain, Yellow rai T4, was virtually immune to natural infection by A. cundidu race 2. Ebrahimi et al. (1976) described the inheritance of resistance to white rust in the USDA accession PI 3476 18. F, progenies from the crosses between resistant and susceptible plants gave a disease reaction similar to that of the resistant parent. However, no data from the F2 have been reported. Bains and Jhooty ( 1979) screened 150 lines/cultivars of B. junceu against mixed infections caused by A. cundidu and Peronosporu purusiticu, but no resistance was observed. Failure to identify resistance to A. cundidu race 2 in B. junceu was also reported by Delwiche and Williams (1974). Tiwari et ul. ( 1988) studied the inheritance of resistance derived from a Russian accession of B. junceu, Vniimk405, to race 2 of A. cundidu. Their data from BC,

Table V Specificity of Isolates of Al6ugo cundi& to the Main Cultivated Crucifers' ~

~~

Pathotypes of A. candida and their homologous hosts

Host species Brassica rapa Brassica nigra Brassica oleracea Brassica juncea Brassica napus Brassica carinata Raphanus sativus

Genomeb

(fw (W

(CC) (AABBi MCC) (BBCC) (RR)

Ac/AAC (Ac~)~

SIRe RIS RIS RIS RIS RIS R

Ac/BB

Ac/CC

(Ac8)

(Ad)

Ac/AABB (Ac2)

RIS SIR RIS RIS RP R? R?

R/? RIS S/? R RIS R? R?

RIS RIS RIS SIR R RIS R

Ac/AACC (unknown)

* *

* *

*

AcIBBCC (A-)

? ? RIS ? RIS SIR? ?

Ac/RR (Ac 1)

R RIS R RIS R RIS SIR

Data from Petrie (1988), I. R. Crute (personal communication), and S. R. Rimmer (unpublished). Capital letters refer to species genome. Ac, Albugo candida; capital letters refer to homologous host genome of isolate. Race designation after Pound and Williams (1963). R, resistant; S, susceptible; SIR, most host genotypes susceptible, some resistant; R/S, most host genotypes resistant, some susceptible; ?, uncertain due to limited testing; *, not applicable-no isolates are known whose homologous host is B. napus.

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

31

and F2 populations strongly support a single dominant gene for resistance in their material. In addition to B. juncea, A. candida race 2 also attacks some genotypes of B. rapa, B. nigra, B. napus, and B. carinata (Pound and Williams, 1963; Petrie, 1988; Verma and Bhowmik, 1989). Because reaction to A. candida race 2 in B. rapa varies among individual plants, ranging from low to high infection type, resistance may be governed by both major and minor genes and quantitatively inherited, according to Edwards and Williams ( 1987). With a rapid-cycling population of B. rapa (CrGC-I), they found that the quantitative resistance conditioned by minor genes could be effectively enhanced by means of mass selection or half-sib family selection. In B. nigra and B. carinata, resistance to race 2 has been reported to be conferred by a single dominant gene (Delwiche and Williams, 1974, 1976, 1977). Verma and Bhowmik (1989) studied resistance in B. napus to an uncharacterized isolate of A. candida from B. juncea (presumably race 2) and showed that two dominant genes occurred in the resistant parent. b. Resistance to Race 7 No information concerning the inheritance of resistance of B. rapa to race 7 has been published. However, the cultivar Tobin contains resistance to this pathotype, derived from wild Mexican populations of B. rapa. The rate of population improvement for resistance from one generation to the next under selection pressure suggests the presence of a single dominant gene for resistance in this cultivar. Recently, in western Canada, a new pathotype of race 7 (race 7v) has occurred that overcomes this resistance. However, resistance to the new pathotype is also present in populations of cultivar Tobin at low frequency, and by recurrent selection, with both race 7 and race 7v, subpopulations with high frequencies of resistance to both pathotypes can be obtained in three or four selection cycles. c. Resistance in Brassica napus All present Canadian and European cultivars are resistant to the indigenous races of white rust. However, inheritance of resistance to A. candida in B. napus was investigated by Fan et al. ( 1983) using a resistant Canadian variety, Regent, and the susceptible Chinese lines, 2282-9 and GCL. The segregation of F, progenies from both crosses, 2282-9 XRegent and GCL X Regent, and their reciprocals suggested that resistance was governed by two independent dominant genes. Resistant plants resulted from the presence of a dominant allele at either of the two loci, and susceptibility would be expressed when the alleles at both loci were homozygous recessive. In addition, the segregation of some families of the F2 from the GCL X Regent cross indicated that a third dominant resistant gene occurs

32

R. K. DOWNEY AND S. R. RIMMER

in Regent. The three resistance genes were designated Ac7- 1, Ac7-2, and Ac7-3. F, plants from 2282-9 X GCL and the reciprocal were all susceptible to white rust. This suggested that the recessive genes camed by 2282-9 and GCL were allelic. These two Chinese lines were found to be somewhat different in their reaction to race 7 (Fan et ul., 1983; Liu et ul., 1989), probably indicative of minor gene effects, 2282-9 generally appearing slightly more susceptible than GCL. Liu and Rimmer (1992) reported that this same line, 2282-9, was resistant to an Ethiopian isolate of A. cundidu from B. curinuta. Canadian cultivars, including Regent and Stellar, were susceptible to this isolate. An inheritance study using crosses of 2282-9 with a doubled haploidderived line from Stellar indicated that a single dominant gene for resistance was present in 2282-9. d. Selection for Resistance Resistance to white rust can be efficiently selected in the cotyledon or seedling stages of plant development. Either oospores or zoosporangia can be used to produce zoospores for inoculum; oospores may be stored almost indefinitely at room temperature under dry conditions. Germination of stored oospores can be achieved by incubating on a shaker for 4-5 days (Verma and Petrie, 1975). Zoosporangia will store for 1 -2 years if collected dry from plants and stored at - 10 to -20°C. Zoosporangia germinate readily in distilled water at 10 to 16°C within 2-4 hr. The zoospores from germinated oospores or sporangia are used for inoculation. Details of inoculation techniques and disease assessment based on the interaction phenotype are described by Williams (1985). Plants that show any symptoms of white rust up to the flowering stage are discarded and the remaining plants are intermated (B. rupu) or selfed (for amphidiploid species, e.g., B. junceu). 3. Sclerotinia Stem Rot

Stem rot, caused by Sclerotiniu sclerotiorum (Lib.) de Bary, is a major yield constraint in many parts of the world, including Canada (Morrall and Dueck, 1982), Germany (Kriiger and Stoltenberg, 1983), and China (Liu ef ul., 1990). The same pathogen causes severe disease on many other dicotyledonous plants, including sunflower, field beans, peas, carrots, and lettuce (Purdy, 1979). Because of its broad host range, the lack of evidence for host specificity among isolates of the pathogen, and its sporadic occurrence due to weather conditions, economic control of this disease has been difficult. Many producers depend on fungicide applications during the flowering period for control and considerable effort has been directed

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

33

toward development of predictive methods to assist producers in determining whether or not fungicide applications would be economical. Sclerotinia sclerotiorum is an example of a broad host range, nonspecialized, polyphagous pathogen. Parleviet ( 1989) has presented a general discussion of the problems related to selection for resistance to such pathogens. Some evidence for variation for resistance to stem rot in oilseed brassicas has been reported. Brun ef al. (1987b) compared different inoculation techniques and demonstrated significant differences in stem rot severity among a number of B. napus accessions. Norin 9, a Japanese accession, showed good resistance to stem rot. Insertion of toothpicks infested with mycelium of the pathogen was shown to be a reliable method for evaluation of resistant materials. Sedun et al. (1989) compared the rate of lesion expansion of stem rot on stems of various Brassica species as a measure of disease resistance. Lesion expansion was slowest on B. carinata and B. napus compared to B. nigra, B. juncea, and B. rapa, which showed the most rapid lesion expansion. An interesting observation was that B. carinata, when inoculated in the leaf axils, frequently exhibited premature leaf abscission before infection could become established and thus consequently escaped stem rot. Liu ef al. (1990) reported that heritability of sclerotinia resistance was high in B. napus, controlled by nuclear genes and unlinked to the low-erucic acid trait. Infection of oilseed brassicas by S. sclerotiorum depends on the utilization by germinating ascospores of senescing petals as an energy substrate in order to colonize the stem (Kriiger, 1975; Lamarque, 1983). Disease escape may thus be possible through the development of apetalous cultivars. An apetalous mutant of B. napus obtained from G. Buzza (Pacific Seeds Pty., Australia) was used to test this hypothesis and was substantially free of stem rot compared to the normal petalous cultivar Westar (Table VI). Fu

Table VI Mean Yield, Disease Incidence, and Severity of Sckrotiniu Stem Rot and Range of Severity on Erusicu napus cv. Westar and an Apetalous Strain of Oilseed Rape in 1987" Cultivar or strain Westar Apetalous Standard error

Disease

Disease

incidence

seventy

Range of seventy

Yield per plot

(%)

(%)

(%)

(g)

19.2 2.1

15.4 I .5

0-45 0-4

151

f 4.0

f 3.5

-

f 32.0

" From S. R. Rirnrner (unpublished data).

I05

34

R. K. DOWNEY AND S. R. RIMMER

ef al. (1 990) studied the inheritance of the apetalous character in B. napus and showed that four recessive genes control this trait. They also observed that the apetalous lines were unaffected by stem rot. Apart from its potential use in avoidance of infection by S. sclerotiorum, there is reason to believe that increased yield may be associated with the apetalous trait (Mendham ef al., 1991). This could be due to the improved light penetrance into the crop and subsequent increase of photosynthesis as substantial light reflectance by petals occurs during flowering of normal types. The major difficulty in developing cultivars with resistance to stem rot has been the lack of convenient reliable techniques for selection of resistant phenotypes among population sizes required for a breeding program. In those climates where reliable infection levels may be obtained consistently, the best method is probably field testing and evaluation. This method is likely to be satisfactory in some parts of China and Europe where cool wet conditions are usual at flowering time. In Europe, where intensive oilseed rape production is widespread, the availability of satisfactory fungicide control has probably inhibited a concerted effort to select for resistance to this disease. It is perhaps not too surprising then that most progress in selection for resistance to stem rot has been made in China, where chemical control is unavailable for most farmers due to the expense and lack of suitable fungicides. Under drier conditions stem rot occurs sporadically and in these situations (for example, in western Canada) field selection for resistance has not been effective. 4. Alternuriu Black Spot

Black spot of oilseed rapes (also called dark leaf and pod spot or Alternaria blight) is caused primarily by Alternaria brassicae (Berk.) Sacc., although depending on environmental conditions, Alternaria brassicicola (Schw.) Wilts., Alternaria raphani Groves and Skolko, and Alternaria alfernata (Fr.) Keissler may also be associated with the disease in some areas. Aspects of this disease related to breeding for black spot resistance have recently been reviewed by Singh and Kolte ( 1993). It is important that a correct diagnosis of the species involved in the etiology of the disease in a specific situation be determined before embarking on extensive work on resistance breeding. The present review will focus on black spot caused by A . brassicae. Alternaria brassicae is the most prevalent causal agent of this disease on oilseed Brussica species in most production areas of the world. Yield losses may range up to 70%, varying from location to location and from year to

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

35

year [for references, see Singh and Kolte ( 1991)]. In Alberta, yield losses of up to 30% have been reported (Tewari and Conn, 1988). No high levels of resistance to black spot have been reported in cultivated oilseed Brassica species; generally, the rank in order of most to least susceptible is B. rapa, B. juncea, B. napus, and B. carinata (Bhowmik and Munde, 1987). Sinapis alba appears to be the most resistant of species closely related to the oilseed brassicas (Brun et al., 1987a; Dueck and Degenhardt, 1975; Rai et al., 1976). Chevre et al. ( 1991) reported interspecific transfer of the S. alba resistance into B. napus. This was accomplished by manual pollination using reciprocal crosses and by somatic hybridization from protoplast fusion. Subsequently, embryo rescue and cytogenetic analysis resulted in their obtaining B. napus plants with 38 chromosomes and with resistance to A. brassicae similar to that of S. alba. The genetic nature of the resistance transfer is currently under investigation. Some indication that cytoplasm may influence the degree of resistance has been obtained by Banga et al. (1984), who provided evidence that alloplasmic lines of B. juncea, with the cytoplasm of B. napus or B. carinata, exhibited a comparatively higher degree of resistance under field conditions than euplasmic lines, whereas lines with cytoplasm of B. rapa were more susceptible. Some variation for pathogenicity among isolates of A . brassicae occurs but no isolates by host genotype interactions have been reported (Mridha, 1983). Although A. brassicae has been reported to produce a host-specific toxin, destruxin B (Bains and Tewari, 1987), no evidence that the toxin is specific to particular genotypes within any Brassica species has been presented. Host-specific toxins are normally defined by their interaction with specific genotypes within a species, e.g., AK-toxin produced by the Japanese pear pathotype of A. alternata is specifically toxic only to cultivars of Japanese pear that are susceptible to the fungus [see Scheffer (1989) for a discussion of this concept]. Alternuria brassicae produces a number of destruxins (Buchwaldt and Jensen, 1991) that are toxic to plants from a number of genera of dicotyledons. Though the sensitivity of species to destruxin B is correlated with their relative susceptibility to infection by the pathogen, the absence of genotypes specifically sensitive to the toxin suggests that the destruxins should be considered as host-selective toxins (Buchwaldt and Green, 1992). Though this may seem a rather fine distinction, the implications of this for in vitro selection for resistance to black spot employing these toxins may be considerable (see Section V1,B). Tewari and colleagues have shown that other more distantly related cruciferous species may be very resistant to black spot (Tewari et al., 1987; Conn et al., 1988). Eruca sativa exhibited a hypersensitive-like reaction to

36

R. K. DOWNEY AND S. R. RIMMER

inoculation with A. brassicae. Camelina sativa appeared most resistant. Germination of spores of the pathogen was reduced on this host and penetration of the leaf was rare. However, transfer of these types of resistance to cultivated oilseed brassicas remains a formidable task. 5. Light Leaf Spot

Light leaf spot of brassicas is caused by the fungus Pyrenopeziza brassicae Sutton and Rawlinson, whose anamorphic state is Cylindrosporium concentricum Grev. This disease is perhaps the most important disease of winter oilseed rape in the United Kingdom and has become an increasingly severe problem in France (McCartney ef al., 1987). A review of the pathogen, including genetics and inoculation techniques, has been given by Courtice et al. (1988). Many brassica crops are affected, including oilseed rape and the vegetable brassicas, especially cauliflower and broccoli, as well as Brussels sprouts, turnip, etc. Variation among isolates of the pathogen for ability to cause disease and for resistance among and within cultivars of B. napus (Rawlinson et al., 1978; Maddock et al., 1981) and for race-specific resistance in B. oleracea and complementary virulence in the pathogen (Simons and Skidmore, 1988) has been demonstrated. Breeding for resistance in oilseed rape in the United Kingdom is in progress. 6. Turnip Mosaic

Oilseed brassicas are susceptible to a number of virus disease problems, and in some areas of China, especially where oilseed rapes are grown adjacent to areas where market garden production of vegetable brassicas is intensive, considerably damage may result. The most important virus disease in China is mosaic, caused, primarily, by turnip mosaic virus, though cucumber mosaic virus (CuMV) and occasionally ribgrass mosaic virus may also be involved (R. Stace-Smith, personal communication). Severe damage to seedlings can occur if the crop is planted at the time of the aphid vector migration. The disease can be avoided by later planting. It has been very difficult to find resistance to all strains of the different viruses present in China. Beet western yellows virus has also been reported to cause some damage in the United Kingdom (Gilligan el al., 1980). 7. VmicifliumWilt

Verticilliumwilt caused by Verticillium dahliae Kleb. has been reported to cause severe damage to oilseed rape in southern Sweden (Jonsson, 1978) and in Germany (Kriiger, 1989). Jonsson reported that variation for resist-

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

37

ance to Verticillium wilt occurred in both spring and winter B. napus lines but that B. rapa lines were susceptible. A description of the symptoms of the disease and techniques for field assessment have been presented by Grontofl ( 1987).

B. DEVELOPMENT OF HERBICIDE-TOLERANT CULTIVARS Selective herbicides provide an inexpensive and effective means of controlling weeds in crop cultivation. The introduction of the herbicide trifluralin in the early 1960s gave producers control over some of the most serious weeds of the Brassica oilseeds, for example, wild oats (Avenafatua L.), lamb’s quarters (Chenopodium album L.), and redroot pigweed (Amaranthus retrofexus L.), However, an effective selective herbicide is needed to control increasing populations of the related cruciferous weeds in canola rotations. 1. Triazine Tolerance

The excessive use of the herbicide atrazine on corn land (Zea mays L.) in eastern Canada resulted in the natural selection of a weedy biotype of B. rapa (Maltais and Bouchard, 1978). This tolerance was found to be cytoplasmically inherited and effective against the S-triazine family of herbicides. Through an interspecific cross and backcross program, the tolerant cytoplasm of B. rapa was combined with the nucleus of B. napus to produce the first triazine-tolerant cultivar, OAC Triton (Beversdorf et af., 1980).

Triazine tolerance has also been found in many other plant species and genera (Le Baron and Gressel, 1982) and is caused by a single base pair substitution in a chloroplast gene (psbA). This mutation results in a single amino acid substitution in the 32-kDa polypeptide, to which the herbicide would normally bind, rendering the herbicide ineffective (Hirschberg et af., 1984; Gressel, 1985; Reith and Straus, 1987). Unfortunately, the substitution of glycine for serine at position 264 on the D, or Qs protein also reduces the efficiency of the photosystem I1 (PSII) electron transfer system (Gressel, 1985), the probable cause of slower growth, lower seed yield, and reduced oil content in OAC Triton and subsequent triazine-tolerant cultivars such as Tribute, Triumph, Tristar, and Stallion (Beversdorf et af., 1988; Forhan, 1988). Thus, although triazine-tolerant B. nupus cultivars are very useful and indeed essential in fields where highly competitive weeds such as wild mustard (Sinapis arvensis L.), stinkweed (Thlaspi arvense L.), and quackgrass (Agropyron repens L.) abound, all evidence

38

R. K. DOWNEY AND S. R. RIMMER

suggests that the growth rate and yield of triazine-tolerant cultivars will always be significantly less than that of their recurrent parent. 2. Chlorsulfuron and Imidazolinone Tolerance

Several other herbicide-tolerant Brussicu plants are now under develop ment resulting from the use of mutagensis or transformation. Swanson and co-workers have applied microspore mutagenesis and selection to produce B. nupus plants tolerant to chlorsulfuron and imidazolinone herbicides. These sulfonylurea herbicides normally target and inhibit the enzyme acetohydroxy acid synthase (AHAS), the first enzyme common to the biosynthesis of leucine, valine, and isoleucine. Mutagenized microspores were cultured on media containing the herbicides of interest and the surviving haploid embryos regenerated. Small haploid plantlets were diploidized with colchicine and herbicide-tolerant doubled haploid plants were recovered (Swanson et ul., 1988, 1989). Progeny of these selected plants are now in Canadian official evaluation trials and could be in commercial use as early as 1993. However, the frequency with which tolerant plants of B. nupus were recovered suggests that similar mutants may exist in the natural weed population. Thus the use of these herbicides will need to be rotated with other herbicide classes and combined with good agronomic weed control practices to ensure their long and effective life. Miki et ul. (1990) produced chlorsulfuron-tolerant transgenic B. nupus plants by transfemng, via A. tumefuciens, the Arubidopsis mutant AHAS gene identified by Haughn et ul. (1988). DuPont has also produced transgenic chlorsulfuron-tolerant strains of B. nupus, but has since discontinued this project (C. J. Mauvais, personal communication). 3. Glyphosate Tolerance

Researchers at Monsanto have developed glyphosate (Roundup)-tolerant B. nupus plants through the A. tumefuciens-mediated transfer of a mutant form of the 5-enolpyruvylshikimate-3-phosphate(EPSP) synthase gene. However, the tolerance levels of the first transformants were less than that required for commercial use, due to the characteristic rapid translocation of the herbicide to the growing tip. After a number of generations of gene constructs, the present gene-promoter combination appears to confer an acceptable level of tolerance to commercial application rates of Roundup (Parker et ul., 1991). Rounduptolerant strains in a Westar cultivar background entered official Canadian performance trials in 1992.

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

39

4. Phosphinotricin Tolerance

Researchers at Hoechst have also used gene transfer to produce B. napus plants tolerant to the nonselective herbicide phosphinotricin (ppt; glufosinate-ammonium), sold commercially as Basta or Ignite. Tolerance was imparted through the transfer and expression of a modified ppt-acetyltransferase gene from Streptomyces hygroscopicus into B. napus plants of the cultivar Topas (Donn et al., 1990; Oelck et al., 1991). Herbicide-tolerant strains derived from these transformants entered Canadian evaluation trials in 1992. 5 . Bromoxynil Tolerance

Brassicu napus plants that are tolerant to the broad-leaved herbicide bromoxynil have also been obtained through Agrobacterium-mediated transformation. This herbicide acts by inhibiting and uncoupling photosynthetic electron transport (Oettmeier et al., 1982). The gene coding for nitrilase, which hydrolyzes bromoxynil to its nonphytotoxic benzoic acid, was isolated from bacteria, Klebsiella pneumoniae subsp. ozaenae (Stalker and McBride, 1987), and transferred to B. napus plants.

IV. FUTURE PROSPECTS The plasticity of oilseed brassicas for genetic manipulation and their amenability to biotechnological methods promise exciting developments for improvements of agronomic characteristics in the future. It is not unreasonable to consider that these developments will only be limited by our skills and creativity in devising them and in the resources applied to them and not by any intractable biological properties of the species themselves. Brassica spp. can be genetically manipulated with relative ease through interspecific hybridization. Methods to produce doubled haploids from anther or microspore culture have been pioneered in these species. Plants can be regenerated from protoplasts and transformed using Ti plasmids or other mechanisms. The intensive molecular genetic studies of the crucifer Arabidopsis thaliana may provide a source of cloned genes that can be inserted and expressed in these crop species. Some of the developments in these areas are discussed below in relation to agronomic improvements.

40

R. K. DOWNEY AND S. R. RIMMER

A. INTERSPECIFICHYBRIDIZATION The possibility of transfemng desirable characteristics from related genera to the oilseed Brassica species has greatly improved as more efficient and effective embryo rescue and protoplast fusion techniques have been developed. In addition, such techniques greatly extend the germ plasm base available to the breeder. Ripley and Arnison ( I 990) used embryo rescue to effect the interspecific cross B. napus X Sinapis alba as did Mohapatra and Bajaj (1987) in making the cross B. juncea X S. alba. Because S. alba exhibits a number of desirable traits, including flea beetle resistance, large yellow seeds, nonshattering seeds, and resistance to white rust and Alternaria, such crosses could be very useful. However, it may prove difficult to induce the desired crossing over among such distantly related genomes. Embryo rescue has also facilitated interspecific crosses between the oilseed brassicas and B. oleracea. Prakash and Raut (1983) used embryo rescue in crossing B. rapa with B. oleracea to resynthesize B. napus plant types better suited to the Indian environment. Chiang et al. (1977) also used embryo rescue to transfer resistance to race 2 of club root (Plasmodiophora brassicae) from B. napus to B. oleracea. Embryo rescue in the cross B. rapa X Eruca saliva resulted in allodiploid plants with reduced susceptibility to Alternaria and aphids (Agnihotri et al., 1990). A number of researchers, using conventional crossing techniques, have attempted to transfer resistance to L. maculans from those species containing the B genome (B. nigra, B. carinata, and B. juncea) to B. rapa and B. napus (genomes AA and AABB, respectively). Roy (1978a, 1984) first reported the successful transfer of resistance from an Indian accession (BJ168) of B. juncea to B. napus. No cytogenetic analysis of the early progeny was made, and in later generations (F6-F,), lines still segregated for disease resistance, suggestive of aneuploidy. Roy reported that Chiang (unpublished data) found the normal chromosome number (2n = 38) of B. napus with regular pairing (19 bivalents) in diakinesis in these advanced lines. However, most lines from Roy’s material, examined in our laboratory (S. R. Rimmer, unpublished data), contained 39 or 40 chromosomes. Subsequently others (Sacristan and Gerdemann, 1986; Sjijdin and Glimelius, 1989a,b; A. M. Chkvre, personal communication) have attempted the introgression of resistance genes from B. carinata, B. juncea, or B. nigra into B. napus or B. rapa. Sacristan and Gerdemann ( 1 986) used both B. juncea and B. carinata accessions as sources of resistance. They observed that B. carinata crossed more readily to B. napus than did B. juncea, in terms of the seed set in the F, and BC, and that B. napus types were more quickly recovered. However, the resistance from B. carinata was lost very quickly. They suggest

AGRONOMIC I M P R O V E M E N T IN O I L S E E D BRASSICAS

41

that introgression of the resistance gene(s) from the B genome may occur more readily in backcrosses to B. nupus by pairing with chromosomes of the C genome, as would be expected in the hybrid of B. junceu X B. napus with the genome formula of AABC, than by pairing with the A genome, as expected in the B. curinutu X B. napus hybrid (ABCC). It seems probable that resistance can be transferred from B. junceu to B. nupus, especially in conjunction with tissue and microspore methods, and if cytogenetic studies are used to ensure the recovery of euploid B. napus genotypes.

B. IMPROVEMENTS BASEDON BIOTECHNOLOGIES 1. In Vitro Haploid Production

The efficient production of haploid and doubled haploid plants from anther or microspore culture has become an important new tool for Brussicu breeders. Anther culture techniques, in which immature pollen (i.e., microspores) is induced to undergo embryo formation, eventually developing into haploid plants, were worked out in the late 1970s and early 1980s (Keller, 1984; Keller et ul., 1987). More recently, researchers have focused on efficient protocols to induce embryogenesis in isolated microspore cultures (Lichter, 1982; Chuong and Beversdorf, 1985; Keller et ul., 1987). Siebel and Pauls ( 1989) consider microspore culture in B. nupus to be more efficient, in terms of embryo yield, than anther culture. If doubled haploids can be produced efficiently they can be used very effectively in plant breeding and related genetic studies. Because the doubled haploids are homozygous, considerable time can be saved in parental identification or even varietal development compared with conventional inbreeding and selection procedures (Ulrich et ul., 1984). Three commercial cultivars have originated from haploid plants, Maris Haplona and Mikado, which originated from natural haploids identified in the field, and Cyclone, a product of microspore culture. An additional advantage of the doubled-haploid technique is the significantly smaller population size required to find the least likely recombinant, particularly when several genes are involved (Rajhathy, 1976). For example, to recover the low-glucosinolate segregate characteristic, conditioned by six recessive genes, an F2population of 12,269 would be needed in order to have a 95% probability of recovering one plant with the desired genotype. In contrast, a population of only 191 doubled-haploid plants would be needed to have the same probability of recovering the required genotype.

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R. K. DOWNEY AND S. R. RIMMER

However, the costs of materials and technical support for doubled-haploid programs are relatively high; thus, to be effective, the efficiency of doubled-haploid production must be high. Strains and cultivars of both B. napus and B. carinata have been highly responsive (Chuong and Beversdorf, 1985), and in the case of a few B. napus cultivars (e.g., Topas), a high yield of haploid embryos can be consistently obtained (up to 10% of cultured microspores) (Huang et al., 1990). Both B. rupa and B. juncea have generally given very low embryo yields, although some progress has been recently made with B. rapu (Baillie et a/., 1992). Effective procedures for colchicine doubling of the haploid plants have been developed (e.g., Coventry and Kott, 1988). Because mutations are immediately exposed in haploid and doubledhaploid plants and the haploid production processes in B. napus are efficient, the system has been used in mutagenic studies. Using a chemical mutagen followed by exposure to the imidazolinone herbicides, resistant B. napus haploids were produced and some of their doubled-haploid progeny are in official Canadian evaluation trials (Swanson et al., 1988, 1989). Similarly, adding the mutagen ethylnitrosourea ( E N ) into the microspore culture media allowed Wong and Swanson (1991) to recover two high oleic acid producing doubled-haploid plants from a doubled-haploid population of 3000. Researchers have also successfully used microsporederived embryos as a recipient cell system for gene transfer wherein macerated embryos and Agrobacterium were cocultivated (Swanson and Erickson, 1989; Oelck et al., 1991). 2. Somaclonal Variation

Microspores, cell cultures, and secondary embryos are excellent targets for in vitro selection for disease resistance, provided that the disease defense system is active in such an early stage of plant development. The fact that some Brassica pathogens (e.g., L. maculans, A. brassicae, and even S. sclerotiorum) produce toxins that may be involved as pathogenicity factors in disease development has resulted in considerable interest in their in vitro use as a selection technique for disease resistance. It is likely that host-specific toxins would be very useful for this purpose, though some argument may be made that resistance to non-host-specific toxins, if obtainable, might prove to be more durable. Using cell and explant culture systems, disease-resistant somaclonal variants and mutants have been selected in regenerated B. napus and B. juncea. By exposing B. napus cell cultures to fungal culture filtrates or partially purified toxins of L. maculans or A . brassicicola. disease-resistant variants have been obtained (Sacristan, 1982; MacDonald and Ingram, 1986; Newsholme et al., 1989). However, New-

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

43

sholme ef ul. (1989) observed that secondary embryoids selected in the presence of sirodesmin PL (a nonspecific toxin produced by L. mucufuns) or with culture filtrates of L. mucufuns lost resistance quickly after removal of the selection pressure. Plants regenerated from embryoids selected for tolerance to the toxic substances were no more resistant to the pathogen than plants regenerated from embryoids not exposed to the toxins when tested in the field. In B. junceu, variants for seed color, oil content, and other agronomic characteristics were regenerated from cotyledon explants (George and Rao, 1983). Salt-tolerant lines of B. junceu have been obtained by screening highly morphogenic cotyledon explant cultures on high-NaC1 media (Jain et af., 1989, 1990, 1991). The regenerated plants and their progeny appeared to retain the acquired salt-tolerant property. 3. Protoplast Fusion

Protoplast fusion, in which living cells with cell walls removed are fused to form new living entities, has also been used successfully to overcome pre- and postfertilization barriers. Protoplast fusions between Brussica and Arubidopsis were reported to yield stable genotypes that incorporated chromosomes or fragments from each species (Hoffman and Adachi, 1981). Gene exchange was also reported in intergeneric somatic hybrids from protoplast fusion between B. nupus and Diplotaxis harm (Klimaszewska and Keller, 1988) as well as between Sinupis turgidu Del. and Brussica (Toriyama et uf., 1987). Protoplast fusion is also the only means of combining two cytoplasmically inherited characteristics in a single genotype. In addition, fusion can result in a reassortment of nuclear and cytoplasmic elements. Thus, it is possible in B. nupus to combine in one cell the nucleus of one parent with foreign chloroplasts and mitochondria genomes, or a mixed foreign and domestic chloroplast - mitochondrion combination, or a choice of chloroplast genomes with a recombined mitochondrial genome. For example, CMS B. nupus plants, containing radish (R.sutivus) cytoplasm, exhibited chlorosis, especially at low temperatures, and contained low chlorophyll levels at high temperatures (Bannerot et ul., 1977; Rousselle, 1982; McCollum, 198I). In addition, these plants had poorly developed nectaries and reduced nectar flow (Mesquida and Renard, 1978). The chloroplast problem was solved through protoplast fusion by replacing the radish chloroplast genome with B. nupus chloroplasts but retaining the radish mitochondria controlling the CMS trait (Pelletier ef uf., 1983). In the resulting hybrids variability of flower morphology was also observed, with some plants having their nectar production restored to 8 1 % of the male

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R. K. D O W N E Y AND S. R. RIMMER

fertile control. These observations indicate that a recombination of the mitochondrial genome resulted in a restoration of nectaries while retaining the male sterility trait (Pelletier, 1990). In B. nupus plants, protoplast fusion and plant regeneration have also made possible the combination of the B. rapa chloroplasts that impart tolerance to the triazine family of herbicides with the mitochondria that control male sterility in the Ogura (Pelletier et ul., 1983; Kao et ul.. I99 I ), Polima (Barsby et al., 1987; Chuong et al., 1988), and nap (Yarrow et al., 1986) CMS systems. Thus two cytoplasmically inherited characteristics were combined in a single genotype. 4. Transformation

Although transgenic plants of B. napus have been obtained by microinjection (Neuhaus et ul. 1987) and electroporation (Guerche et af., 1987), Agrobacteriurn-mediated transformation is now the method of choice for this and other oilseed brassicas (Mehra-Palta et al., 199 1). Indeed, biotechnologists have made B. napus a target species for transformation, although B. rapa has also been successfully transformed (Mehra-Palta et al., 1991). As indicated in Section III,B, herbicide-tolerant transgenic B. napus cultivars will probably be one of the first transgenic products moving in international trade. Because refined edible vegetable oils contain no protein and oilseed brassica meal is only used in animal feeds, regulatory hurdles for such products could be minimal. Pollen control systems that rely on inserted genes are also well advanced, as indicated in Section II,A,3,e, but foreign genes for control of many other characteristics are now entering laboratory and field trials. a. Oil Quality As previously noted, the market value of a vegetable oil is largely determined by its fatty acid composition. To increase the value and versatility of Brassicu seed oil, several modifications to the original composition of rapeseed oil have been made through conventional and mutation breeding. Recently, Calgene has announced further modifications using foreign genes to modify the normal fatty acid biosynthetic pathway (Fig. 2). Using a seed-specific antisense gene construct to inhibit stearoyl-ACP desaturase activity, Knutzon et al. (1992) increased the level of stearate in developing embryos of B. rapa and B. nupus. In the B. rapa plants, stearate level in the seed was increased from a normal of less than 2% to as high as 40%. Unfortunately, the transformed B. rupa plants had very low oil contents and gemination was adversely affected. On the other hand, some B. napus transformants had stearic acid levels of over 30%, with oil content and

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

45

mCWH Capric

I

(12:O)

/vAAMlCWH Lauric

1

(14:O) ~

c

o

o

H

Palmitic

(16:0)

J.

Stearic

(18:O)

--f

Oleic

1

(183)

m C O O H + - c m n

Linoleic

(18:2)

Linolenic

(18:3)

-coon Eicosenoic

(20:l) J.

COOH

I

Erucic

(22:l)

DESATUAATION

Figure 2. Biosynthetic pathway for major fatty acids in vegetable oils.

germination levels comparable to nontransformed control plants. Calgene expects high-stearate canola oil to have an advantage in the manufacture of 100% canola margarine and in the confection trade. Voelker et ul. (1992), also at Calgene, has reported the development of transgenic B. nupus plants that produce a seed oil containing high levels (- 40%) of the C 12 :0 fatty acid, lauric acid. Laurate is normally not found in North American or European oilseeds but is an important constituent of tropical oils, such as coconut and palm. To obtain this new B. nupus oil composition the Calgene team purified the lauroyl-ACP thioesterase enzyme from the seed of the California bay tree and demonstrated in vivo that this enzyme interfered with the fatty acid chain elongation system. The gene controlling this enzyme was then transferred to B. nupus plants under the control of seed-specific promoters. Provided that transformants have a high seed and oil yield, the high-laurate oil could find a market in the manufacture of detergents, soaps, lubricants, and other industrial products. It now appears as if every step in the fatty acid biosynthetic pathway (Fig. 2) is open to manipulation. The modifications noted here are but the forerunners of many specialty oils yet to come. b. Oil Quantity The value of a vegetable oil is usually about twice that of the residual meal. Thus attempts are underway to apply gene transfer technology to

46

R. K. D O W E Y AND S. R. RIMMER

increase the oil content of the seed. Because metabolites produced in glycolysis are used to synthesize fatty acids, the enzymatic reactions in glycolysis are believed to be important in regulating carbon flow into oil biosynthesis (D. T. Dennis, personal communication). One of the key regulating enzymes for carbon metabolism of oilseeds is pyruvate kinase. The gene for this enzyme was isolated from potato and transferred to B. napus plants in overexpression constructs via an Agrobacterium transfer system. Of these transgenic lines, 20 were under field evaluation in 1992 to determine if oil content has been increased (W. A. Keller, personal communication). c. Protein Quality The amino acid profile of oilseed or cereal protein is an important factor in determining the nutritional and monetary value of a feed. Lysine and methionine are two of the most valuable amino acids because they are needed in oilseed meal to supplement the low levels of these essential amino acids in grain from cereals and maize. Two research teams transferred the gene expressing the methionine-rich 2s albumin seed storage protein from the Brazil nut (Bertholletia excelsa Humb. & Bonpl.) to B. nupus plants. Unfortunately, the level of expression was low, 0.02-0.1% of the seed protein (Guerche ef al., 1990; De Clercq et al., 1990).Subsequently, Altenbach et al. (1992) transferred the same gene into both spring and winter forms of B. napus and obtained between 1.7 and 4.0% of the total seed protein as the heterologous methionine-rich protein, up to 33% more methionine than normal B. nupus storage protein. These researchers suggest that the high level of expression in their transformants could be due to their use of a stronger promoter. Although increased levels of lysine in oilseed brassicas have yet to be reported, it is almost certain that such research is underway (Vanderkerckhove et al., 1989). d. Insect Control A number of insects belonging to the Lepidoptera and Cofeoptera genera attack Brussicu oilseed crops. Several gene-based insect control systems are under study but none have reached the commercial stage of development. Some of the toxins produced by Bacillus thuringiensis (Bt) are known to be effective against a number of lepidopterous species that attack the North American Brassica crops. The only report on the transformation of B. napus and B. rapa plants that express a Bt toxin is that of Mehra-Palta et af. (1991). None of the Bt toxins tested to date has proved to be effective against the flea beetle (Phyllotreta spp.), the most serious and persistent pest of oilseed brassicas in North America.

AGRONOMIC IMPROVEMENT IN OILSEED BRASSICAS

47

e. Disease Resistance Plants can activate a number of defensive mechanisms when attacked by pathogenic fungi, including the production and accumulation of proteinease inhibitors and lytic enzymes such as chitinase. Chitinase provides protection to the plant by catalyzing the hydrolysis of chitin, a major cell wall component of most fungi. By transfemng a 35s endochitinase gene from bean to B. nupus, Broglie el ul. (1991) recorded an elevated chitinase activity in the leaves and roots of the transformed plants. These plants were more resistant to attack by the AG4 group of Rhizoctoniu solani in that the onset of disease symptoms was delayed and the severity of the disease was reduced. Insertion and expression of viral coat protein genes in plants susceptible to the virus have resulted in some cases in the plants becoming resistant. Examples of this are resistance of potatoes to potato leafroll virus (Kawchuk et ul., 1991), and tobacco with tobacco mosaic virus (TMV) coat protein gene expression resistance to TMV and other tobarnoviruses (Nejidat and Beachy, 1990). This might be a useful strategy for develop ment of resistance in Brussicu spp. to turnip mosaic virus strains. Useful resistance to this viral pathogen has been difficult to achieve.

f. Heavy Metal Sequestering It has been suggested that the development of plants that are tolerant to, or capable of sequestering, heavy metals in nonconsumed plant tissues would be useful in detoxifjmg mine sites and other such contaminated areas. To develop such a plant, Misra and Gedamu (1989) used an Agrobacterium-mediated transfer system to insert a chimeric gene, containing a cloned human metallothionin-I1 gene, into B. nupus plants. The seeds from the transformed and nontransformed plants were germinated on media containing toxic levels of cadmium (up to 100 pA4 CdCl,). Seed of the nontransformed plants showed growth inhibition and leaf chlorosis whereas the root and shoot growth of the seed from the transformed plants were unaffected. g. Molecular Farming

It may be possible to use Brussicu oilseed crops as factories to synthesize highly valuable chemical products or pharmaceuticals. Vanderkerckhove er al. (1989) demonstrated that the small 2s albumin proteins of B. nupus can be altered to produced the pharmacological neuropeptide, leu-enkephalin, although the yield was only 15 to 75 g per hectare. These researchers believe that much higher yields could be obtained with higher gene expression in new transformants or by turning off the 12 to 19 competing 2s albumin genes normally expressed in developing B. nupus seed. They also suggest that by substituting antigenic peptide epitopes for the variable

48

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region in the 2 S protein, large-scale isolation of vaccines should be feasible.

C. USESOF DNA MARKERS Plant breeders have long desired markers to aid in the selection for important traits. Generally, morphological markers linked to important agronomic traits are rare, but the use of isozymes and, more recently, DNA polymorphisms now offers better prospects for the development of reliable markers for selection purposes. Although, the concept of using restriction fragment length polymorphisms ( RFLPs) as neutral codominant markers linked to important phenotypic traits is less than 15 years old (Botstein et al., 1980), the use of RFLP maps in plant breeding programs is already common. Most large private and public plant breeding organizations are using such maps or are actively developing them. However, such maps in themselves are of limited usefulness to the breeder until important agronomic traits can be positioned on the map, a procedure that can require a significant amount of resources. RFLP mapping programs endeavour to develop a saturated linkage map based on a set of DNA polymorphic probes and to associate the mapping of particular probes to agronomic traits of interest. Applications of these basic techniques to plants have been described by Landry and Michelmore ( 1987). Tanksley et al. (1989) have reviewed the numerous potential advantages and applications of using RFLP maps and RFLP markers linked to agronomic traits for plant breeding purposes. A map of B. napus based on RFLPs detected with cDNA probes has been published recently (Landry et al., 1991). A good set of probes linked with useful traits can be valuable in parental selection for crossing, and for screening later segregating populations. Disadvantages of using RFLP markers are that the method requires a molecular genetics laboratory fully equipped for sophisticated DNA manipulation, the use of radioactive labeled probes, and large amounts of DNA extracted from the plant materials. The use of random amplified polymorphic DNAs ( RAPDs) using PCR technology potentially avoids these problems. This technique also can be useful for mapping purposes and considerable interest in the method is developing (Deragon and Landry, 1992). 1. Linkage Mapping

RFLP genetic maps have been published for B. rapa (Song et al., 1990; Chyi et al., 1992), B. nupus (Landry et al., 199 I ) , and B. oleracea (Slocum

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et al., 1990; Landry et al., 1992). Landry et al. (1992) have obtained RFLP probes that are linked to resistance to the clubroot pathogen, Plasmodiophora brassicae, in B. oleracea. In addition, two dominant quantitative trait loci (QTL) designated CR2a and CR2b were located on linkage groups 1 and 8, respectively. Other mapped traits were a locus for leaf morphology and one for biennial flowering. Yield, oil, and protein content of seed can also be mapped if relatively few loci with major effects on the traits in question are involved (Lander and Botstein, 1989; Knapp, 1991). It is to be expected that the use of these techniques will become a major component of oilseed brassica plant breeding programs in the near future.

V. SUMMARY AND CONCLUSIONS There can be little doubt that the oilseed brassica crops are destined to play an ever-increasing role in supplying the world’s food, feed, and industrial needs in the decades to come. Not only do these crops offer the best opportunity to meet the increasing oil requirements of many developing nations, such as China, and those of the Indian subcontinent, but they also offer the chance for crop diversification in predominantly cereal-producing countries such as Argentina, Uruguay, Spain, and parts of the United States. There is no evidence in the literature or production statistics that suggest breeders are encountering a seed yield plateau or that an upper limit has been approached for either oil or protein content. Indeed, the potential for hybrid oilseed brassicas has not yet been fully exploited. However, a recent policy change in the way commodity support prices are calculated in the European Economic Community will likely result in producers reducing their investment in crop inputs, e.g., fertilizer and pest control, and a corresponding drop in the average yield of European rapeseed. The major constraints to continued expansion and concentration of production are the many insects, diseases, and weed pests with which the crop must contend. The advent of herbicide-tolerant Brassica plants via biotechnology may well eliminate the weed problem, but the insect and disease problems remain a formidable challenge despite the advances made to date. The present high interest in using rapeseed oil as a hydraulic fluid replacement or. additive to diesel fuel in Europe could result in a much expanded industrial use market. Similarly, the emerging ability to tailormake specific oil compositions for niche markets, as well as to elevate the

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nutritional value of brassica seed products, opens a whole new array of opportunities. The wide range in form and product now made possible through genetic manipulation ensures that these crops will continue to grow in importance regardless of the demands that nature or the market may impose.

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POPULATION DIVERSITY GROUPINGS OF SOYBEAN BRADYRHIZOBIA Jeffry J. Fuhrmann Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware I97 1 7

I. Introduction 11. Genotypic Groupings

111. Phenotypic Groupings A. Serology B. Intrinsic Antibiotic Resistance C. Uptake Hydrogenase D. Dissimilatory Nitrate Reduction E. Rhizobitoxine F. Surface Polysaccharides G. Protein Profiles H. Rhizobiophage Typing I. Plant Growrh-Regulating Substances J. Other Phenotypes IV. Summary of Phenotypic and Genotypic Relationships V. Taxonomic Status of Brodyrhiwbium japoninrm VI. Concluding Remarks References

I. INTRODUCTION Substantial research progress has been documented for many N,-fixing systems, including the symbiosis between soybean [Glycine max (L.) Merr.] and the nodulating bacterium Brudyrhizobium juponicum (Gresshoff et ul., 1990; Stacey et al., 1992). However, as noted for leguminous symbioses in general (Bottomley, 1992), progress in the ecology of B. juponicum has been less impressive than that realized for most other A h 8 I i+ A w y , Vd. I0 Copyright 0 1993 by Acrdemic Press, Inc. All rights of reproduction in any form reserved.

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aspects of the soybean symbiosis. Although the inherent complexity of the soil - soybean environment has certainly contributed to this impasse, ecological research has also been hampered by a persistent uncertainty of how best to assess the symbiotic diversity present within populations of B. juponicum. The literature abounds with studies demonstrating diversity among strains of B. juponicum, but relatively few studies have assessed the potential symbiotic significance of the resulting groupings. In a recent review, Bottomley (1992) emphasized that researchers need to develop a unified approach to the study of rhizobial ecology so as to facilitate comparison and synthesis of experimental results. Clearly, the identification of coherent strategies for the characterization of indigenous populations of B. japonicum would constitute a major step toward achieving this goal for the soybean symbiosis. The objectives of this review are to summarize the present literature characterizing population diversity in B. juponicum and to draw attention to established or possible relationships among the various diversity groupings, particularly with respect to the symbiotic performance of the associated strains. Apparent gaps in current knowledge will also be noted as appropriate. Emphasis will be placed on characteristics that may be considered traditional by current standards, but this simply reflects the relative depth of the scientific literature involving these groupings. For the same reason, discussion will primarily address research conducted in North America, although the literature that is available from other geographical regions suggests that many of the relationships to be discussed are generally applicable.

11. GENOTYPIC GROUPINGS Research has clearly established that the species B. juponicum is composed of at least two major genetic groupings. Hollis et ul. (198 1) identified two major DNA homology groups based on DNA - DNA hybridization studies, and proposed that the species B. juponicum (then Rhizobium juponicum) be reserved for one of these groups (I/Ia) and that the second group (11) represented a distinct species. Two highly divergent groups, consistent with those proposed by Hollis ef ul. (1981), have also been observed based on molecular genetic studies (Minamisawa, 1990; Stanley el ul., 1985). In a recent report, Kuykendall ef ul. (1992) analyzed a number of B. juponicum strains using DNA hybridization probes and confirmed the groupings proposed by Hollis ef ul. (198 1).

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As documented in subsequent sections of this review, there is mounting evidence that the two major DNA homology groupings are highly correlated with many characteristics of soybean bradyrhizobia of potential importance to symbiotic performance. These strong correlations also appear to permit one to infer the DNA homology grouping to which a given strain of B. japonicurn belongs based on certain readily obtained phenotypic characteristics [e.g., Fuhrmann (1990) and Kuykendall et al. (1988)l. Therefore, the use of phenotypic traits to assign soybean bradyrhizobia indirectly to established genetic groupings may represent a valuable strategy for initiating characterizations of unidentified soybean bradyrhizobia, such as those comprising indigenous soil populations.

111. PHENOTYPIC GROUPINGS A. SEROLOGY Serological reaction is probably the most commonly encountered means of characterizing soybean bradyrhizobia, particularly for surveys of indigenous B. japonicurn. Similarly, it is often conveniently used as a basis for comparison with other phenotypic characteristics. The reader is directed to Vincent ( 1982) for a general introduction and historical perspective on the use of serological techniques in the Rhizobiaceae. 1. Diversity of Serological Phenotypes

Serological analyses are primarily used to provide empirical groupings of bradyrhizobia having value for reference purposes. Therefore, serogroups of B. japonicurn are typically characterized according to their reactions with antisera produced against strains having an extensive research history. In North America, the serogroups most commonly referred to are those derived from strains present in the Rhizobium Culture Collection maintained by the United States Department of Agriculture (Beltsville, Maryland) (Keyser and Griffin, 1987). Much of the present-day framework for the serological study of soybean bradyrhizobia was established by Damirgi et al. (1967), Date and Decker (1965), Johnson and Means (1963), and Koontz and Faber (196 1). A number of serological methods are currently used to characterize soybean bradyrhizobia. The most common of these are agglutination (Date and Decker, 1965; Means et al., 1964; Wollum, 1987), fluorescent

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antibody techniques (Bohlool, 1987), and various forms of the enzymelinked immunosorbent assay (Asanuma et al., 1985; Ayanaba et al.. 1986; Fuhrmann and Wollum, 1985; Kishinevsky and Jones, 1987). Polyclonal antisera have been used almost exclusively, although the successful use of monoclonal antibodies with soybean bradyrhizobia was reported by Velez er al. ( 1988). Agglutination is historically the most prominent of the three methods and can probably be considered the standard for comparison. In reality, however, method selection is based primarily on familiarity, and there are no published studies providing critical comparisons of the common serological techniques. Serological studies of indigenous B. juponicum have revealed considerable diversity within and among geographical locations. It has been possible in some instances to correlate the presence of particular serogroups within a restricted region to soil properties such as pH (Damirgi er al., 1967; Ham ef al., 1971) or total nitrogen (Bezdicek, 1972). On a regional basis in the United States, strains aligning with serogroup 123 are especially prominent in the upper midwest states (Damirgi et al., 1967; Ham et al., 1971; Kamicker and Brill, 1986; Keyser er al., 1984; Weber el al., 1989), whereas strains correlating to serogroups 3 1,76, and 94 are common in the southern and mid-Atlantic states (Caldwell and Hartwig, 1970; Fuhrmann, 1989, 1990 Keyser el al., 1984; Mpepereki and Wollum, 1991; Weber et al., 1989). Although the prevalence of serogroups 31 and 123 may be a result of their use in early soybean inoculants, there is no known historical explanation to account for the high frequencies of serogroups 76 and 94 (Weber et ul., 1989). 2. Correlation with Symbiotic Performance

One common goal of serological characterizations of rhizobia is to identify groups that have practical significance to the management of a particular symbiosis. However, Vincent (1982) cautioned that serological results should not be indiscriminately extrapolated to other properties such as symbiotic effectiveness, and warned against allowing the distinction between a serogroup and the corresponding serotype strain to become blurred. Studies have revealed significant variation within standard strains of B. japonicum maintained at different laboratories, further suggesting that generalizations based on serotype strains should be approached with caution (Mullen and Wollum, 1989). Yet, although many studies have documented serological diversity within rhizobial populations, relatively few have assessed the value of the resulting groupings in predicting symbiotic performance.

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One common problem with using serology to characterize soybean bradyrhizobia is the presence of strains that are nonreactive with all antisera tested. The frequency of nonreactive strains is often significant, particularly in soils from the southern and mid-Atlantic United States (Fuhrmann, 1990; Mpepereki and Wollum, 1991). Moreover, there is evidence that rhizobia in this grouping can be symbiotically diverse (Fuhrmann, 1990). There is clearly a need to identify and document additional reference strains for characterizing indigenous soybean bradyrhizobia. Serological analyses commonly reveal strains that cross-react with antisera derived from two or more reference strains. The best documented example of this is the suite of soybean bradyrhizobia that constitute serocluster 123 (serogroups 123, 127, and 129)(Schmidt et al., 1986). Although related serologically,the serogroups comprising this serocluster are known to exhibit physiological and symbiotic diversity (Gibson et al., 1971; Hickey et al., 1987; Sadowsky et al., 1987). Given the possibility that additional seroclustersexist, currently recognized serogroups may inadvertently serve to mask significant diversity among member strains. Studies have documented large differences in symbiotic effectiveness among soybean bradyrhizobia in a single serogroup. Basit et al. (199 1) found threefold differences in shoot weights and N contents among 37day-old soybean plants nodulated by 34 serogroup 1 10 strains. Other work found that the mean effectiveness of indigenous isolates within serogroups was inconsistently related to that of the corresponding serotype strain (Fuhrmann, 1990). In the later study, grouping the indigenous isolates according to their reactions with 12 antisera accounted for only 59% of the variation in N contents of 42day-old soybean plants (Fuhrmann, 1990). Certain soybean genotypes are known to restrict nodulation by particular serogroups or strains of soybean bradyrhizobia (Caldwell, 1966; Caldwell et al., 1966; Vest, 1970; Vest and Caldwell, 1972), although there is recent evidence that some of these reactions may be only coincidentally related to serology (Devine et al., 1991). Host genotype-specificnodulation may have value in altering nodulation competition by displacing undesirable indigenous strains from root nodules in exchange for more effective indigenous or inoculant strains (Caldwell and Vest, 1968; Devine and Breithaupt, 1980a; Ishizuka et al., 1991a,b; Kvien et al., 1981; Weiser et al., 1990). In particular, this possibility has been explored as a means of developing soybean genotypes that exclude strains belonging to serocluster 123 (Cregan and Keyser, 1986; Cregan et al., 1989a,b; Sadowsky et al.. 1987; Schmidt et al., 1986). Indigenous strains in serocluster 123 are generally considered to be relatively ineffective in N2 fixation, although support for this contention comes primarily from research conducted with

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the serotype strain USDA 123 (Caldwell and Vest, 1970) or other indirect evidence (Kvien et af., 1981). 3. Relationship to DNA Homology Groupings

A strong correlation exists between DNA homology groupings in B. japonicum (Hollis et af., 198 1) and their corresponding serogroups. Documented serogroups in group I/Ia include 6, 38- 1 15, 58, 62, 1 10, 122, and 123, whereas group I1 includes 31, 46, 76, 86, 94, and 130. Additionally, Keyser and Griffin (1987) listed three serogroups (4, 125 - 126, and 135) which have apparently not been analyzed as to DNA homology. Keyser and Griffin (1987) also listed two cross-reaction groups: 1 10- 1 15, in which both serotype strains are in group I/Ia, and 76- 123, in which the serotype strains are in groups I1 and I/Ia, respectively. In addition to this 76- 123 grouping, Weber et al. (1989) reported two cross-reaction serogroups (6276 and 94 - 1 10) derived from serotype strains belonging to both group I/Ia and 11. These appear to be the only reported serological groupings that bridge the DNA homology groups. It is noteworthy that only group I/Ia contains serotype strains considered to be highly effective (Keyser and Griffin, 1987).

B. INTRINSIC ANTIBIOTIC RESISTANCE Numerous studies have used intrinsic antibiotic resistance (IAR) as a means of characterizing rhizobia, including soybean bradyrhizobia, taken from both established culture collections and indigenous populations (Eaglesham, 1987). Certainly one reason for its common use is the relative ease with which IAR can be determined, especially when the bradyrhizobia of interest are available as pure cultures. The reader is directed to Eaglesham (1987) for general information concerning the use of IAR in rhizobial research. 1. Diversity of

IAR Phenotypes

As with serological analyses, characterization of bradyrhizobia by IAR is used primarily to obtain empirical reference groupings that can be compared with other traits of interest. Although Eaglesham (1987) described a number of methods for determining IAR, a review of the literature shows

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that the one most commonly used is the agar dilution technique in which antibiotic-amended plates are spotted with cultured bradyrhizobia and later examined for growth. Approaches to the use of IAR to characterize bradyrhizobia vary greatly among laboratories. Most studies have employed several individual antibiotics, each at a single concentration, that concentration being derived from preliminary studies with a representative subsample of strains [e.g., Hickey et al. (1987), Kuykendall et al. (1988), and Thompson et al. (1991)l. Others have used several individual antibiotics, each over a range of concentrations (Cole and Elkan, 1979; Sawada et al., 1990; Young and Chao, 1989), a single antibiotic at many different concentrations (Pankhurst et al., 1982), or selected combinations of antibiotics (Mueller et al., 1988). Comparison of IAR results among studies is further complicated by a lack of standardization of antibiotics employed. The more commonly used antibiotics for soybean bradyrhizobia are kanamycin, nalidixic acid, rifampicin, and streptomycin, although carbenicillin, chloramphenicol, erythromycin, neomycin, novobiocin, penicillin, polymyxin, spectinomycin, tetracycline, and vancomycin have also been used by several investigators. Eaglesham (1987) noted that the diversity detected within a given population is often positively correlated with the number of antibiotics against which the population is tested. For similar reasons, it may be desirable to select antibiotics that differ in their modes of antimetabolite action (Sinclair and Eaglesham, 1984). Large differences also exist among studies with regard to the concentrations used for a particular antibiotic. It is not uncommon for antibiotic concentrations to differ by more than 10-fold among studies [e.g., Hickey et al. (1987), Kuykendall et al. (1988), Mueller et al. (1988), and Thomp son et al. (1991)l. Because inhibition by antibiotics is concentration dependent, it is often unclear whether apparent differences in IAR among studies represent differences in methodology, actual population differences, or a combination of the two. Despite the difficulties noted above, a number of studies have demonstrated unambiguous differences among populations by means of IAR. In particular, Mueller et al. (1 988) determined the IAR of nodule bacteroids taken from eight cultivars and three locations in South Carolina and found considerable differences in IAR patterns among the sampling combinations. This study also concluded that, overall, the native bradyrhizobia displayed high resistance to streptomycin and low resistance to rifampicin, kanamycin, and nalidixic acid in various combinations. In contrast, bradyrhizobia indigenous to Iowa soils were shown to be very susceptible to a much lower concentration of streptomycin than that used in the South

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Carolina study (15 versus 500 pg/ml) (Hickey ef al., 1987). Similarly, Thompson ef al. ( 199 1) reported that bradyrhizobia isolated from soybean in Thailand were much less resistant to several antibiotics than were the United States populations studied by Mueller et al. (1988). 2. Correlation with Symbiotic Performance

Possible relationships between IAR phenotype and symbiotic performance have not been adequately explored to allow for meaningful generalizations to be made. Particularly lacking are studies in which IAR group ings of bradyrhizobia have been subsequently tested for their associated N,-fixing abilities. Thompson ef al. ( 199 1) concluded that IAR was useful for separating isolates for subsequent effectiveness testing, but did not attempt to correlate the two characteristics. However, as described in the subsequent section, there is evidence that IAR groupings are correlated to DNA homology in soybean bradyrhizobia (Kuykendall ef al., 1988). Thus, to the extent that DNA homology is correlated with symbiotic effectiveness, groupings identified by IAR may indirectly indicate N, fixation potential. The presence of IAR in soybean bradyrhizobia raises the question of its possible ecological role, especially with respect to saprophytic survival and nodulation competitiveness. Eaglesham ( 1987) cites a number of studies that provide indirect support for the hypothesis that IAR may represent an ecological advantage in many soil systems. Here again, however, there is an obvious need for additional studies investigating the role of IAR in the dynamics of bradyrhizobial populations. 3. Relationship to DNA Homology Groupings

Kuykendall ef al. (1988) reported a nearly perfect correlation between the DNA homology groupings of Hollis ef al. (198 1) and intrinsic resistance to a suite of antibiotics. In general, group I1 strains exhibited high levels of resistance to carbenicillin ( 5 0 0 pg/ml), chloramphenicol (500 pg/ml), erythromycin (250 pg/ml), nalidixic acid (50 pg/ml), rifampicin (500 pg/ml), streptomycin (100 pg/rnl), and tetracycline (100 pg/ml), whereas strains in group I/Ia were susceptible. Support for these findings can be found in the work of Pankhurst et al. (1982) in which B. japonicum strains in group I1 (based on serological correlations) were much more resistant to rifampicin than were strains from group I/Ia. Kuykendall ef al. (1988) concluded that differentiation based on IAR profiles should prove

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valuable in genetic and phenotypic characterizationsof soybean bradyrhizobia, including studies of indigenous populations.

C. UPTAKE HYDROGENME The reduction of protons to hydrogen gas is an integral component of biological N, fixation (Arp, 1992; OBrian and Maier, 1988): N,

+ 8H+ + 8e- + 16ATP

-

2NH,

+ 16ADP + 16Pi + H,

(1)

Although the stoichiometric relationship between N, reduced and H, produced can vary, it is not possible to eliminate entirely energy allocation to proton reduction. The amount of energy diverted to H, evolution constitutes 25% or more of the total electron flux allocated to N, fixation (Evans et ul., 1987). Therefore, H2 evolution potentially represents a substantial inefficiency in the N, fixation process. Certain rhizobia, including some soybean bradyrhizobia, possess a hydrogenase system that catalyzes the oxidative release of energy from H,:

H,

-

2H+

+ 2e-

(2) Rhizobia exhibiting this capability have traditionally been referred to as hydrogen uptake positive (Hup+) and often produce root nodules that show a complete lack of H, evolution. This recycling of H, and energy is generally thought to increase the efficiency of N, fixation, and it has been suggested that the Hup+ phenotype is one criterion that may directly indicate superior symbiotic effectiveness of soybean bradyrhizobia. 1. Diversity of Hydrogenase Phenotypes

In reviewing the literature regarding hydrogenase phenotypes in soybean bradyrhizobia, it is important to consider the methods used to detect hydrogenase activity. Soybean bradyrhizobia have traditionally been categorized simply as Hup- or Hup+ by means of either in vitro or in vivo techniques. However, recent research has demonstrated the presence of an additional host-regulated phenotype (Huphr) in which hydrogenase activity is only exhibited by bacteroids while in symbiosis with cowpea [ Vigna unguiculuta (L.) Walp.] or certain soybean genotypes (van Berkum, 1990; van Berkum and Sloger, 1991). Differential expression of hydrogenase between soybean and cowpea had been previously reported for soybean bradyrhizobia (Keyser et al., 1982). Thus, there are currently three documented hydrogenase phenotypes in soybean bradyrhizobia: Hup +,hydro-

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genase activity expressed in vitro and in vivo with common North American soybean genotypes; Huphr, hydrogenase activity exhibited only in vivo with certain host genotypes; and Hup-, lack of demonstrable hydrogenase activity under any of the above conditions. Many studies of soybean bradyrhizobia have relied solely on in vitro methods for the detection of hydrogenase activity. Some early studies employed a tritium exchange method (Lim, 1978), but there is evidence that this method may erroneously indicate the presence of an active hydrogenase system in Hup- strains (Robson and Postgate, 1980; van Berkum et af., 1985). A common alternative technique monitors H, disap pearance from the headspace of cultures grown either chemotrophically or heterotrophically under hydrogenase-inducing conditions (van Berkum, 1987). Although there is no evidence that this latter method produces false positive results, it would apparently fail to detect those bradyrhizobia possessing the Huphr phenotype (van Berkum, 1990; van Berkum and Sloger, 1991). Although relatively cumbersome and time-consuming when compared with in vitro techniques, assays employing intact nodules or bacteroid suspensions are able to yield definitive information concerning the hydrogenase activity of a particular host -strain combination. Conversely, assays conducted with a single host genotype, or in the absence of an accompanying in vitro assay, are unable to distinguish between the Hup+ and Hup-hr phenotypes. Evidence to date, however, indicates that expression of the Huphr phenotype in soybean may be limited to symbioses formed with hosts genetically distant from common North American soybean lines (van Berkum and Sloger, 1991). A number of studies have examined the frequency of Hup+ strains in indigenous populations of soybean bradyrhizobia. Early studies assayed bradyrhizobia taken from nodules collected from throughout much of the eastern and midwestern United States (Keyser ef al., 1984; Lim et af., 1981; Uratsu et al., 1982). These studies concluded that the frequency of Hup+ strains ranged from 20 to 25% overall and from 0 to 90% on a regional basis. All of these studies were conducted using the tritium exchange method (Lim, 1978) and, therefore, may have overestimated the frequency of Hup+ phenotypes (van Berkum et al., 1985). More recently, a survey of soybean bradyrhizobia indigenous to Delaware indicated that 14 of 92 nodule isolates examined (1 5%) were Hup+ (Fuhrmann, 1990). Hydrogenase activity was determined in vitro using a modified heterotrophic growth technique (van Berkum, 1987) and, therefore, would not have detected the presence of the Huphr phenotype. Sawada et al. (1989) found that nodules formed by 34% of 85 isolates of

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bradyrhizobia indigenous to Japanese soils evolved little or no H, in symbiosis with soybean, thereby indicating the presence of an active hydrogenase. 2. Correlation with Symbiotic Performance

The literature contains conflicting evidence regarding the symbiotic effect of hydrogenase activity by nodulating rhizobia. Arp (1992) discussed the mechanisms by which it is thought that the presence of an active Hup system is beneficial to N,-fixing symbioses: 1. The energy released by H, oxidation can be recycled and used to produce supplemental ATP for nitrogen fixation or other cellular processes (Emerich et al., 1979). 2. In aerobic N,-fixing systems such as those involving rhizobia, recovery of the energy released by H, oxidation is coupled to 0, consumption via the electron transport chain. This 0, consumption may serve to help protect the nitrogenase enzyme from 0,-induced inactivation (Emerich et al., 1979). 3. Hydrogen oxidation may reduce H,-induced inhibition of nitrogenase by decreasing the partial pressure of the gas in the nodule (Rasche and Arp, 1989).

Additionally, it has been suggested that Hup+ nodules may have a greater functional longevity than Hup- nodules (Zablotowicz et al., 1980) and that the Hup+ phenotype may be an ecological advantage to soybean bradyrhizobia existing chemoautotrophically in low 0, environments, such as aggregate interiors (Ozawa et al., 1989; Viteri and Schmidt, 1989). Many studies have investigated the symbiotic response of the soybean plant to nodulation by wild-type or nonisogenic mutant strains differing in Hup phenotype. These studies have reported both beneficial (Albrecht et al., 1979; Hanus et al., 1981; Zablotowicz et al., 1980) and neutral (Basit et al., 1991; Hume and Shelp, 1990; Kimou and Drevon, 1989) responses to nodulation by Hup+ bradyrhizobia relative to Hup- strains. In a greenhouse study, Fuhrmann (1990) found that the Hup+ phenotype was either beneficial or neutral to the soybean symbiosis, depending on the accompanying colony morphology of the isolate under examination. This same study found that hydrogenase phenotype was a relatively poor indicator of the symbiotic effectiveness of indigenous soybean bradyrhizobia when compared with groupings based on serology. However, interpretation of all of these studies is hampered by possible confounding effects of other differences between the two groups other than hydrogenase phenotype.

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Other investigations have employed isogenic strains of soybean bradyrhizobia differing only in Hup expression so as to eliminate possible confounding effects. The weight of these experiments indicates that hydrogenase activity is beneficial to the symbiosis (Arp, 1992; Evans et al., 1987; Hungria et al., 1989), although detrimental effects were observed in a short-term study using hydroponic plant culture (Drevon el al., 1987). Evans et al. (1987) proposed a number of precautions that should be observed in studies of hydrogenase effects: strains isogenic except for Hup phenotype should be used, the Hup+ phenotype should be stable and vigorous, the hydrogenase should be effectively coupled to ATP formation, and the plants should be grown to maturity under normal environmental conditions. 3 . Relationship to DNA Homology Groupings

Hydrogenase phenotype in soybean bradyrhizobia has been shown to be strongly correlated to DNA homology groupings. Essentially all Hup+ strains appear to belong to group I/Ia, although not all strains in this grouping are Hup+ (Fuhrmann, 1990; Minamisawa, 1990). One apparent exception to this is a Hup+ strain from serogroup 31, which would be expected to align with group I1 based on its serological classification (Hollis et al., 1981; van Berkum et al., 1985; van Berkum, 1990). In contrast, all strains reported to exhibit the Huphr phenotype are from serogroups correlating to group 11, but, again, not all group I1 strains have the Hup-hr phenotype (van Berkum, 1990; van Berkum and Sloger, 1991).

D. DISSIMILATORY NITRATE REDUCTION Dissimilatory nitrate reduction (DNR) refers to the use of the anionic nitrogen oxides (NO; and NO;) as alternative terminal electron acceptors in the absence of oxygen, thereby permitting respiration to occur under anaerobic conditions. Denitrification specificallyrefers to the dissimilatory reduction of one or both anionic oxides to gaseous nitrogen compounds (NO, N,O, or N2) (OHara and Daniel, 1985). Although denitrifying ability has been detected in other members of the Rhizobiaceae, its relatively frequent occurrence in B. japonicum has resulted in a correspondingly greater number of studies for the species. Additionally, many strains of B. japonicum are capable of nitrate respiration, i.e., the reduction of NO? to NO? without further reduction to gaseous products (OHara and Daniel, 1985). Although technically not denitrification, nitrate respiration is often

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studied and discussed in conjunction with denitrification due to its similar metabolic role. 1. Diversity of DNR Phenotypes

Studies conducted in a number of laboratories have uniformly identified three patterns of DNR in diverse collections of B. japonicurn: (1) denitrification resulting in the release of nitrogenous gases, (2) nitrate respiration resulting in the accumulation of nitrite, and (3) no detectable DNR (Breitenbeck and Bremner, 1989; van Berkum and Keyser, 1985; Zablotowicz et al., 1978). Researchers working with less diverse collections have also detected phenotypes correlating with one of these three major patterns (Daniel et al., 1980; Neal ez al., 1983; Smith and Smith, 1986). Investigations of DNR phenotypes in large collections of B. japonicum are rare (van Berkum and Keyser, 1985; Zablotowicz et al., 1978). Of these, the study by van Berkum and Keyser (1985) is particularly noteworthy in that over 300 isolates of soybean bradyrhizobia, representing three collections taken from the United States and China, were examined for DNR. The vast majority of the isolates were capable of anaerobic growth on NO,, regardless of their geographic origin. A striking exception to this was a complete lack of DNR activity in isolates from serogroup 135. Among those isolates rated positive for DNR, bradyrhizobia collected from soils in the United States were approximately equally divided between denitnfiers and nitrate respirers. Those indigenous to China, however, were much more likely to exhibit denitrifying activity. 2. Correlation with Symbiotic Performance

The significance of DNR activity by B. japonicum to the soybean symbiosis and to the bacterium itself is unresolved. Because it permits respiration in anoxic environments, it is often hypothesized that DNR may enhance bradyrhizobial survival in waterlogged soils and anaerobic microsites, and that it may improve symbiotic performance in the 0,-limited nodule environment (Breitenbeck and Bremner, 1989; Neal et al., 1983; O’Hara and Daniel, 1985). It is also possible that DNR may reduce the potential for inhibitory effects of NO; on nitrogenase activity in the nodule (O’Hara and Daniel, 1989, although recent studies of nitrate relationships in root nodules would tend to discount this hypothesis (Giannakis et al., 1988; Sprent et al., 1987). Studies have demonstrated that energy derived from DNR can support N2 fixation (Rigaud et al., 1973; van Berkum and Keyser, 1985), but this potential benefit must be balanced against any associated loss of combined N for plant growth (Smith and Smith, 1986).

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3. Relationship to DNA Homology Groupings

Possible relationships between DNR activity and DNA homology have not been specifically addressed. However, based on reported correlations between DNR and serological phenotypes (van Berkum and Keyser, 1985), it is possible to infer that a reasonably strong relationship exists between DNR and genetic groupings. In general, strains in serogroups associated with DNA homology group I/Ia were able to denitrify, whereas those identified with group I1 exhibited nitrate respiration. A small number of exceptions to the correlation between DNR and serogroup were noted by van Berkum and Keyser (1985). Additionally, strains in serogroup 135 were uniformly deficient in DNR, but these strains are of uncertain DNA homology.

E. RHIZOBITOXINE Certain strains of B. japonicum produce the phytotoxin rhizobitoxine (RT), which causes a distinctive chlorosis of newly formed leaves of susceptible soybean genotypes. Although the chlorosis was first reported in the 1950s (Erdman et al., 1956), the significance of RT production to the symbionts remains unclear. Similarly, although the chlorosis-inducing properties of RT are well documented, the physiological basis for the chlorosis has not been determined. Research initiated in the 1960s by L. D. Owens and colleagues established that RT is a basic amino acid (Keith et a/., 1975; Owens et al., 1972). A closely related ethoxy analog of RT, aminoethoxyvinylglycine (AVG), also possesses many of the biological properties of RT (Devine and Breithaupt, 1980b). Unlike RT, AVG is commercially available and has therefore found application in RT-related research [e.g., Devine and Breithaupt ( 1980b) and Ruan and Peters ( 199I)]. 1. Diversity of RT Phenotypes

Soybean bradyrhizobia have generally been characterized as either RT or RT- based on their ability to induce foliar chlorosis in susceptible soybean genotypes (Erdman et al., 1956; Devine and Weber, 1977; Fuhrmann, 1990; La Favre and Eaglesham, 1986; Owens and Wright, 1965a). These bioassays usually detect RT production within root nodules (Owens and Wright, 1965a),although other soybean bioassays rely on RT production in vitro (La Favre and Eaglesham, 1984). It has been observed that some chlorosis-inducing strains may not produce RT in vitro (Owens and +

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Wright, 1965b) and that colony-type variants within a strain may differ in RT production in culture but not in planfa (La Favre er al., 1988). Detection of RT production in the root nodules of susceptible soybean genotypes, however, appears to be a reliable technique (Minamisawa and Kume, 1987; Minamisawa, 1989; Ruan and Peters, 1991). Early work revealed that soybean genotypes differ in their susceptibility to RT+ strains (Erdman ef al., 1957; Johnson and Means, 1960). This variation may result from differences in RT production by the bradyrhizobia within the nodules of contrasting host genotypes (Johnson and Clark, 1958), but other work has indicated that soybean genotypes may possess intrinsic differences in their tolerance to, or in their ability to detoxify, exogenously applied RT (Owens and Wright, I965a). Recent studies employing a highly sensitive enzyme inhibition assay for RT found low levels of the toxin in nodules of an RT-resistant soybean cultivar (Ruan and Peters, 199I), but did not differentiate between reduced production and enhanced detoxification of RT as the causative mechanism. Interestingly, strains of bradyrhizobia that cause RT-induced chlorosis in soybean have not been shown to do so in symbiosis with cowpea (Eaglesham and Hassouna, 1982; Keyser er al., 1982). Expression of RT symptoms is often more severe and less transient in greenhouse-grown soybeans than in field-grown plants. Unlike field-grown plants, the former are often grown in greenhouse potting media in the absence of combined forms of N so as to emphasize the contribution of N, fixation to plant growth. Nitrate applied at low rates was recently shown to reduce the negative effects of RT+ bradyrhizobia on soybean productivity in the greenhouse (Teaney, 1991). Presumably, a similar effect may occur in the presence of inorganic N in field soils. As documented below, studies have revealed that essentially all soybean bradyrhizobia aligning with DNA homology group I1 of Hollis ef al. (198 1) produce RT. regardless of whether the level of production is sufficient to induce acute foliar chlorosis. This observation is potentially of extreme importance because it indicates that RT+ strains are a prominent component of many indigenous bradyrhizobial populations (Devine er al., 1988; Fuhrmann, 1990; Minamisawa, 1989, 1990). For example, a survey of B. japonicirrn in Delaware indicated that over 37% of the 360 isolates sampled were in group I1 (Fuhrmann, 1990). The same study found that up to 80% of the nodules formed by soybean plants in a given field were formed by group I1 strains and that over 80% of these isolates induced foliar chlorosis. In a survey of B. japonicum indigenous to I2 states, serogroups correlating to group I1 were prominent in Arkansas, Delaware, Florida, Kansas, Louisiana, Mississippi, North Carolina, and Pennsylvania (Keyser ef al., 1984). When averaged across all 12 states, over 45% of the isolates belonged to

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JEFFRY J. FUHRMANN

group 11. In general, RT+ bradyrhizobia appear to be particularly common in the southern and mid-Atlantic states. 2. Correlation with Symbiotic Performance

Rhizobitoxine confers no known benefit to strains of B. japonicum capable of producing the compound (La Favre and Eaglesham, 1986). Similarly, there is a lack of studies examining the impact of RT-producing strains on soybean productivity under field conditions. However, because RT symptoms are generally transitory in the field, associated yield effects have generally been assumed to be negligible (Erdman ef af., 1956, 1957; Devine and Weber, 1977). As an analogue of cystathionine, RT irreversibly inhibits j3-cystathionase in the transsulfuration pathway of methionine biosynthesis in both bacteria (Owens ef af., 1968) and plants (Giovanelli ef af., 1971). The reaction catalyzed by &cystathionase is the conversion of cystathionine to homocysteine, pyruvate, and ammonium. An alternative means of methionine biosynthesis in plants (direct sulfhydration pathway) is apparently not affected by RT (Giovanelli ef af., 1972), but evidence indicates that this latter pathway may be of minor importance in plants (Giovanelli et af., 1980). Although no mechanism has been proved to cause RT-induced chlorosis, the need for methionine in the production of various compounds related to chlorophyll synthesis suggests that inhibition of j3-cystathionase may be involved. Rhizobitoxine is also known to inhibit ethylene production from methionine, but this effect does not appear to be responsible for the production of RT symptoms (Owens et af., 1971). Other studies have indicated that inhibition of ethylene production with AVG can enhance nodulation of leguminous plants other than soybean, particularly in the presence of nitrate (Fearn and LaRue, 1991; Peters and Crist-Estes, 1989; Zaat ef af., 1989). However, a recent study with isogenic RT mutants of B. japonicum did not support possible RT involvement in stimulation of soybean nodulation (Ruan and Peters, 1992). Minamisawa ef af. (1990) reported that RT inhibited de novo synthesis of uptake hydrogenase in B. japonicum. The activity of existing hydrogenase was not affected by RT. Cystathionine and methionine strongly prevented the inhibition, suggesting that the inhibition involved the level of sulfur-containing amino acids in the cell. Few studies have examined quantitatively the effect of RT+ strains of B. japonicum on soybean growth and symbiotic performance. Based on short-term greenhouse studies, characterization of indigenous B. japonicum by their ability to cause RT-induced chlorosis was inferior to alterna-

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tive techniques in predicting symbiotic effectiveness (Fuhrmann, 1990). Nevertheless, similar greenhouse studies have revealed that nodulation by wild-type RT+ B. juponicum can result in reductions in leaf chlorophyll, leaf protein, shoot dry weight, N, fixation, and nodular leghemoglobin when compared with plants nodulated by wild-type RT- strains (Teaney and Fuhrmann, 1993). Furthermore, a recent field study found that high initial levels of nodulation by USDA 94 (an RT+ strain) decreased soybean seed yield by 39%, N, fixation by 44%, and total shoot N content by 42% relative to plants nodulated by only the indigenous strains (Vasilas and Fuhrmann, 1992). It is not known whether these effects resulted from RT production, inferior N, fixation, or a combination of these or other factors linked with RT production. The possible direct involvement of RT can only be resolved by conducting studies utilizing isogenic strains of B. juponicum differing in RT production (Ruan and Peters, 1992). Although RT production confers no known competitive advantage to B. juponicum, the literature does contain reports of possible relevance to this topic. For example, it has been reported that RT+ strains exhibit enhanced nodulating abilities with soybean plants having the rj, rj, (nonnodulating) genotype when compared with RT- strains (Devine and Weber, 1977). However, subsequent research found that coapplication of B. juponicum and AVG did not increase nodulation of the restrictive host (Devine and Breithaupt, 1980b). Other researchers have also been unsuccessful in confirming enhanced nodulating ability resulting from RT production (La Favre and Eaglesham, 1984; Ruan and Peters, 1992). Conversely, Devine et ul. (1990) reported that the Rj, gene in soybean may preferentially exclude nodulation by DNA homology group 11. Other research has provided evidence that RT production may be correlated with enhanced nodulation competitiveness with increasing soil temperatures (Kluson et ul., 1986; Weber and Miller, 1972), but this may reflect more fundamental genetic differences among the bradyrhizobia. 3. Relationship to DNA Homology Groupings

Devine et ul. ( 1983, 1988),working with recognized strains of B. juponicum,were the first to note a correlation between chlorosis induction and DNA homology. All of the recognized RT+ strains in their studies belonged to group 11, but not all group I1 strains produced chlorosis in soybean when used as inocula. A more recent survey of indigenous B. juponicum confirmed these observations (Fuhrmann, 1990). In all of these studies, RT phenotype was determined using a soybean bioassay of RT production in root nodules. Concurrent with the above studies, research conducted in Japan found

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that all group I1 strains examined produced RT in symbiosis with the soybean host, regardless of any associated chlorosis-inducing ability (Minamisawa, 1989, 1990). One strength of the Japanese studies is that RT phenotype was determined by detecting RT production in plant root nodules by means of an amino acid analyzer (Minamisawa and Kume, 1987), a more sensitive and reliable assay than that afforded by induction of host chlorosis. It thus appears that all group 11 strains tested to date produce measurable amounts of RT, but that induction of host chlorosis requires some minimum threshold concentration of RT.

F. SURFACEPOLYSACCHARIDES Research on the surface polysaccharides(SPSs) associated with B. japonicum has primarily addressed the quantity and composition of the acidic extracellular polysaccharides ( EPSs) produced by various strains, although other SPSs, including lipopolysaccharides(LPSs) and capsular polysaccharides (CPSs), have also received some attention. One common manifestation of differences in SPSs among B. japonicum is the development of distinct colony morphologies on agar media. In fact, it is this morphological diversity that has been most commonly documented in the literature. This is certainly due in part to the ease with which morphological data can be collected during the routine culture of bradyrhizobia on solidified laboratory media. 1. Diversity of SPS Phenotypes

Several studies have documented diversity of B. japonicum colony morphologies. Both interstrain diversity (Basit et al., 1991; Fuhrmann, 1990; Minamisawa, 1989; Upchurch and Elkan, 1977) and intrastrain diversity (Aganwal and Keister, 1983; Basit et al., 1991; La Favre et al., 1988; Mullen and Wollum, 1989; Kuykendall and Elkan, 1976; Mathis er al., 1986) have been observed. Similar diversity has been observed for other Bradyrhizobium spp. (Hadad and Loynachan, 1986; Hemdge and Roughley, 1975; Sinclair and Eaglesham, 1984; Sylvester-Bradley et al., 1988). Colonies produced under standardized incubation conditions are typically Characterized based on mean colony diameter and other more subjective qualities, including elevation, margin, viscosity, and opacity. Most characterizations of B. japonicum colonies have been made using a yeast extract mannitol agar medium and a 7- to 14-day incubation period at 25 to 30°C [e.g., Basit et al. (199 I), Fuhrmann (1990), Kuykendall and Elkan (1976),

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La Favre et al. ( 1988), and Mathis et al. ( 1986)]. Three colony groups have been most commonly reported: ( I ) small (< 1 mm), entire colonies of variable opacity, (2) large (2I mm), convex, entire, mucoid, and translucent to opaque colonies, and (3) large (21 mm), flat, irregular, watery, and translucent colonies. These classifications will subsequently be referred to as SD (small dry), LM (large mucoid), and LW (large watery), respectively (Fuhrmann, 1990). Major factors influencing colony morphology are the quantity and nature of SPS produced by a given strain. Some differences in colony morphology have been attributed to contrasting levels of EPS production (La Favre et al., 1988; Upchurch and Elkan, 1977). Differences in diameter among the colony morphology groupings reflect in part the nutritional characteristics of the bacteria involved [e.g., Agarwal and Keister (1983)l. In general, strains unable to utilize fully the nutrients provided in a medium will grow more slowly than those that are nutritionally more adapted. This has been demonstrated to best advantage in studies characterizing colony derivatives of B. japonicum strain USDA 1 10 (Kuykendall and Elkan, 1976; Mathis et al., 1986). In other cases, morphological differences have been correlated primarily to qualitative differences in EPS. For example, Minamisawa (1989) confirmed that B. japonicum produces two types of EPSs (Huber et al., 1984), and also noted that differences in EPSs were reflected in colony morphologies. Strains forming colonies similar to the LM classification produced type A EPS, which is composed of glucose, mannose, galactose, 4-0-methylgalactose, and galacturonic acid. Conversely, strains forming LW-like colonies produced type B EPS, composed of rhamnose and 4-0-methyl glucuronic acid. However, it should be noted that recent research revealed that nodular polysaccharides produced in planta by the two homology groups were less divergent than were EPS produced in vitro (Streeter et al., 1992). A limited number of studies have used colony morphology to characterize soybean bradyrhizobia. In a characterization of indigenous B. japonicum, Fuhrmann (1990) found that LM, LW, and SD colony types represented 45, 37, and 18%of isolates examined, respectively. The same study found large differences among sampling sites in the frequency of the various colony types. In a survey of cowpea bradyrhizobia, Sinclair and Eaglesham ( 1984) reported encountering two colony types resembling the SD and LW colony types of Fuhrmann (1990). 2. Correlation with Symbiotic Performance

Studies with colony derivatives of strain USDA 110, and derivatives from a limited number of other strains of B. japonicum, revealed that those

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forming large mucoid colonies were symbiotically inferior to those producing small nonmucoid colonies on a yeast extract - mannitol medium (Kuykendall and Elkan, 1976; Upchurch and Elkan, 1977). The basis for this symbiotic divergence was not determined, although differences in colony morphologies were attributed to contrasting nutritional characteristics and EPS production rates. However, Kuykendall and Elkan (1976) reported preliminary evidence that this correlation between colony morphology and symbiotic effectiveness may not extend to derivatives produced from other strains of B. japonicum. Also, Mathis et al. ( 1986) found that mannitol utilization, as reflected in colony morphology, was not necessarily related to symbiotic effectiveness in strain USDA 110. A more recent study compared small and large colony-type derivatives of an RT+ strain of B. japonicum (USDA 76) for differences in RT production (La Favre et al., 1988). Only the small (nonencapsulated) colony-type derivative produced RT in vitro, whereas both the small and large (encapsulated) types induced host chlorosis as bacteroids. The authors speculated that the in vitro results may indicate possible differences between the derivatives in RT production in the host rhizosphere, although no direct evidence was presented in support of this hypothesis. Interstrain differences in colony morphology may have value in the characterization of indigenous populations of soybean bradyrhizobia. In one study, classification according to colony morphology was found to be as useful a predictor of symbiotic effectiveness as was serological analysis (Fuhrmann, 1990). This study found that isolates producing LW colony types fixed substantially less N2than did either the LM or SD types. Basit et al. (1991), in an investigation of strain diversity within serogroup 1 10, also found that SD and LM colony types were similar to each other with respect to symbiotic effectiveness. It is generally conceded that SPSs may be important in the early developmental stages of leguminous symbioses, although there is mounting evidence that the relative importance of different types of SPSs can vary depending on the symbiosis being examined (Kijne, 1992; Law er a!., 1982; Stacey et al., 1991). Interestingly, Stacey et al. (1991) found that an LPS-defective mutant was able to form “empty” pseudonodules with certain soybean genotypes, suggesting that some diffusible factor (rather than infection) was necessary for the formation of nodule primordia. Other researchers have reported that a particular B. japonicum strain produces a depolymerase that is active on its own EPS, but the possible symbiotic role of this enzyme has not been resolved (Dunn and Karr, 1990). Clearly, additional research is needed to elucidate fully the role of SPS in the soybean -B. japonicum symbiosis.

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3. Relationship to DNA Homology Groupings

Research has established that a strict correlation exists between EPS phenotype in vitro and DNA homology grouping (Huber et al., 1984; Minamisawa, 1989, 1990). Strains of B. japonicum in DNA homology group I/Ia all produce type A EPS, whereas those in group I1 produce type B EPS. Analogous differences in other types of SPSs among strains of B. japonicurn have not been documented. Because colony morphology is correlated with differences in SPS production and composition (Minamisawa, 1989), it follows that colony morphology should provide a convenient means of infemng DNA homology groupings. Evidence to data indicates that colony types LM and SD correlate with DNA group I/Ia and that colony type LW correlates with group I1 (Basit et al., 1991 ;Fuhrmann, 1990).

G. PROTEIN PROFILES Perhaps no technique has revealed greater diversity within soybean bradyrhizobia than has the analysis of cellular proteins by electrophoresis. Protein analyses commonly involve physical disruption of the bacterial cells, protein denaturation, treatment with sodium dodecyl sulfate (SDS), and subsequent electrophoretic separation and staining on polyacrylamide gels (PAGE) as described by Laemmli ( 1970). Essentially all studies to date with B. juponicum have employed one-dimensional gels, although twodimensional electrophoresis has been useful in detailed investigations of the Rhizobiaceae (Roberts et al., 1980). 1. Diversity of SDS - PAGE Phenotypes

The literature contains only a handful of studies that have applied SDS- PAGE to extensive collections of soybean bradyrhizobia (Hickey et ul., 1987; Kamicker and Brill, 1986; Noel and Brill, 1980). Additionally, Sadowsky et al. (1987) analyzed a limited number of field isolates of serogroup 123 by SDS-PAGE, and Eaglesham et al. (1987) used the technique to characterize an extensive collection of bradyrhizobia isolated from cowpea root nodules. The general conclusion of the above studies is that SDS-PAGE is a more sensitive and discriminating procedure than essentially any of the alternative techniques employed, including both IAR (Hickey et al., 1987; Sadowsky et al., 1987) and serology (Kamicker and Brill, 1986; Noel and

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Brill, 1980). For example, Hickey ef al. ( 1987) analyzed 176 indigenous isolates from serogroup 123 and identified 74 unique strains belonging to two major gel groups. Similarly, analysis by SDS-PAGE divided 543 isolates into 29 strains, whereas serological analysis revealed only five identifiable groupings (Noel and Brill, 1980). As noted by Noel and Brill (1980), the relatively great discriminating power of SDS- PAGE can be disadvantageous. Specifically, absolute classification of bradyrhizobia is complicated by an inability of the procedure to provide unambiguous plus-or-minus results. Balanced against this difficulty is the technique’s ability to provide a positive characterization of all isolates examined, thereby avoiding complications analogous to the nonreactive grouping commonly encountered in serological analyses. 2. Correlation with Symbiotic Performance

Although the analysis of cellular proteins by SDS- PAGE is apparently superior to many alternative techniques in detecting population diversity, essentially nothing is known concerning the symbiotic relevance of the resulting groupings. It is suggested that future studies should build on the foundation provided by the studies cited above, not by simply confirming that SDS-PAGE can detect diversity, but by exploring the practicality and predictive power of various grouping strategies. 3. Relationship to DNA Homology Groupings

No studies have specifically addressed whether DNA homology groupings are reflected in groupings obtained with SDS- PAGE. However, in an experiment conducted by Noel and Brill (1980), one gel group (A) correlated with the group I1 serogroups 3 1 and 46 (Keyser and Griffin ( 1987), whereas the remaining gel groups (B-D) aligned with serogroups belonging to group I/Ia. Hickey et al. (1987) reported two major gel groups but did not indicate whether these were correlated to DNA homology groupings or related classifications.

H. RHIZOBIOPHAGE TYPING Many strains of B. juponicurn, like other members of the Rhizobiaceae, are vulnerable to attack and lysis by bacteriophages. Reactions between phages and strains of B. juponicum are generally very specific (Kowalski et ul., 1974). This specificity is the basis for phage typing of B. juponicurn,

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which refers to the characterization or identification of bradyrhizobia according to their susceptibility to lysis by various viruses. 1. Diversity of Rhizobiophage Phenotypes

Rhizobiophage typing is in many ways analogous to serological analysis. Use of phage typing requires that the rhizobiophages used in the analysis be previously isolated from soil or root nodules. Individual strains or isolates of bradyrhizobia are rated positive or negative for lysis when tested against a range of phages of known origin. Possible phenotypes include reaction with a single phage, cross-reactions with more than one phage, and no reaction (Basit ef al., 1991; Kowalski et al., 1974). Characterization of B. japonicum by phage typing is generally considered to be more discriminating than is serology. Kowalski ef al. (1974) found that phage typically lysed only those strains belonging to the same serogroup as the strain originally used for phage isolation, but that up to 80% of the isolates constituting their serogroup 123 were unaffected by phage active against USDA 123. This low frequency of lysis may reflect the diversity that is now recognized to exist within serocluster 123 (Schmidt ef al., 1986). However, Basit et al. ( 1991 ) reported high diversity in rhizobiophage phenotypes in serogroup 110, a serogroup that is not recognized as being part of a more extensive serocluster. 2. Correlation with Symbiotic Performance

The ability of rhizobiophage typing to predict the symbiotic effectiveness of the associated B. japonicum has received little attention. One exception is the study by Basit ef al. (1991), which found no apparent correlation between rhizobiophage phenotype and symbiotic effectiveness among strains within serogroup 1 10. Rather, most research with rhizobiophages has focused on their potential ecological impact on susceptible strains (Hashem and Angle, 1988, 1990; Hashem ef al., 1986; Vidor and Miller, 1980). In general, these studies have indicated that rhizobiophages can reduce nodulation and yield of soybean grown under simplified experimental conditions. However, apart from possible effects on nodulation competition among strains (Hashem and Angle, 1990), yield effects under conditions typical of most soybean-producing areas are thought to be minimal. 3. Relationship to DNA Homology Groupings

No studies have examined the correlation of rhizobiophage phenotypes with DNA homology. Because phage typing appears to be more discrimi-

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nating than most other commonly used phenotypic analyses, one can speculate that it is highly correlated to DNA homology. However, this high specificity, and resulting propensity for yielding negative reactions (Basit et ul., 1991; Kowalski et ul., 1974), would likely limit its value for predicting the DNA homologies of uncharacterized strains of B. juponicum.

I. PLANTGROWTH-REGULATING SUBSTANCES Plant growth-regulating substances (PGRSs) are generally thought to play a significant, but as yet unresolved, role in the development of leguminous N,-fixing symbioses. Involvement may be particularly important during the early stages of symbiotic development, such as root infection and nodule primordium formation (Appelbaum, 1990; Kijne, 1992; Sobral ef ul., 1991). Certainly one major source of PGRSs is the host plant itself. However, studies have also demonstrated that B. juponicum and other members of the Rhizobiaceae can produce various PGRSs, potentially at levels sufficient to induce a host plant response (Phillips and Torrey, 1972). Other research suggests that certain strains of B. juponicum may play a role in the degradation of PGRSs (Egebo ef ul., 1991). 1. Diversity of PGRS Phenotypes

Many studies of PGRS phenotypes in B. juponicum have focused on the production and degradation of auxins, particularly indole-3-acetic acid (IAA). Recent research has clearly established that some but not all strains of B. juponicum can produce IAA in vitro (Kaneshiro and Nicholson, 1990; Minamisawa and Fukai, 1991), and there is evidence that these differences among strains are reflected in IAA concentrations in nodules (Kaneshiro and Nicholson, 1990). Other investigators have shown that B. juponicum strain USDA 1 10 possesses an IAA degradative pathway (Egebo et ul., 1991). Research with mutants of specific strains of B. juponicum has shown that both tryptophan catabolic mutants (Kaneshiro and Kwolek, 1985; Kaneshiro and Nicholson, 1990) and 5-methyltryptophan-resistant mutants (Hunter, 1987, 1989) produce elevated levels of IAA and related compounds in culture and as bacteroids. Production of cytokinins by soybean bradyrhizobia has also received considerable attention by researchers. Phillips and Torrey ( 1970, 1972), working with a single strain of B. juponicum, demonstrated the in vitro production of a zeatin-like compound. More recent studies revealed that a minimum of three cytokinin-active substances were excreted into culture media by various strains of B. juponicum (Sturtevant and Taller, 1989; Taller and Sturtevant, 1991). No research to date has demonstrated pro-

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duction of cytokinins by B. japonicum in either the rhizosphere or root nodules of soybean. 2. Correlation with Symbiotic Performance

It is presently not known whether PGRSs, especially those of bradyrhizobial origin, play a significant role in the development of the soybean symbiosis. However, based on their effects on plants in general, auxins may be anticipated to affect the soybean symbiosis via regulation of cell enlargement and division, vascular tissue differentiation, root initiation, assimilate partitioning, and ethylene production (Davies, 1987). Cytokinins may be primarily important in the stimulation of cell division (Bauer ef al., 1985; Sobral ef al., 1991; Taller and Sturtevant, 1991). The modifying effect of auxin on ethylene production is noteworthy because ethylene is involved in the regulation of root growth and differentiation and in the expression of plant wounding responses (Chadwick and Burg, 1967; Reid, 1987). Mutants exhibiting enhanced production of IAA have been shown to both inhibit (Hunter, 1987) and enhance (Kaneshiro and Kwolek, 1985) soybean nodulation and N, fixation. These contrasting results may indicate that nodulating bradyrhizobia must produce an optimum amount of IAA (or a related compound) in order to maximize root nodulation (Minamisawa and Fukai, 1991). Hunter and Kuykendall (1990) suggested a similar mechanism in reporting that a prototrophic revertant of a tryptophan-requiring auxotrophic mutant was symbiotically superior to the original parent strain of B. japonicum. Application of IAA is known to increase the release of ethylene from plant roots, thereby potentially leading to an inhibition of root growth (Chadwick and Burg, 1967). Research with leguminous species other than soybean indicates that root nodulation can also be adversely affected by ethylene production (Drennan and Norton, 1972; Goodlass and Smith, 1979;Grobbelaar ef al., 1971; Zaat ef al., 1989). Nitrate-induced evolution of ethylene may be partly responsible for decreased nodulation of alfalfa under high N conditions (Ligero ef al., 1987). There is evidence that all of these ethylene-induced effects on plant growth can be reversed by application of AVG, the structural analog of rhizobitoxine discussed earlier (Ligero et d.,1991; Zaat ef al., 1989). 3. Relationship to DNA Homology Groupings

Recent studies by Minamisawa and Fukai (1991) revealed that group I1 strains of B. japonicum consistently produced IAA in vifro at concentrations ranging from 2 1 to 70 pA4, whereas group I/Ia strains exhibited no

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such ability. Similar results were reported by Kaneshiro and Nicholson (1 990). Analogous studies examining possible correlations between the production of other PGRSs and genetic groupings have not been reported in the literature. Although RT is not considered to be a PGRS in the usual sense, it is interesting to note that production of both IAA and RT appears to be limited to DNA homology group 11, and that these compounds are thought to actively stimulate and inhibit ethylene production, respectively.

J. OTHER PHENOTYPES A number of phenotypic characteristics having uncertain relevance to the soybean symbiosis have been shown to correlate with the DNA homology groupings of Hollis et uf. (1981). These characteristics are briefly described below. 1 . Ex Planta Nitrogenase Activity

The expression of ex pfunta nitrogenase activity by certain strains of B. japonicum under microaerophilic conditions is well documented (Agarwal and Keister, 1983; Huber et af.,1984; Pankhurst et af., 1982). The study by Huber et al. (1984) specifically addressed the relationship between nitrogenase phenotype and DNA homology and revealed that only strains in DNA homology group I1 expressed significant ex planta nitrogenase activity. In contrast, earlier investigations found that colony-type derivatives of USDA 110 (group I/Ia) expressed substantial nitrogenase activity in vitro (Kuykendall and Elkan, 1976; Upchurch and Elkan, 1977), yet Huber et af. (1984) detected no such activity for USDA 110. The cause of this discrepancy is not clear but may involve differences in the methods used to culture the bradyrhizobia (Huber et al., 1984). 2. Hemoproteins

Keister and Marsh ( 1990) characterized the hemoproteins of 17 strains of B. japonicum having known DNA homologies. No differences were noted among the homology groupings when the bacteria were cultured in the laboratory. However, distinct hemoprotein phenotypes were observed for bacteroids recovered from soybean root nodules. Particularly noteworthy was the observation that hemoproteins could be used to distinguish group I from group Ia strains, as well as the combined group I/Ia strains from those in group 11. None of the other phenotypes discussed in this review, with the exception of serology, have been shown to be capable of

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detecting this distinction. Bacteroids from both groups I and Ia generally produced only trace amounts of the particulate hemoprotein designated aa3, whereas only the former produced the soluble protein designated P-422. Conversely, bacteroids from group I1 produced substantial amounts of aa3 but no P-422. 3. Fatty Acids A limited number of studies have examined the fatty acid composition of B. japonicum (Kuykendall et al,, 1988; MacKenzie et al., 1979), and only Kuykendall et al. (1 988) have examined the correlation between fatty acid phenotypes and DNA homology. This study revealed that group I/Ia strains were easily distinguished from those in group I1 based on their relative amounts of 19 :0 cyclopropane and I8 : 1 fatty acids.

IV. SUMMARY OF PHENOTYPIC AND GENOTYPIC RELATIONSHIPS It is apparent that many phenotypic characteristics of B. japonicum are strongly correlated with each other and with the DNA homology groupings of Hollis et al. (1981). The majority of these interrelationships are summarized in Table I. Certain other characteristics, such as protein profiles and phage reactions, can be inferred to correlate with various phenotypes but are not easily categorized in tabular form.

V. TAXONOMIC STATUS OF Bradyrhizobium japonicum In the original proposal to establish Bradyrhizobium as a genus distinct from Rhizobium, it was recommended that only one species, B. japonicum, be recognized pending further studies of taxonomic relationships within the genus (Jordan, 1982). Essentially concurrent with its establishment as a species, however, it was first reported that B. japonicum consists of at least two highly divergent genetic groupings (Hollis et a/., 1981). As documented earlier in this review, numerous studies have subsequently confirmed the presence of genetic and phenotypic divergence within the species (Devine et a/., 1988; Fuhrmann, 1990; Huber et a/., 1984; Kaneshiro and Nicholson, 1990; Keister and Marsh, 1990; Kuykendall et al., 1988; Minamisawa, 1989, 1990; Minamisawa and Fukai, I99 1; Stanley et

Table I Summary of Relationships among Selected Phenotypes and DNA Homology Groupings of Bdyrhizobium jujumicum

Phenotype(s) exhibited by DNA homology grouping Phenotype category Serogroup

6 (including 24) 38- 1 I5 58

62 110

Hydrogenase

I1

I/Ia

I22 I23 Hup+ Hug

Intrinsic antibiotic Sensitive resistance Colony morpholog4 large mucoid Small dry

Ref.

31 46 76 86 94 I30

Hollis et a/. (198 I ) and Keyser and Griffin (1987)

Huphr Hup(HuP+)'

Fuhrmann (1990), Minam isawa ( 1990). and van Berkum and Sloger ( I99 1) Kuykendall et al. (1988) Basit et a/. ( 199 I ), Fuhrmann (1990), and Minamisawa (1989) Huber et a/. (1984) and Minamisawa (1989) Minamisawa and Fukai (1991) Fuhrmann (1990) and Minamisawa (1989) van Berkum and Keyser ( 1985)

-

Resistant Large watery

IAA production

IAA-

IAA+

Rhizobitoxine productionb

RT-

RT+

Dissimilatory nitrate reduction" Ex planta nitrogenase activity

Denitrification DNR(Nitrate respiration) Inactive (Active)

Nitrate respiration, DNR(Denitrification) Active

Hemoproteins

Low aa3,P-422"-

High aa3,P-422-

Fatty acids

Low 19:O cyclopropane, high 18: I

High I9:O cyclopropane, low 18: I

Huber et al. (1984), Kuykendall and Elkan (1976), and Upchurch and Elkan (1977) Keister and Marsh ( 1990) Kuykendall et al. (1988)

" Relationshipsderived indirectly from other phenotype-genotype correlations. RT production based on direct analysis for RT rather than on induction of host chlorosis.

'Phenotypes in parentheses occur at relatively low frequencies (see references).

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al., 1985). This mounting evidence of divergence recently culminated in a proposal to reclassify soybean bradyrhizobia in DNA homology group I1 as a new species, Bradyrhizobium elkanii (Kuykendall et al., 1992). It is interesting to note that numerous studies have provided evidence that many B. japonicum, particularly those in DNA homology group 11, are often similar to bradyrhizobia isolated from cowpea. Morphologically, the “large watery” colony type of B. juponicum (Fuhrmann, 1990) resembles the “wet” colony type described for cowpea bradyrhizobia (Sinclair and Eaglesham, 1984). Pankhurst et al. (1982) found a similar correlation between rifampicin resistance and ex pluntu nitrogenase activity for bradyrhizobia from both soybean and cowpea. Serological analyses have revealed strong cross-reactionsbetween antisera produced against group I1 B. juponicum and antigens from cowpea bradyrhizobia (Koontz and Faber, 196 1). Ahmad et al. (198 1) reported that cowpea bradyrhizobia with “dry” colony types strongly reacted with antisera to B. juponicum RCR 3407, a strain that is probably in DNA homology group I/Ia based on its placement in serogroup 122 (Keyser and Griffin, 1987). Many bradyrhizobia isolated from either cowpea or soybean are able to nodulate both hosts (Keyser et al., 1982). Yet, although bradyrhizobia isolated from both hosts can produce RT in culture and in symbiosis with soybean, none has been shown to induce chlorosis in the cowpea host (Eaglesham and Hassouna, 1982; Eaglesham et al., 1987; La Favre and Eaglesham, 1986). Similarly, strains of B. juponicum exhibiting the Huphr phenotype commonly express hydrogenase activity in symbiosis with cowpea but only rarely with soybean (Keyser et al., 1982; van Berkum, 1990; van Berkum and Sloger, 1991).

VI. CONCLUDING REMARKS This review has described known and suspected relationships among various diversity groupings of soybean bradyrhizobia. These relationships clearly demonstrate that the species B. japonicum contains two fundamentally distinct subgroups that coincide with established DNA homology groupings. It is also evident that these subgroups possess unique characteristics of potential importance to the soybean symbiosis, and that many of these interrelated characteristics may have significance to the general ecology of B. japonicum. Based on these observations, it is suggested that population diversity groupings based on DNA homology may represent a valuable and unifying concept in research of soybean bradyrhizobia, particularly for those studies concerned with the characterization of indigenous populations.

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ACKNOWLEDGMENT Paper No. 145 I of the Delaware Agricultural Experiment Station.

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Intrinsic antibiotic resistance in Bradyrhizobium japonicum. Soil Boil. Biochem. 20, 879-882. Mullen, M. D., and Wollum II., A. G . (1989). Variation among different cultures of Bradyrhizobium japonicum strains USDA I10 and 122. Can. J. Microbiol. 35,583-588. Neal, J. L., Allen, G. C., Mom, R. D., and Wolf, D. D. (1983). Anaerobic nitratedependent chemolthtrophic growth by Rhizobium japonicum. Can. J. Microbiol. 29, 3 16-320. Noel, K. D., and Brill, W. J. (1980). Diversity and dynamics of indigenous Rhizobiurn juponicum populations. Appl. Environ. Microbiol. 40,93 1-938. OBrian, M. R., and Maier, R. J. (1988). Hydrogen metabolism in Rhizobiurn-Energetics, regulation, enzymology, and genetics. Adv. Microb. Physiol. 29, 1 - 52. OHara, G . W., and Daniel, R. M. (1985). Rhizobial denitrification: A review. Soil Biol. Biochem. 17, 1-9. Owens, L. D., and Wright, D. A. (1965a). Rhizobial-inducedchlorosis in soybeans: Isolation, production in nodules, and varietal specificity of the toxin. Plant Physiol. 40,927-930. Owens, L. D., and Wright, D. A. (1965b). Production of the soybeanchlorosis toxin by Rhizobiurn japonicum in pure culture. Plant Physiol. 40,931 -933. Owens, L. D., Guggenheim, S., and Hilton, J. L. (1968). Rhizobium-synthesized phytotoxin: An inhibitor of fiyctathionase in Salmonella typhimurium. Biochim. Biophys. Actu 158,219-225. Owens, L. D., Lieberman, M., and Kunishi, A. (197 I). Inhibition of ethylene production by rhizobitoxine. Plant Physiol. 48, 1-4. Owens, L. D., Thompson, J. F., Pitcher, R. G., and Williams, T. (1972). Structure of rhizobitoxine, an antimetabolic enol-ether amino-acid from Rhizobium japonicum. J. Chem. Soc. Chem. Commun..7 14. Ozawa, T., Fukushima, K.,and Komai, Y. (1989). Beneficial effect of hydrogen uptake ability on survival of Bradyrhizobium japonicum in soil aggregate. Soil Sci. Plant Nutr. (Tokyo) 35,527-534. Pankhurst, C. E., Scott, D. B., and Ronson, C. W. (1982). Correlation between rifampicinresistance of slow-growing Rhizobium strains and their ability to express nitrogenase activity in culture. FEMS Microbiol. Lett. 15, 137- 139. Peters, N. K., and Crist-Estes, D. K. ( I 989). Nodule formation is stimulated by the ethylene inhibitor aminoethoxyvinylglycine.Plant Physiol. 91,690-693. Phillips, D. A., and Torrey, J. G. (1970). Cytokinin production by Rhizobiurn. Physiol. Plant. 23, 1057- 1063. Phillips, D. A., and Torrey, J. G. (1972). Studies on cytokinin production by Rhizobium japonicum. Plant Physiol. 49, 1 I - 15. Rasche, M. E., and Arp, D. J. (1989). Hydrogen inhibition of nitrogen reduction by nitrogenase in isolated soybean nodule bacteroids. Plant Physiol. 91,663-668. Reid, M. S. (1987). Ethylene in plant growth, development, and senescence. Planf Horrn. Their Role Plant Growth Dev.,257-279. Rigaud, J., Bergersen, F. J., Turner, G . L., and Daniel, R. M. (1973). Nitrate dependent anaerobic acetylene-reduction and nitrogen fixation by soybean bacteroids. J. Gen. Microbiol. 77, 137 - 144. Roberts, G. P., Leps, W. T., Silver, L. E., and Brill, W. J. (1980). Use of two-dimensional polyacrylamide gel electrophoresis to identie and classify Rhizobium strains. Appl. Environ. Microbiol. 39,4 14 -422. Robson, R. L., and Postgate, J. R. (1980). Oxygen and hydrogen in biological nitrogen fixation. Annu. Rev. Microbiol. 34, 183-207. Ruan, X., and Peters, N. K. (1991). Rapid and sensitive assay for the phytotoxin rhizobitoxine. Appl. Environ. Microbiol. 57,2097-2 100.

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Ruan, X., and Peters, N. K. (1992). Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum. J. Bacteriol. 174, 3467 - 3473. Sadowsky, M. J., Tully, R. E., Cregan, P. B., and Keyser, H. H. (1987). Genetic diversity in Brudyrhizobium japonicum serogroup 123 and its relation to genotype-specific nodulation of soybean. Appl. Environ. Microbiol. 53, 2624-2630. Sawada, Y., Miyashita, K., Tanabe, I., and Kato, K. (1989). HUP phenotype and serogroup identity of soybean-nodulatingbacteria isolated from Japanese soil. Soil Sci.Plant Nuir. ( T o ~ Y o )281-288. ~~, Sawada, Y., Miyashita, K., and Yokoyama, T. (1990). Diversity within serogroups of Japanese isolates of Bradyrhizobium japonicum as indicated by intrinsic-antibiotic resistance. Soil Sci. Plant Nutr. (Tokyo) 36, 50 I - 504. Schmidt, E. L., Zidwick, M. J., and Abebe, H. M. (1986). Bradyrhizobium japonicum serocluster 123 and diversity among member isolates. Appl. Environ. Microbiol. 51, 1212-1215. Sinclair, M. J., and Eaglesham, A. R. J. (1984). Intrinsic antibiotic resistance in relation to colony morphology in three populations of west African cowpea rhizobia. Soil Biol. Biochem. 16,247 -25 I . Smith, G. B., and Smith, M. S. (1986). Symbiotic and free-living denitrification by Bradyrhizobium japonicum. Soil Sci. Soc. Am. J. 50, 349- 354. Sobral, B. W. S., Honeycutt, R. J., Atherly, A. G., and Noel, K. D. (1991). Recognition and infection in legume nodulation. Biol. Eiochem. Nitrogen Fixation. 229-258. [See also Sobral, B. W. S., Honeycutt, R. J., Atherly, A. G., and Noel, K. D. (1991). Recognition and infection in legume nodulation. Sfud.Plant Sci. I, 229-258.1 Sprent, J. I., Giannakis, C., and Wallace, W. (1987). Transport of nitrate and calcium into legume root nodules. J . Exp. Eot. 38,1121 - 1 128. Stacey, G., So, J.-S., Roth, L. E., Lakshmi S. K. B., and Carlson, R. W. (1991). A lipopolysaccharide mutant of Brudyrhizobium japonicum that uncouples plant from bacterial differentiation. Mol. Plant- Microbe Interact. 4, 332-340. Stacey, G., Bums, R. H., and Evans, H. J., eds. (1992). “Biological Nitrogen Fixation.” Chapman & Hall, New York. Stanley, J., Brown, G. G., and Verma, D. P. S. (1985). Slow-growing Rhizobium japonicum comprises two highly divergent symbiotic types. J. Bacteriol. 163, 148 - 154. Streeter, J. G., Salminen, S. O., Whitmoyer, R. E., and Carlson, R. W. (1992). Formation of novel polysaccharides by Brudyrhizobium japonicum bacteroids in soybean nodules. Appl. Environ. Microbiol. 58,607 - 6 1 3. Sturtevant, D. B., and Taller, B. J. (1989). Cytokinin production by Bradyrhizobium japonicum. Plant Physiol. 89, 1247- 1252. Sylvester-Bradley,R., Thornton, P., and Jones, P. (1988). Colony dimorphism in Bradyrhizobium strains. Appl. Environ. Microbiol. 54, 1033- 1038. Taller, B. J., and Sturtevant, D. B. (1991). Cytokinin production by rhizobia. Adv. Mol. Genet. Plant- Microbe Interact. Proc. Int. Symp., 5th, 1990, 1,2 15 - 22 1. [Seealso Taller, B. J., and Sturtevant, D. B. (1991). Cytokinin production by rhizobia. Curr. Plant Sci. Biotechnol. Agric. 10, 2 15 - 22 I .] Teaney 111, G. B. ( 199 I ). Physiological response of soybean to nodulation by rhizobitoxineproducing strains of Bradyrhizobium japonicum and factors influencing the expression of rhizobitoxine-indud chlorosis. M.S. Thesis, University of Delaware, Newark, Delaware. Teaney 111, G. B., and Fuhrmann, J. J. (1992). Soybean response to nodulation by bradyrhizobia differing in rhizobitoxine phenotype. Plant Soil 145,275-285. Thompson, J. A., Bhromsiri, A., Shutsrirung, A., and Lillakan, S. (1991). Native root-nodule

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bacteria of traditional soybean-growing areas of northern Thailand. Plant Soil 135, 53-65. Upchurch, R.G., and Elkan, G. H. (1977).Comparison of colony morphology, salt tolerance, and effectiveness in Rhizobium japonicum. Can. J. Microbiol. 23, 1 I I8 - 1 122. Uratsu, S. L., Keyser, H. H., Weber, D. F., and Lim, S. T. (1982). Hydrogen uptake (HUP) activity of Rhizobium japonicum from major U.S. soybean production areas. Crop Sci. 22,600-602. van Berkum, P. (1987). Expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of Bradyrhizobium japonicum. J. Bacteriol. 169,4565 -4569. van Berkum, P. (1990). Evidence for a third uptake hydrogenase phenotype among the soybean bradyrhizobia. Appl. Environ. Microbiol. 56, 3855 - 3860. van Berkum, P., and Keyser, H. H. (1985). Anaerobic growth and denitrification among different serogroups of soybean rhizobia. Appl. Environ. Microbiol. 49, 772-777. van Berkum, P., and Sloger, C. (1991). Hydrogen oxidation by the hostcontrolled uptake hydrogenase phenotype of Bradyrhizobium japonicum in symbiosis with soybean host plants. Appl. Environ. Microbiol. 57, I863 - 1865. van Berkum, P., Keyser, H. H., and Weber, D. F. (1985). Examination of Hup expression by soybean bradyrhizobia belonging to different serogroup phenotypes. Nitrogen Fixation Res. Prog. Proc. Int. Symp., 6th 361. Vasilas, B. L., and Fuhrmann, J. J. (1993). Field response of soybean to nodulation by a rhizobitoxine-producing strain of Bradyrhizobium. Agron. J. 85, 302 - 305. Velez, D., Macmillan, J. D., and Miller, L. (1988). Production and use of monoclonal antibodies for identification of Bradyrhizobium japonicum strains. Can. J. Microbiol. 34, 88 - 92. Vest, G. (1970). Rj,-A gene conditioning ineffective nodulation in soybean. Crop Sci. 10, 34-35. Vest, G., and Caldwell, B. E. (1972). Rj,-A gene conditioning ineffective nodulation in soybean. Crop Sci. 12,692-693. Vidor, C., and Miller, R. H. (1980). Relative saprophytic competence of Rhizobiurn japonicum strains in soils as determined by the quantitative fluorescent antibody technique (FA). Soil Biol. Biochem. 12,483-487. Vincent, J. M. (1982). Serology.Nitrogen Fixation 2,235-273. Viteri, S . E., and Schmidt, E. L. (1989). Chemoautotrophy as a strategy in the ecology of indigenous soil bradyrhizobia. Soil Biol. Biochem. 21,461 -463. Weber, D. F., and Miller, V. L. (1972). Effect of soil temperature on Rhizobium japonicum serogroup distribution in soybean nodules. Agron. J. 64,796-798. Weber, D. F., Keyser, H. H., and Uratsu, S. L. (1989). Serological distribution of Bradyrhizv bium japonicum from U.S. soybean production areas. Agron. J. 81,786-789. Weiser, G . C., Skipper, H. D., and Wollum 11, A. G. (1990). Exclusion of inefficient Bradyrhizobium japoncium serogroups by soybean genotypes. Plant Soil 121,99 - 105. Wollum 11, A. G. (1987). Serological techniques for Bradyrhizobium and Rhizobium identification. In “Symbiotic Nitrogen Fixation Technology” (G. H. Elkan, ed.),pp. 149- 155. Dekker, New York. Young, C. C., and Chao, C. C. (1989). Intrinsic antibiotic resistance and competition in fastand slow-growing soybean rhizobia on a hybrid of Asian and US cultivars. Biol. Fertil. Soils 8.66 - 70. Zaat, S. A. J., VanBrussel, A. A. N., Tak,T., Lugtenberg, B. J. J., and Kijne, J. W. (1989). The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by R h i z v bium leguminosarum biovar. viciae on Vicia saliva subsp. nigru by suppressing the ‘thick and short roots’phenotype. Planta 177, I4 I - 150.

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Zablotowicz, R. M., Eskew, D. L., and Focht, D. D. (1978). Denitrification in Rhirobium. Can. J. Microbiol. 24, 757 - 760. Zablotowicz, R. M., Russell, S. A., and Evans, H. J. (1980). Effect of the hydrogenase system in Rhizobium japonicum on the nitrogen fixation and growth of soybeans at different stages ofdevelopment. Agron. J. 72, 555-559.

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CROPRESPONSESTO CHLORIDE Paul E. Fixen Potash & Phosphate i Institute, Brookings, South Dakota 57006

I. Introduction 11. Chloride in Plants A. Biochemical Functions B. Osmoregulatory Functions C. Uptake and Interaction with Other Nutrients D. Disease Interactions E. Crop Development 111. Yield and Quality Kesponses to Chloride A. Wheat and Barley B. Oats C. Corn D. Soybeans E. Potatoes F. Other Crops IV. Chloride Sources, Losses, and Application A. Chloride Sources and Losses B. Chloride Fertilizers and Application V. Predicting Crop Response to Chloride A. Plant Analysis B. Soil Testing C . Other Factors Influencing Response VI. Summary and Future Research Needs References

I. INTRODUCTION One of the earliest reports on crop response to chloride (Cl-) concerned the use of common salt (NaCl) as a fertilizer in the mid-1800s (Tottingham, 1919). For example, in England, barley (Hordeurn vulgure L.) Aducnrr~in Apvnm~y,Vd.I0 Copyright (3 1993 by Academic Press, Inc. AU rights of reproduction in any form rrxrved.

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was topdressed with common salt for the purpose of stiffening the straw. In his report, Tottingham concluded that NaCl served directly as a fertilizer and that C1- was the active element. In 1954, Broyer et al. (1 954) offered sufficiently convincing evidence to cause the general acceptance of C1- as a plant essential element. However, for over 20 years it was generally believed that field-grown crops would not benefit from C1- additions because of the ubiquitous presence of C1- in the environment. The potential role of C1- in cropping systems was not seriously considered until the 1970s, when research in the Philippines (von Uexkull, 1972), Europe (Russell, 1978), and the northwestern United States ( Powelson and Jackson, 1978) clearly showed that C1- could play an important role in crop management. These studies stimulated interest by other researchers to further investigate the role of C1- in crop management. Yield increases from application of C1- were verified in the field during the 1980s. In Oregon, yields of soft white winter wheat (Tritium aestivum L.) fertilized with NH,Cl exceeded those of ( NH,)tSO,-fertilized wheat by 1243 kg grain ha-' averaged across three sites (Chnstensen et al., 1981). In South Dakota, CaCl, and KC1 applications produced the same hard red spring wheat yield at equivalent C1- rates, and both produced 240 kg ha-' more grain than KN03 when averaged across 5 site-years (Fixen et al., 1986b). Barley grain yield with KCl applied exceeded an equivalent rate of K as K2S04by 299 kg ha-' at one of five sites in North Dakota (Timm et al., 1986). The numerous field responses to C1- likely involve the unique roles C1plays in plants, beyond biochemical functions. Such roles include osmoregulatory functions, plant development, and interaction with other nutrients and diseases. Critical C1- concentrations for these roles appear to be much higher than for biochemical functions.

11. CHLORIDE IN PLANTS

Chloride has a diverse set of functions in plants. Some involve intracellular processes that are very specific while others involve how plants interact with the environment. Because the biochemical functions of C1- require no more than 100 mg kg-', C1- is classified as a micronutrient. However, much higher concentrations, in the range of 2000-20,000 mg kg-', are normally present in plants, indicating that C1-, unlike other micronutrients, is relatively nontoxic at high concentrations. In fact, many of the nonbiochemical roles of C1- appear to require concentrations com-

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parable to those of macronutrients. Understanding the diverse functions of CI- in plants is critical to unders~ndingcrop responses to Cl-.

A. BIOCHEMICAL FUNCTIONS 1. Deficiency Symptoms

Physiological C1- deficiency symptoms in plants grown in nutrient solutions have been well characterized. Symptoms have been described for several species. a. Tomato (Lycopersicon esculentum Mill.) Chloride deficiency in tomato plants starts with a wilting of the leaflet blade tips, followed progressively by chlorosis, bronzing, and necrosis (Broyer el al., 1954). Lateral roots branch extensively but are stubby with club tips (Johnson et al., 1957). b. Spinach (Spinacia oleracea L.) After 3-4 weeks in a solution without added CI-, shoot growth of spinach was reduced 70% while root growth was reduced approximately 50% (Robinson and Downton, 1984). Leaves were smaller, narrower, had curled edges, and appeared wrinkled. The stem and lower leaf petioles had prominent accumulation of a n t h ~ y a n i n while s the leaf blades had higher chlorophyll levels. Roots were smaller and had a “herring bone” appearance.

c. Sugar Beets (Beta vulgaris L.) A “hemng bone” root pattern was also observed on C1-deficient sugar beets (Terry, 1977). Young leaves developed small yellowish spots with interveinal areas remaining greener than areas close to veins. Some c u p ping of the leaf blade at the midrib was noted. Johnson ef al. (1957) observed premature wilting due to C1- deficiency. d. Lettuce (Lactuca saliva L.) The first symptom observed by Johnson et al. (1957) was wilting. Restricted root growth with stubby, club-tipped laterals was prominent. e. Cabbage (Brassica oleracea L.) Increased va~abilityamong plants was apparent as was pr~maturewilting of the tips and margins (Johnson et al., 1957). Slowed expansion of the inner leaf sections caused cupping. Leaves cupped both upward and down-

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ward on the same plant. As the deficiency progressed, newly formed leaves became chlorotic. f. Barley (Hordeum vulgare L.) The most distinctive symptom was general chlorosis of newly emerging leaves (Johnson ef al., 1957). The leaves remained wrapped in tubular form longer than normal, were slower growing, smaller, more fragile than normal leaves, and eventually became necrotic. g. Alfalfa (Medicago sativa L.) Leaflet tips were split along the midrib and irregular chlorotic blotches appeared in the leaflet center (Johnson ef al., 1957). h. Corn (Zeu mays L.) Premature wilting was the only deficiency symptom observed by Johnson et al. (1957). i. Beans (Phaseolus vulgaris L.) Increased nastic movement of the leaves was the only symptom observed by Johnson ef al. (1957). Leaves of Cl--deficient plants were notably more oriented toward the sun at sunset. j. Potato (Solanurn tuberosurn L.) The symptom first observed was a lighter green color, with new growth tending to have a “pebbled” appearance described as vertical protrusions on the upper side of leaflets (Gausman et al., 1958a). As the deficiency intensified, margins of terminal leaflets curled upward and chlorosis developed on terminal leaflet tips and eventually extended approximately onefourth of the distance down the leaflet margins. A “purplish bronzing” of the older chlorotic areas was the final symptom to develop.

k. Coconut Palm (Cocus nucifera L.) Trees deficient in C1- were reported to have older leaves with yellowing and/or orange mottling and dried up leaf tips and edges (von Uexkull and Sanders, 1986). Also noted were reduced growth rates, fewer nuts set, reduced nut size, droopy leaves, signs of moisture stress, and stem cracking and bleeding. 2. Photosynthesis

The direct role of CI- in photosynthesis has been the focus of numerous studies since Warburg and Luttgens (1944) showed that 0,evolution by

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isolated chloroplasts required CI-. Other researchers concluded that CIwas involved in the splitting of water molecules in photosystem I1 (Izawa et al., 1969) and, more specifically, CI- acts as a cofactor of an NH,OH-sensitive, Mn-containing, 02-evolving enzyme (Kelley and Izawa, 1978). Using isolated thylakoids, Critchley et al. (1982) suggested that CI- facilitates electron transport by reversible ionic binding to the 0,-evolving complex or to the thylakoid membrane. A later study by Critchley (1983) offered evidence that this effect was indeed due to CI- and not to the accompanying cation. Terry ( 1977) questioned the methodology involved in investigations of the role of CI- in photosynthesis. He concluded that the in vitro photosynthesis responses to CI- may not have a physiological basis but instead may be due to the isolated condition of the chloroplasts. The principal cause of a 60% reduction in sugar beet growth from CI- deficiency in his study was lower cell multiplication rates in leaves, not a reduction in in vivo photosynthesis rates. It was shown by Robinson and Downton (1984) that the C1- content of chloroplasts is highly regulated. These authors reported a 70% reduction in spinach growth and leaf CI- concentration due to CI- deficiency, but no significant change in CI- concentration in the chloroplasts. Thus, photosynthetic rates may not be affected even though CI- is severely deficient. Robinson and Downton suggest that Terry (1977) measured a reduction in CI- concentration in isolated chloroplasts due to the unreliability of the nonaqueous technique used. The characteristic compartmentalization of CI- and accumulation in chloroplasts under deficient conditions would result in a very low critical leaf CI- concentration for photosynthesis. The severely CI--deficient spinach with unaffected photosynthetic rates reported by Robinson and Downton (1984) had a leaf CI- concentration of 131 mg/kg dry weight. The agronomic critical level of C1- in crop plants is clearly set by processes other than photosynthesis. 3. Enzyme Activation

Several enzymes are known to be stimulated by CI-. ATPase, located on tonoplasts and other sealed vesicles, appears to be stimulated by C1-, mostly by a direct effect on the enzyme but partly via dissipation of electrical potential (Churchill and Sze, 1984). This stimulation is preferentially inhibited by nitrate. a-Amylase, the enzyme that hydrolyzes starch to sugars, requires CI- for activation (Metzler, 1977). Asparagine synthetase is also stimulated by CI- (Rogness. 1975).

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B. OSMOREGULATORY FUNCTIONS The ability of CI- to move rapidly across cell membranes and its biochemical inertness are two important properties that allow CI- to serve as a key osmotic solute in plants (Maas, 1986). Chloride serves in this capacity at relatively low energetic cost to the plant (Sanders, 1984). When C1- is in short supply, plants may use more energy-costly organic salts for turgor control. Chloride is located primarily in the cell’s central vacuole as a component of a simple salt solution involved in cell expansion. 1. Counterion for Cation Transport

Because CI- is very mobile and tolerated at high concentrations, it is ideally suited to maintain electrical charge balance when cations such as K+ move across cell membranes. 2. Osmotic Adjustment

The process of osmotic adjustment occurs when solutes such as C1accumulate within a cell, causing the water potential within the cell to decrease below the external potential. The resulting water potential gradient causes water to enter the cell and the plasmalemma to expand against the rigid cell wall, resulting in an increase in cell turgidity. Jensen and Tophoj (1985) conducted an outdoor pot study on barley that showed that application of KCI increased leaf water content and improved plant water status during soil water stress. Grain yield was highly correlated with leaf water content. Tissue concentrations of K+ and CIwere increased similarly by the KCI additions, indicating that both ions contributed to osmotic adjustment. Potassium chloride application to wheat in a field study resulted in more negative plant water potentials (Maurya and Gupta, 1984). Christensen et a/. (1981) showed in a field study on winter wheat that NH,Cl application resulted in more favorable plant water status than application of (NH,),SO,. The role of C1- in reducing the effects of moisture stress under field conditions is still uncertain and will be discussed in more detail in later sections. 3. Stomata1 Operation

Stomata open when water moves into the guard cells, causing them to become more turgid. The influx of water is caused by an increase in solute concentration, which in turn causes the intracellular water potential to

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become more negative. Usually the major solutes involved are K+, C1-, and malate (Maas, 1986). If CI- is in short supply, malate typically is synthesized within the guard cells from starch produced in chloroplasts and is used as a balancing ion (Allaway, 1981). However, Assman and Zeigler (1986) have made calculations that indicate there may be no energetic advantage to using C1- as the balancing ion instead of malate. The amount of C1- used as a counterion to balance K+ vanes with species and the level of C1- in the environment. In his review of physiological response to C1-, Maas (1986) illustrated differences among species in counterion dependency as follows: onion (Allium cepa L.), an absolute requirement for C1- (Schnabl and Raschke, 1980); broad bean (Viciafaba L.), mostly organic acids (Outlaw and Lowry, 1977);corn and Commelina, both CI- and malate (Penny et al., 1976; Ratschke, 1975). Palm trees were at one time thought to be similar to onion in having an absolute requirement for C1- because their guard cells appeared devoid of chloroplasts (von Uexkull and Sanders, 1986). von Uexkull and Sanders (1986) suggested that some of the CI- deficiency symptoms discussed earlier, such as frond breakage and stem bleeding, were due to improper stomata1 functioning. However, von Uexkull (1985) reported results showing that many palm guard cells contain both chloroplasts and starch and that the physiological role of C1- in palms is still not fully understood. 4. Leaf Movement

Orientation adjustments of leaves are due to turgor changes of motor cells (Satter and Galston, 1981). Such changes appear to be made by the same mechanism discussed earlier for guard cells. Researchers have noted that leaf orientation on wheat in field plots can be influenced by C1treatment (Taylor and Jackson, 1980).

C. UPTAKE AND INTERACTION

WITH OTHER NUTRIENTS

Plants absorb CI- from the soil solution via at least two mechanisms. For barley, the first mechanism approaches its maximum uptake rate at C1concentrations near 0.1 -0.2 mM. The second mechanism operates only at CI- levels of 0.5 mM and above (Elzam and Epstein, 1965). Uptake is likely metabolically controlled and sensitive to temperature and metabolic inhibitors. Chloride uptake by excised corn root segments increases with decreasing pH down to a pH of 5.5, due possibly to the involvement of a

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protonated camer (Lin, 1981). Illumination increases uptake due to an increased ATP supply that serves as an energy source for the active uptake process (MacDonald et al., 1975). Uptake of CI- is competitively inhibited by Br-, NO,-, and but not by F or I- (Elzam and Epstein, 1965; Murarka et al., 1973; Mengel and Kirkby, 1987). 1. Nitrogen Interactions

Nitrogen and C1- interact via several mechanisms including both soil and plant processes. Rates of some steps in the mineralization of soil organic matter are affected by C1-. This can influence the form of N absorbed by crop plants. At the root surface, NO,- and C1- ions are known to compete with each other in the uptake process. a. Nitrification Inhibition Numerous studies have demonstrated that C1- inhibits nitrification in acid soils (Hahn et al., 1942; Agarwal el al., 197 1 ; Heilman, 1975; Golden et al., 1979; Christensen and Brett, 1985; Roseberg et al., 1986). Christensen and Brett (1985) reported that in laboratory studies of other investigators, concentrations of 46- 152 mg C1- kg-I soil were necessary for measurable nitrification inhibition. In their own studies of unlimed soil (pH 5 . 9 , where NH,Cl had been applied and inhibition of nitrification occurred, concentrations in the surface 10 cm of soil were 235 and 17 1 mg C1- kg-I soil, respectively, 1 and 7 weeks after application. Under these conditions the NH4+ N:NO,- N ratio remained above 3: 1 for 12 days longer with NH,Cl than with (NH,)2S04. Increasing soil pH to 6.6 with lime eliminated the nitrification inhibition properties of the C1- treatments. Most studies evaluating the effects of CI- salts on nitrification were not designed to separate general osmotic effects from effects specific to C1-. However, Roseberg et al. ( 1986) determined that at any given soil solution osmotic potential, CI- salts resulted in a lower rate of nitrification than did SOZ-salts. They concluded that inhibition of nitrification by CI- salts is due to a combination of specific C1- ion effects and low soil osmotic potential. They also suggested that the mechanism of inhibition could involve either direct or indirect effects of C1- on nitrifying organisms. b. Form of N Effects Effect of N form on C1- response by take-all-infected soft white winter wheat in Oregon was evaluated by Taylor et al. ( 1983) at two planting dates and soil pH levels of 5.6, 6.0, and 6.2. Yield response to 40 kg C1- ha-’ applied in the fall as KCI was nonsignificant (p = 0.05) when (NH,)2S0, was the N source, regardless of soil pH and seeding date. When Ca(NO,),

CROP RESPONSES TO CHLORlDE

11s

was the N source, chloride response averaged 1370 kg ha-' for pH levels of 6.0 and 6.2 at the late seeding date. Chloride response was nonsignificant at other pH and planting date combinations. Split application of a much higher rate of C1- (86 kg ha-' fall 342 kg ha-' spring) as NH,Cl produced 1550 kg ha-' more grain than (NH,),SO, at the late planting date and pH of 5.6. Imgated hard red spring wheat in Montana inoculated with the take-all fungus at a soil pH of 7.9 showed greater yield response to Cl- fertilization when ammoniacal N forms were applied than when NO3- was used (Engel and Mathre, 1988). Grain yield response to 45 kg C1- ha-' as NaCl with NH,+ was 5 17 - 869 kg ha-' while it was nonsignificant where no N was applied or when NaNO, was used.

+

c. Competition with NO3Nitrate and C1- compete with each other for uptake in many species (Harward ef al., 1956; Meyer ef al., 1957; James et al., 1970a; Murarka et al., 1973; Fuqua et al., 1974; Glass and Siddiqi, 1985; Christensen and Brett, 1985; Coos ef al., 1987). Increasing the supply of either one tends to reduce the tissue concentration of the other. Murarka ef al. (1973) showed in a greenhouse study that even though applications of 100 or 200 mg C1kg-' soil reduced NO3- concentrations in the potato plant, they did not reduce the amount of protein or dry matter yield. However, the authors pointed out the potential problems that varying soil C1- levels could have on use of plant NO,- levels as a diagnostic tool for N management. Under some conditions, positive interactions between NO,- and C1have been measured. Application of C1- increased N concentration in leaves of coconut palm in the Philippines (von Uexkull and Sanders, 1986). At soil C1- levels greater than 19 mg kg-' soil, spring wheat C1concentrations in South Dakota increased with increasing soil NO3- levels (Fixen ef al., 1987). Below 19 mg C1- kg-' soil, they decreased with increasing soil NO,-. As in other studies, C1- application consistently reduced plant NO,- concentrations. 2. Phosphorus Interactions

Chloride appears to interact with P in a complex manner. In some cases P availability has been increased by elevated C1- while in other cases it has been decreased or not affected. a. Chloride Effects on P Uptake Pot experiments with Caribou loam soil using ,*P on potatoes lead Gausman ef al. (1958b) to suggest that an optimum or critical level of C1existed for maximum P uptake to occur, with uptake decreasing on either

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PAUL E. FIXEN

side of this level. In their studies the optimum level appeared to be 300 to 450 mg C1- kg-' soil. A later paper from the same research project, but using potatoes grown in washed sand in the greenhouse, reported increased 32Pactivity at flowering with 100 mg C1- kg-' but reduced uptake above this concentration. No significant effect was measured at harvest. Solution culture studies using white clover (Trifoliurn repens L.) in Australia suggested that an optimum C1- concentration exists for P uptake, but that the optimum changes with solution P concentration (Rogan, 1977). The reduction in P uptake at C1- levels exceeding the optimum was attributed to anion competition. Soft white winter wheat infected with take-all responded to P when applied with 428 kg C1- ha-' as NH4Cl and KCl but not when applied with (NH.J2S04and 40 kg C1- ha-' as KCI (Taylor et al., 1983). The authors suggested that the addition of P overcame the competitive inhibition of P uptake caused by the high CI- concentration in the root zone. Others have found no effect of C1- on P uptake. A study of 20-day-old corn seedlings in solution culture at pH 4 showed no effect of CI- on 32P uptake during a l-hr period (Carter and Lathwell, 1967). Similar results were reported for lima beans by Kretschmer ef af. (1953). b. Phosphorus Effects on C1- Uptake Field experiments in central Washington on a slit loam soil naturally low in C1- showed that application of P fertilizer had a pronounced synergistic effect on uptake of C1- by potatoes from KCl applications (James et al., 1970b). No effect of C1- on P uptake was measured. Fine and Carson (1954) found that application of P fertilizer in both greenhouse and field experiments alleviated salt injury symptoms of oats and barley growing in saline soil and gave marked yield increases. They suggested that the function of P may have been to reduce the excessive quantities of CI- and sulfate accumulating in leaves. It is feasible that at low soil C1- levels, P application may tend to increase response to CIadditions, whereas at very high soil C1- levels (as encountered in some saline soils), P application may reduce CI- uptake and the negative effects of high salts. The literature is not clear on these interactions. 3. Manganese Interactions

Application of chloride-containing salts to acid soils has increased the Mn concentration of plants. Researchers in Oregon studied CI effects on Mn uptake by bush beans and sweet corn in poorly drained soils (pH values from 4.7 to 5.3) that contained Mn concretions (Jackson et al.. 1966). Chloride application increased Mn uptake of both crops and re-

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sulted in Mn toxicity symptoms on trifoliate leaves. The authors offered Mn toxicity as a possible explanation for the yield reductions on acid soils sometimes observed from band application of KCl at planting time. Additional laboratory studies by the Oregon researchers explored the mechanism by which CI- increases soil Mn availability (Westerman er af., 1971). They suggested that CI- enhances the reduction of some Mn oxides in acid soils and in the process increases extractable Mn. Potassium chloride increased Mn release from 14 soils collected from six countries located in temperate and subtropical regions (Krishnamurti and Huang, 1987). Release from the calcareous vertisol was the lowest even though its total Mn content was the highest. The researchers indicated that redox, complexation, and exchange reactions appeared to be involved with the observed Mn release. Lindsay ( 199 1 ) calculated the solution species of Mn in equilibrium with manganite and pyrolusite at a pe pH of 16.6 when CI- and SO:- are at 0.001 A4 and CO, is at 10-3.52atm. Under these conditions Mn2+ is the dominant solution form. However, the activity of MnCP increases 10-fold with every 10-fold increase in CI- activity. Therefore, when the CI- activity increases to approximately 0.25 M the activities of MnCI+ and Mn2+ are equal. At 0.025 M Cl-, MnZ+ activity would be 10 times the MnCP activity. If a volumetric soil water content of 0.25 is assumed, 0.025 M C1- is equivalent to I68 mg kg-I soil CI- or 302 kg CI- ha-' distributed evenly in the top 15 cm of soil. If the same 302 kg ha-' were mixed with only 10%of the top 15 cm of soil, the MnCP activity in the fertilized zone could be equal to the Mn2+activity and essentially double the quantity of total Mn in solution. Therefore, it would seem that fertilization with CI- could significantly increase Mn availability via direct complexation. However, this effect would be temporary due to the mobility of CI- in soil.

+

D. DISEASE INTERACTIONS In recent years, the most studied effects of C1- on crop plants have been those that relate to crop diseases. Chloride application has suppressed or reduced the effects of numerous diseases on a variety of crop species. A partial list of such occurrences is given in Table I. Although CI- interactions with crop diseases are well documented, the mechanisms involved in these effects are not well defined. Generally, proposed mechanisms fall into two categories, either suppression of the pathogen or an increase in host tolerance. In the following discussion a third category will be included for those situations where no disease-related effect on the pathogen or the host was detected.

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PAUL E. FIXEN Table 1

Plant Diseases with Reported Suppression Using CI- Fertilized Crop

Diseases

Winter wheat Spring wheat Barley Durum wheat corn Pearl millet Coconut palm Potatoes Celery Rice

Take-all root rot, tanspot, stripe rust, Septoria, leaf rust Common root rot, tanspot, leaf rust, Septoria Common root rot, Fusarium root rot, spot blotch Common root rot Stalk rot Downy mildew Gray leaf spot Hollow heart, brown center Fusarium yellows Stem rot, sheath blight

Adapted from Fixen ef a/. ( 1987).

1. Suppression of Crop Pathogens

a. Take-all Root Rot Numerous studies across diverse soils have demonstrated suppression of the wheat take-all fungus (Gaeumunnomyces gruminis var. tritici) by ammoniacal fertilizers. In a review paper, Powelson et af. (1985) cited 12 references that document such suppression, with the first published in 1941. The mechanism most often suggested is that plant uptake of NH4+ instead of NO,- decreases the pH of the rhizospere. Christensen and Brett (1 985) used data from the literature to determine that an NH4 N :NO, N ratio of 3: 1 or greater is required for rhizosphere acidification to occur. The reduced pH offers a competitive advantage to acid-tolerant microorganisms, such as the fluorescent Pseudomonas, and decreases the growth of G. gruminis hyphae along the root (Cook, 1981). The nitrification inhibition properties of C1- discussed earlier may be partially responsible for the suppression of take-all by C1- (Christensen and Brett, 1985). The studies leading the Oregon researchers to this hypothesis showed that C1- application slowed nitrification, reduced take-all severity, and increased winter wheat grain yield especially on moderately acid soils. Take-all incidence (percentage of plants infected) was unaffected by C1- in these studies (Christensen el af., 1987). Powelson et uf. (1985) have suggested that water stress could reduce the efficacy of NH4+ and C1- fertilizers for take-all control. They referred to reviews by Cook ( 1981), Rovira and Wildermuth ( 198l), and Schroth and Hancock ( 1982), pointing out that root-colonizing epiphytic bacteria do

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not tolerate moisture stress situations and are less likely to be effective suppressors under low soil moisture. Of course, such an effect would only be relevant if the biology of the rhizospere is involved with disease suppression by C1-. Huber and others have published extensively on the role of Mn in control of plant diseases, especially take-all (Huber and Wilhelm, 1988; Huber, 1989, 1990). Manganese is usually lower in tissues susceptible to fungal, viral, and bacterial pathogens than in resistant tissues. Huber has suggested that increased Mn availability by C1- may be one of the mechanisms responsible for the suppressive effects of C1- on several diseases. b. Common Root Rot The effect of C1- on common root rot may be indirect and related to its effect on plant nitrate (Timm et al., 1986; Goos et al., 1987, 1989). Chloride reduced the number of barley plants infected with common root rot (primarily Cochliobulus sativus) at the boot stage and the disease severity index at the dough stage at three out of five sites in 1983 in North Dakota (Timm ef al.. 1986). Culm NO3- concentrations were lowest with the high C1- rate at four out of five sites and suggested that a reduction in NO,- concentration may be involved in the root rot suppression mechanism. Further studies in 1984 using two barley cultivars differing in common root rot susceptibility showed reductions in common root rot severity from applications of 46 and 188 kg KCl ha-' at all three sites tested (Goos et al., 1987). Culm NO3- concentration at the early boot stage was also dramatically reduced by KCl application for both cultivars at all three sites. A singular relationship existed between culm NO3- concentration and common root rot severity for both cultivars at each of the three sites (Fig. 1). The lower plant NO3- concentration apparently reduced plant predisposition to common root rot. Differences in cultivar reaction to common root rot may be due, in part, to differences in NO,- accumulation. Experiments on common root rot suppression on barley were conducted at six additional sites in 1986 (Goos et al., 1989), bringing the total number of sites for the 3 years to 14. Chloride application consistently reduced plant nitrate levels and increased plant C1- levels. Common root rot was reduced significantly at 8 of the 14 sites. Usually application of 45 - 50 kg KCI ha-' was adequate to reduce the disease. c. Foliar Diseases Yellow rust (Puccinia striiformis) in winter wheat was one of the first foliar diseases reported to be suppressed by C1- (Russell, 1978). Studies conducted in England in the greenhouse and field from 1972 to 1977

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PAUL E. FIXEN 3.5-

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2000

3000

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6000

Tissue Nitrate, mg N/kg

Figure 1. Relationship between common root rot severity of two barley cultivars and tissue nitrate concentration at three sites (Gooset al.. 1987).

showed that application of either NaCl or KCI greatly reduced the severity of yellow rust symptoms on all six winter wheat cultivars evaluated at two locations in three successive years. Interestingly, application of a combination of NaCl and KC1 reduced yellow rust to a greater extent than an equivalent amount of CI applied completely as NaCI. Yellow rust was encouraged by applications of NaNO,. Though rates applied to the soil ranged from 376 to 2260 kg ha-', even the highest rate failed to affect adversely growth or yield of winter wheat in these experiments. Field studies in Oregon have demonstrated similar suppression of stripe rust (Puccinia striformis) on winter wheat (Christensen et al., 1982). The percentage of leaf area attacked by the stripe rust disease was significantly reduced on 381 cultivars by a spring topdressing of 340 kg CI- ha-' as NH,CI when compared to (NH4),S04. Stripe rust infection type and apparent rate of disease progression, based on two sampling dates (12 May

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and 22 May), were unaffected by CI- application. These data confirm the results of Russell ( 1978) showing C1- suppression of yellow or stripe rust. Christensen ef al. ( 1982) also concluded that their results suggest that CIdelays stripe rust epidemics by lengthening the latent period (time from inoculation to sporulation) or by reducing the amount of effective inoculum across the diverse cultivars tested. Oregon research by Scheyer ef al. ( 1987)assessed stripe rust severity later in the season and on more dates (26 May and 2, 10, 16, and 22 June). Using this more sensitive assessment methodology, application of Cl- was found to reduce the apparent rate of stripe rust progression. The lowest CIrate in the study, 76 kg ha-', was as effective in slowing disease progression as the higher rates. These data also led the authors to conclude that even though C1- suppression of stripe rust was statistically significant, the effects were small and probably too limited to serve as an effective means of disease control. A potentially important interaction between moisture stress and CIsuppression of foliar diseases has been noted by Powelson ef a/. (1985). They observed that the best responses from ammoniacal N and CI- occurred under conditions where moisture had not been limiting. For example, in Oregon, where moisture was nonlimiting throughout the growing season, good suppression of take-all, stripe rust, and septoria leaf and glume blotch was obtained with application of ammoniacal N and C1-. In a similar season, except for dry weather during the boot to dough stages, good suppression of take-all occurred with C1-, but no significant reductions in severity of P. striiformis or Sepforia spp. were detected. The researchers suggested that the moisture stress conditions in late spring already limited pathogen activity beyond a point that could be further modified by uptake of C1-. Plant water relationships may be involved in the mechanism of foliar disease suppression by C1- (Powelson ef al., 1985). Water potential of the plant can have a substantial effect on fungal development (Cook, I98 I). The effects of C1- on plant water relationships were discussed earlier. It is possible that these effects reduce the ability of the pathogen to attack the plant (Powelson rt al., 1985). The hypothesis suggested is that an elevated CI- content in plant cells increases leaf turgor potential and may result in slower growth and activity of the pathogen due to the reduced turgor gradient. The reductions in attack severity by P. sfriiformis, Puccinia recondita, and Sepforia sp. because of Cl--containing fertilizers may be due in part to this mechanism (Christensen ef al., 1982; Cunfer ef a/., 1980; Hashim and Russell, 1982; Russell, 1978). South Dakota studies on hard red spring wheat have shown CI- suppression of leaf rust. The primary leaves of five spring wheat cultivars and five

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near-isogenic lines were inoculated with leaf rust isolates of known virulence in a growth chamber study (Rizvi et al., 1988). Chloride application dramatically reduced rust intensity on cultivars and lines and reduced pustule type from susceptible to resistant or intermediate. Chloride a p peared to affect the phenotypic expression of the resistance genes and cultivars. Additional growth chamber experiments were conducted to study the genetics of leaf rust resistance in spring wheat and C1- response (Rizvi, 1990; Rizvi et af.,1990). The investigators concluded that application of Cl- increased the number of genes involved in resistance by one compared to plants without C1. However, further work did not support this conclusion, although the possibility for such an effect has not been ruled out (G. W. Buchenau, personal communication). In another growth chamber study by the same investigators, five spring wheat cultivars were inoculated with tanspot conidia (Pyrenophora triticirepentis) (Buchenau et al., 1988b).Chloride reduced tanspot rating and the number of lesions per leaf. Chloride application in field studies conducted in South Dakota from 1984 to 1987 resulted in reduction of leaf rust, tanspot, and Septoria on spring wheat (Buchenau et al., 1988a; Fixen et al., 1986a). Significantly fewer leaves from C1- treatments developed tanspot conidia or Septoria pycnidia in moist chambers compared to untreated leaves. Leaf rust pustule size and number were reduced. For example, leaf rust severity on Marshall spring wheat was reduced from 65% in untreated plots to 25% where KCI or CaCl, was applied. Texas research has shown C1- suppression of leaf rust on winter wheat at some locations and years (Miller, 1992). Leaf rust rating on cultivar 2 158 during grain fill was reduced from 80% on the check to 5 -45% where 45 kg C1- ha-' was applied. Other sites have shown no effect of C1- on leaf rust. The researcher noted several differences between sites, including cultivar, rainfall. and soil test C1- levels. 2. Enhanced Host Tolerance

Recent investigations indicate that enhanced host tolerance may be more important than previously thought. Christensen et af. (1990) concluded that enhanced host tolerance, as well as inhibition of nitrification in acid soils, may be responsible for higher grain yields when take-all-infected wheat is fertilized with NH,Cl. In their recent studies, plots fertilized with NH,CI and (NH,)2S04 did not differ in take-all incidence, take-all severity, or NH,+:NO,- ratios in the soil, but differed significantly in yield. Furthermore, application of dicyandiamide (DCD), an effective nitrification

123

CROP RESPONSES TO CHLORIDE

-

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Figure 2. Winter wheat grain yield as a function of number of crown roots with take-all for NH,N03 (AN), (NH4)2S04(AS), NH,CI (AC), urea (UR), and NH,NO, urea DCD (DCD) (Christensen et a/., 1990).

+

+

inhibitor, increased severity of take-all. Because the slope of the relationship between yield and take-all severity was less negative for NH,Cl than the other N sources evaluated (Fig. 2), it appears that NH4Cl increased the ability of plants to tolerate severe take-all. A suggested mechanism for enhanced host tolerance is that C1- uptake lowers leaf osmotic potential, resulting in greater plant turgor (Christensen ef al., 1990). Linkages between plant disease and water relationships have been observed and reported by many investigators over nearly 70 years (Weiss, 1924; Chupp, 1946; Griffin, 1963; Cook and Papendick, 1972; Christensen et al., 1981). Direct effects of C1- on plant water relationships were discussed earlier. In a pot study of healthy and take-all-infected spring wheat, Trolldenier ( 1985) reported that transpiration of flag leaves declined with higher incidence of take-all and with low soil moisture contents. Transpiration was also lower for wheat fertilized with K2S04than with KCl. The difference between S04-2 and C1- fertilization was relatively greater for the more severely infected plants, especially those with lower soil moisture. Plants fertilized with C1- had flag leaves that remained green for a longer period and were higher yielding. than did those fertilized with Montana field studies of irrigated spring wheat inoculated with the take-all fungus showed that the effect of fertilizer N source and C1- on root disease scores and percentage of white heads was not as great as the effect on grain yield and test weight (Engel and Mathre, 1988). Application of C1- generally had little effect on disease severity indices regardless of N

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source. However, because disease was assessed only once during the growing season, it is possible that effects were missed. Yield and test weight were significantly increased by CI- fertilization when NH,OH or urea was applied but not when NaNO, or no N was used. Therefore, N form appeared to influence response to CI- but not through a measurable effect on take-all severity. These results may support an enhanced host tolerance mechanism provided significant disease severity effects were not missed by the sampling methodology. 3. No Disease-Related Effect on Host or Pathogen

Growth chamber and field studies were conducted in Maryland to evaluate the effects of C1- soil amendments on the incidence and severity of powdery mildew on soft red winter wheat (Thier et al., 1986). Application of CI- at a rate of 280 kg ha-’ as either KCI or CaCI, failed to significantly affect grain yield, test weight, heading date, plant height, lodging, and incidence or severity of powdery mildew. Montana field experiments were camed out on hard red winter wheat inoculated with Fusarium culmorum, one of the pathogens causing common root rot (Engel and Grey, I99 I ). Neither of the two cultivars showed any effect of CI- additions on disease severity at the soft dough stage during the 2 years of the study. Grain yield was significantly increased by C1addition in the first year but not in the second year. The effect of CI- on common root rot of hard red spring wheat was evaluated in five field studies in northwestern Minnesota (Windels et al., 1992). Fertilization with CI- tended to increase forage yield and significantly increased grain test weight; however, grain yield and common root rot incidence and severity were not significantly decreased.

E. CROPDEVELOPMENT Chloride effects on plant development have been detected from emergence through plant maturity in both growth chamber and field studies. Emergence of “Butte” hard red spring wheat seedlings grown in sand culture in the growth chamber was increased by CI- addition but emergence of “Guard” was not (Buchenau and Rizvi, 1990). Numerous field studies in South Dakota have shown that “Guard” does not respond to CIfertilization. If the sand was infested with Fusarium graminearum, emergence was not affected by CI- addition. Field studies in South Dakota on hard red spring wheat have detected CI- effects on spikelet primordia development, heading, or culm elongation, and date of anthesis (Schumacher, 1990). Formation of the double

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ridge and the terminal spikelet in plots of “Marshall” wheat treated with 135 kg KCI ha-’ occurred 2 days earlier in 1988 and 1 day earlier in 1989 compared to the control plots. Application of KNO, had no effect on plant development. The total number of spikelet primordia was not affected by KCI fertilization; however, date of anthesis was consistently advanced by 1 day. Physiological maturity, defined as the date of maximum kernel dry matter accumulation, was not affected by the treatments. Because the grain fill period started earlier with KCI-treated plots and ended at the same time as the control, the duration of the grain fill period was increased by an average of 1.5 days. Similar results were obtained for barley in Denmark in an outdoor pot study (Jensen and Tophoj, 1985). The duration of the grain fill period of barley was increased 3 to 6 days by application of KCI in a situation in which it appeared that both K+ and CI- were involved in the response. The lengthened grain fill period of spring wheat reported by Schumacher (1990) resulted in a 4% increase in kernel weights. Much greater increases in kernel weights due to CI- fertilization have been reported in other studies in the northern Great Plains. Increases of up to 14 and 12% have been reported for spring and winter wheat, respectively (Cholick et al., 1986; Engel ef al., 1992). Final kernel weight and grain yield were limited by both temperature and water stress in the studies by Schumacher (1990). Ongoing investigations of six hard red winter wheat cultivars in Montana appear to be giving somewhat different results (Engel er al., 1992). In these studies kernel weight has been increased by CI- fertilization; however, the increase appears to be due more to an increase in rate of kernel growth than to duration of the grain fill period. The impact of crop development effects of CI- on crop yield or quality will likely vary with the environment of the specific growing season. The timing of temperature, the moisture, and other crop stresses relative to crop growth stage are critical to the eventual yield response. For example, a 25% increase in grain yield of barley when KCI was applied rather than K,SO, in Saskatchewan was attributed to maturity enhancement by CI(Rennie et al. 1983). The CI--fertilized barley was more advanced when an early frost caused major yield reductions in the region. Such data indicate the substantial potential for CI- by environment interactions.

111. YIELD AND QUALITY RESPONSES TO CHLORIDE Crop species differ in their potential for response to C1- application under field conditions. Although CI- is an essential nutrient for all crops,

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some crops and cropping situations appear to have a greater potential than others to benefit from its application.

A. WHEAT AND BARLEY Since the late 1970s, extensive C1- research has been conducted on wheat and barley in the northwestern United States and the Great Plains of North America. Many of these studies have shown beneficial effects of C1-, and others have shown none. Factors influencing response probability other than crop or cultivar will be discussed in Section V. 1. Cultivar Differences

It is clear that cereal crops not only differ in their potential for response to C1- in the field (Fixen ef al., 1986b), but cultivars also differ in responsiveness. Average C1- responses across 5 site-years spanning 3 years for three hard red spring wheat cultivars in South Dakota were 4 1 1, 330, and 7 kg grain ha-’ for “Butte,” “Marshall,” and “Guard,” respectively (Fixen, 1987). A screening of 15 cultivars at two locations resulted in response at least at one location by 12 of the cultivars. “Guard” was again one of the three cultivars that showed no response. Some but not all of the response differences were explainable based on disease susceptibility. Spring wheat cultivar differences have also been measured in Manitoba (Mohr, 1992). Average grain yield responses to 50 kg C1- ha-’ across four sites were 150, 137, 116, and - 16 kg ha-’ for “Biggar,” “Roblin,” “Marshall,” and “Katepwa” cultivars, respectively. “Katepwa” had also failed to respond to C1- at eight sites in earlier studies and, like “Guard” in South Dakota, appears to be a C1--nonresponsive cultivar. The Cl- responsiveness of five spring barley cultivars was evaluated at different sites for 2 years in eastern South Dakota (Gelderman ef al., 1988). Average grain yield responses were 222, 200, 200, 49, and I 1 kg ha-’ for “Robust,” “Hazen,” “Morex,” “Bowman,” and “Primus 11” cultivars, respectively. Similar to spring wheat, some barley cultivars seem nonresponsive to C1- fertilization. Similar barley cultivar differences in C1- response have been measured in the Manitoba research referred to earlier (Mohr, 1992). Average response across four sites varied from 239 kg ha-’ for the most responsive cultivar, “Heartland,” to -257 kg ha-’ for the least responsive cultivar, “Bedford.” “Bedford” had responded positively at two out of eight sites in earlier studies with an average grain yield response across all eight sites of 187 kg ha-’.

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Differences in CI- response of hard red winter wheat cultivars have been measured in Kansas (Bonczkowski, 1989). In a study of “Arkan,” a cultivar moderately resistant to leaf rust, and “Newton,” a leaf rust susceptible cultivar, a C1- by cultivar interaction was detected in 1 out of 3 years. Grain yield responses to 50 kg C1- ha-’ were 667 and 47 kg ha-’ for the moderately resistant and susceptible cultivars, respectively. It appeared that the leaf rust resistance in the moderately resistant cultivar was enhanced by C1- application. 2. Magnitude and Frequency of Yield Response

The largest reported wheat or barley yield increases to CI- fertilizers have occurred on take-all-infested winter wheat grown in the northwestern United States on moderately acid soils. For example, nine grain yield increases reported in several studies conducted from 1980 to 1983 ranged from 470 to 2 150 kg ha-’, with an average of 1076 kg ha-’ (Christensen et al., 1981; Christensen and Brett, 1985; Powelson et al., 1985; Scheyer et al., 1987). These responses were all determined by comparing (NH4)2S0, to a rate of NH4Cl application giving an equivalent amount of N. Addition of KCI to soils testing very high in available K in Saskatchewan, Canada, resulted in average yield increases of 342 kg ha-’ across 6 winter wheat sites, 192 kg ha-‘ across 18 spring wheat sites, and 128 kg ha-’ across 9 spring barley sites (Wang, 1987). Durum yields were not increased when averaged across 3 sites. Yield response was statistically significant at 8 of the 36 sites. Wang (1987) suggested that response may have been due to the lower osmotic potential caused by C1- uptake and the resulting increase in plant turgor under moisture stress. Wheat and barley grain yield responses to CI- fertilization over five states in the Great Plains of the United States were recently summarized by Engel et al. ( 1 992). The summary showed that significant C1- response occurred in 42% of the 169 episodes over a wide range of environments and yield potentials (Fig. 3). It is important to note that this summary includes cultivars such as “Guard” spring wheat, “Primus 11” barley, and “Newton” winter wheat, which are proved nonresponding or limitedresponding cultivars. Also included are sites testing high in soil C1-. Responses in the Great Plains have generally been modest compared to those measured in the northwestern United States (Engel et af.,1992). The average significant response in Engel’s summary was 304 kg ha-’ whereas the average response for all sites was 1 I7 kg ha-’. The average response for all sites in the Saskatchewan studies ( Wang, 1987)was 184 kg ha-’. Thus it is economically beneficial to be able to predict responsive situations. That will be the topic of a later discussion in this review.

PAUL E. FIXEN

128

0

2Ooo

4Ooo

Boo0

Control yield, kg/ha Figure 3. Chloride yield response relative to control yields in the Great Plains. 0, Nonsignificant; 0, significant (>67 kg ha-'). Each data point represents a cultivar X site X year episode (Engel et a/.. 1992).

3. Mechanisms Involved in Yield Responses

Many of the responses summarized above were due to documented suppression of root or foliar diseases or enhancement of host tolerance to diseases, as discussed earlier. However, in many other cases responses could not be attributed to disease effects (Bonczkowski, 1989; Engel and Mathre, 1988; Engel and Grey, 1991; Fixen, 1987; Fixen et al., 1986b; Goos et al., 1989; Mohr, 1992). The nondisease mechanisms involved in these responses have not been clearly demonstrated. However, possible mechanisms include effects of Clon plant development and resulting stress avoidance as well as potential effects on plant water relationships. These were discussed earlier. 4. Quality Effects

Great Plains studies have demonstrated frequent improvements in wheat and barley quality due to CI- application. Parameters influenced include kernel weight, plumpness or volume, and test weight (Cholick ef al., 1986; Engel and Mathre, 1988; Engel ef al., 1992; Mohr, 1992; Schumacher, 1990; Windels et a!., 1992). In some cases quality effects, espe-

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cially kernel weight, have been more common than yield effects. In North Dakota studies, barley kernel volume was increased in 6 out of 13 siteyears by application of KCl in situations wherein response to K+ was very unlikely (Zubriski ef al., 1970). It has been observed in South Dakota that CI- application can reduce the severity of late-season lodging in spring wheat. Lodging reduction could be one mechanism for improvement of grain quality.

B. OATS Oat responsiveness to broadcast application of 187 kg KCl ha-’ was compared to that in spring wheat and barley at 6 site-years in eastern South Dakota (Fixen et al., 1986b).Wheat and barley responded significantly at 4 and 3 sites, respectively, but no oat responses were detected. Soil and plant analysis as well as studies conducted the followingyear at nearby sites using KNO, and CaCI, all indicated that responses were due to the C1- in the KCI fertilizer. The authors concluded that oat was less responsive to C1than was wheat or barley but cautioned that the results could have been influenced by the specific cultivars used for each species. More intensive studies on the effects of C1- on oat cultivars were conducted by Gaspar (1988). The average grain yield responses to 64 kg CIha-’ across 5 site-years in eastern South Dakota were 160, 140, 110, 10 (nonsignificant, NS), and -40 (NS) kg ha-’ for the cultivars “Moore,” “Benson,” “Ogle,” “Lancer,” and “Froker,” respectively. Yield responses were due to increases in kernel weight. Neither the number of tillers nor seeds per tiller were influenced by CI-. Parameters unaffected by CI- were crown rust (Puccinia coronafa) infection, leaf relative water content, leaf water potential, and stomata1 conductance. Adjacent experiments evaluating the interaction of N and CI- showed a slight reduction in crown rust infection from C1- at all 5 site-years and a reduction in lodging in most cases, especially at high N rates (Gaspar, 1988). The reduction in crown rust was only 1% of the leaf area and was considered agronomically unimportant. Solute potential and solute potential adjusted for full turgor were decreased by C1- addition, although the decreases did not seem related to yield.

c. CORN Published accounts of CI- effects on corn date back at least to 1958 (Younts and Musgrdve, 1958b).In field and pot experiments in New York,

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PAUL E. FIXEN

increasing KCl rates decreased stalk rot incidence of corn whereas increasing K2S04or KP03 application had little or no effect. Later studies in Wisconsin by Martens and Amy (1967a,b) showed that both KCl and NH4Cl decreased pith levels of reducing sugars, decreased natural root necrosis, and delayed death of corn plants, but increased stalk rot when the stalks were inoculated with Diplodiu muydis (Berk). The observation of delayed plant death is consistent with the findings of Younts and Musgrave (1958b) that KCI reduced natural stalk rot. However, Martens and Amy ( 1967b) attributed the reduction to both K and C1, because the effects of KC1 were much greater than the effects of NH,Cl. The increased stalk rot in inoculated plants due to C1- application reported by Martens and Amy (1967b) was thought to be due to the direct injection into the stalk, which bypasses the normal advance of the pathogen from the roots. The researchers proposed that fungal growth in the root and its advance into the stalk is delayed by both K+ and C1-, but subsequent growth in the stalk is more rapid. Field studies in Wisconsin by Liebhardt and Munson ( 1976) evaluated the effects of K+ and C1- on corn lodging where no KCI had been applied for the previous 4 years. Potassium application resulted in an I800 kg ha-' increase in grain yield and a reduction in corn lodging of 4896, whereas C1application had no effect on either yield or lodging. Lack of significant yield response to C1- by corn was also measured in South Dakota studies (Schumacher and Fixen, 1989). Broadcast-incorporated rates of C1- up to 5 10 kg ha-' failed to give significant yield response for the year of application or the 2 years following the application year. Grain moisture at harvest was significantly increased by C1- during the third year. In the same study, spring wheat yields were significantly increased for the first 2 years. Banding Cl--containing fertilizers near the corn row at rates higher than those normally used has reduced grain yield and delayed maturity. Corn growth in New York was delayed if KCI was applied in a band located 7.5 cm beside and 2.5 cm below the seed at C1- rates exceeding 50 to 100 kg ha-' (Younts and Musgrave, 1958a).Jackson el ul. ( 1 966) observed reduced sweet corn growth and vigor following C1- application on a poorly drained acid soil that contained Mn concretions in the Willamette Valley of Oregon. The negative effect of C1- was attributed to an increase in Mn availability and resulting Mn toxicity. Research in Ohio showed no effect of NH4Cl applied in a band located 4 cm to the side and 4 cm below the seed at rates up to 100 kg ha-' (Teater ef ul., 1960). A rate of I35 kg C1- ha-' decreased yields below those when an equivalent amount of N was applied, but from non-C1- sources, in one of two years. Broadcast application avoids these negative effects of C1- on

CROP RESPONSES TO CHLORIDE

131

corn even at much higher rates of 500 to 600 kg ha-' (Schumacher and Fixen, 1989; Teater ef al., 1960). Studies in the greenhouse and field were conducted on poorly drained Atlantic coastal soils to determine if C1- toxicity could be a problem for corn (Parker ef al., 1985). No detrimental effects were measured even at the highest rates in the studies (728 mg C1- kg-' soil in the greenhouse and 340 kg CI- ha-' in the field). Studies evaluating the interaction between N and K sources suggest that C1- increases the yield response of corn to enhanced ammonium nutrition (Dibb and Welch, 1976; Teyker et al., 1992). In a 2-year field study Teyker ef al. (1992) found no response to enhanced ammonium nutrition with KHCO, or K2S04 as K sources, although grain yield was increased by 380 to 560 kg ha-' when KCI was used. A greenhouse study by Dibb and Welch ( I 976) showed that the importance of KCI supplementation increased substantially when corn was grown with ammonium nutrition. Teyker et al. (1992) suggest that the apparent higher CI- requirement under enhanced ammonium nutrition compared to nitrate nutrition is perhaps due the role of C1- as a counterbalancing ion and major osmolyte. Although corn yield increases from C1- have been measured, there appears to be less potential for corn to benefit from CI- application in the field than is the case for cereal crops. When positive responses occur they have been limited in magnitude.

Although data are limited, soybean and soybean diseases do not appear to be responsive to application of CI- fertilizer. Studies in Missouri exploring the potential of CI- in KCI fertilizer for reducing soybean cyst nematode damage or cyst counts in the soil showed no reductions (Hanson ef al., 1988). Likewise, Kansas experiments on the effects of K and CI- on charcoal rot severity showed a lack of treatment effects (Granade et al., 1988). The major concern with CI- and soybeans is the potential for toxicity in sensitive cultivars when grown in high CI- environments that accumulate soluble salts. Considerable research has been camed out on this problem on the poorly drained soils of the Atlantic Coast Flatwoods area of Georgia (Parker and Gaines, 1987; Parker et al., 1986a,b, 1987). The major cause of the problem is a water table that remains within I or 2 m of the surface and the fact that soil moisture is lost primarily by evaporation and transpiration, which leaves CI- in the rooting zone. The

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PAUL E. FIXEN

sandy texture and high K fertilizer requirements of the soils in the region amplify the problem, which is worse in dry years than in wet years. Dramatic differences in sensitivity to C1- toxicity exists among soybean cultivars, with the sensitive cultivars being C1- accumulators and the tolerant cultivars being CI- excluders. Leaf scorch due to toxicity is just detectable at leaf concentrations of 8600 mg kg-' or seed C1- concentrations of 18,100 mg kg-l (Parker et af., 1986b). Growers have solved the problem by use of Cl--tolerant cultivars.

E. POTATOES Reduction in tuber specific gravity by CI-, especially when applied in bands, has been a major issue in potato production (Berger ef af., 1961). For this reason sulfate sources have often been the preferred form of K in potato production. Recent pot experiments have demonstrated that application of KCI as compared to K2S0, delayed tuber development, increased plant turgor pressure, and enhanced shoot growth (Beringer et af., 1990). These effects suggested a less competitive shoot sink for K,SO,-treated plants and somewhat greater assimilate translocation to tubers. Chloride application can also have beneficial effects on potato production. Oregon research has shown reductions in hollow heart with KCl compared to K2S04and accompanying increases in yield and grade (Jackson and McBride, 1986). This research also revealed that a french fry evaluation of tubers from experiments across 3 years showed that application of CI- did not affect uniformity of color or apparent "sugar ends." Other studies have demonstrated reduction in frost damage in plots receiving 225 kg KCl ha-', presumably due to the 50,000 to 70,000 mg kg-l Cllevels in plant tissues (Jackson et af., 1982). The role of CI- application in potato culture is likely dependent on a number of factors, including end use of the tubers, disease pressures and other stresses, and rate of K needed. Local research and experience on the magnitude of the various potential effects of C1- need to be considered.

F. OTHERCROPS Soils high in C1- have reportedly produced better quality flax (Linum usitatissimum L.) (Brioux and Jouis, 1938). Recent studies with five flax cultivars resulted in a significant seed yield increase of 157 kg ha-' for one cultivar and significant yield decreases of 119 and 144 kg ha-' for two other cultivars from broadcast application of 135 kg KCl ha-' (Grady et al., 1988). No consistent yield effects were measured during the second

CROP RESPONSES TO CHLORIDE

133

year of the study. However, oil content was increased from 408 g kg-I for the control to 4 12 g kg-I for the CI- treatments. Disease incidence was very low for both years. High soil nitrate levels late in the season reduce the concentration of recoverable sugar in sugar beets (Beta vufgaris L.). The uptake antagonism sometimes observed between CI- and nitrate offers a possible mechanism for reducing the negative effect of high nitrate on recoverable sugar in beets (Ludwick et al., 1980). Research in Washington has shown that C1- inhibition of nitrate uptake in sugar beets can lead to increased beet sugar percentages (James ef af., 1970a). Colorado research showed no effect of CI- fertilization on sugar concentration of irrigated beets; however, C1available to the crop from the soil and imgation water varied from 930 to 3810 kg ha-' for the sites used (Ludwick et al., 1980). Sugar beets accumulate large quantities of C1- and uptake has been highly correlated with soil C1- to a depth of 120 cm. In research conducted in the Red River Valley of central North America, C1- uptake varied among five sites and five N rates from 28 to 226 kg ha-' and 5 to 23 kg ha-' for tops and roots, respectively (Moraghan, 1987). Chloride uptake generally increased as fertilizer N rate increased. Moraghan pointed out that the redistribution of soil C1- from below 60 cm to the surface soil by the beet tops could influence the C1- relationships of subsequently planted crops. Coconut and oil palms have responded markedly to C1- fertilization on low-C1- soils, typically occurring at distances greater than 20 to 25 km from the sea (Ollagnier and Olivin, 1984; Ollagnier and Wahyuni, 1984; von Uexkull and Sanders, 1986). Nut yield increases as high as 50% have been measured. Optimum tissue C1- levels of 6000 to 7000 mg kg-l have been proposed.

IV. CHLORIDE SOURCES, LOSSES, AND APPLICATION The only form of chlorine found in nature is C1-. Although other oxidation states are possible and are important in some industrial processes, they are all unstable in the environment.

A. CHLORIDE SOURCES AND LOSSES Four basic factors determine the amount of C1- available to crops growing in well-drained soils (Goos, 1987). They are (1) soil solution C1-, (2) exchangeable C1- in the soil, (3) atmospheric deposition of C1-, including irrigation water, and (4) fertilizer or manure C1-.

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PAUL E. FIXEN

Due to the high solubility of C1- salts, most soil C1- is found in the soil solution. Although some soil minerals contain very small amounts of C1-, they generally do not contribute significantly to plant nutrition. Several studies demonstrating high correlations between soil solution C1- and C1uptake by crops will be discussed later. Acid soils with clay mineralogy dominated by 1 : 1 clays and oxides may have significant anion-exchange capacity. Significant C1- can be held in anion-exchange sites in such soils (von Uexkull, 1990). Atmospheric deposition of Cl- varies greatly with geographic location. Precipitation near ocean shores can annually contribute as much as 100 kg CI- ha-' and drop to 20 kg ha-* just 200 km inland (von Uexkull and Sanders, 1986). Atmospheric contribution in the midwestern United States and Great Plains has been reported as being only 0.6 to 1.2 kg C1- ha-' per year (Junge, 1963). Atmospheric inputs often increase near heavily industrialized areas where large quantities of coal are burned. Some imgation waters contribute substantial C1- to crops (Fixen el al., 1986c) whereas others contribute very little. Chloride in the soil parent material, in precipitation, in soil drainage, historical crop removal, and C1- applied in animal manure or fertilizers all influence the quantity of C1- found in a particular soil. In North Dakota, C1- levels are often high in soils derived from marine geological deposits, in saline soils, where a shallow water table exists, and in low spots below long slopes (Richardson et al., 1988). The mobility of C1- in soils makes leaching the primary mode of C1- loss (Schumacher and Fixen, 1989). As with any solute, actual leaching loss in individual years is dependent on several interacting factors. Therefore, persistence of C1- from fertilizer applications is difficult to predict except in extreme situations. Crop removal of C1- when only grain is harvested is typically very low and has been reported as low as 0.05% of the dry matter in wheat grain (Knowles and Watkin, 1931). Green forage, however, can remove substantial quantities of C1-, especially when growth occurs in a high-C1environment. At peak accumulation, spring wheat contained 18 and 6 1 kg C1- ha-' on sites testing low and high in CI-, respectively (Schumacher, 1988). By crop maturity, C1- in the aboveground portion of the plant had dropped to 50 and 43% of these values, respectively.

B. CHLORIDE FERTILIZERS AND APPLICATION Fertilizer source comparisons have indicated that all common C1sources are equally effective (Fixen ef al., 1986b; Mohr, 1992). Common

CROP RESPONSES TO CHLORIDE

135

fertilizer sources of CI- include KCI, NH,CI, CaCl,, and MgCI,, with approximate CI contents of 47, 66, 65, and 74%, respectively. Considerable placement flexibility appears to exist for CI- fertilizers. Comparisons of preplant broadcast, band, and early spring topdress applications for cereals have given very similar results (Fixen et al., 1986b; Mohr, 1992). Due to the potential for salt injury, optimum rates may not be possible if CI- fertilizer is banded in direct seed contact along with other fertilizers. Foliar application of Cl--containing fertilizers on winter wheat has been evaluated in the United Kingdom (Kettlewell et af., 1990). Late foliar sprays of KCI reduced head and foliar Septoria nodorurn in both years of the study, but no yield effects were measured. The authors speculated that repeated applications may be necessary to control septoria sufficiently to impact yield.

V. PREDICTING CROP RESPONSE TO CHLORIDE In 19 19, Tottingham wrote “On the whole, it appears quite possible that further investigation may lead to the development of practical rules for the use of chlorides in agriculture in such ways as to increase and improve certain crops, due account being taken of those crops injured by these salts, as well as climatic and soil conditions.” The objective of this section is to summarize current knowledge of the “practical rules” for use of CI- in crop production, including the traditional diagnostic tools of plant and soil analysis. Soil and plant analysis methods will not be discussed here. Instead, readers are referred to Fixen et af. (1988) and Johnson and Fixen ( 1990).

A. PLANTANALYSIS Chloride uptake by plants is sensitive to the amount of CI- in the soil and the amount of CI- applied in fertilizer. For example, C1- concentration in shoots of spring wheat at early heading across 12 sites varied from less than 1000 mg kg-’ to over SO00 mg kg-’, depending on soil C1- content of the site (Fixen et al., 1986a). Chloride application has increased plant CIconcentration at early heading from 2000 mg kg-I, where no CI- was applied, to 14,000 mg kg-I, where 90 kg C1- ha-’ had been applied (Fixen et af.,1987). Application of 355 kg CI- ha-’ to winter wheat increased CIconcentration of flag leaves from 1800 mg kgg’ in the check to over

136

PAUL E. FIXEN

CI (kg ha-')

.O -

i

255 *1SE

*

head emeraen

10 -

r-

-\

& n5-

0 -. 0

20

40 60 Days from Emergence

80

100

Figure 4. Seasonal chloride concentration in spring wheat at two CI- levels (Schumacher, 19881.

10,000 mg kg-' (Christensen et al., 1981). Soil CI- contents of five study sites caused the C1- content of sugar beet tops at harvest to vary from 28 to 148 kg ha-' (Moraghan, 1987). The coefficient of determination between C1- uptake by sugar beet tops plus roots and soil C1- to 120 cm was 0.79. As with many elements, plant C1- concentration varies greatly with plant age and plant part. The CI- concentration of the above-ground portion of Marshall spring wheat in a South Dakota field study (Fig. 4) generally increased with time until 1 or 2 weeks prior to heading and then declined until maturity (Schumacher, 1988). It is apparent from these data that C1concentration in the plant is more dynamic over time in high-C1- environments than where C1- supply is limited. Chloride concentration can also vary markedly among plant parts. For example, the collar regions of flag leaves of spring wheat have tested at 3000 mg C1- kg-l whereas the top one-third of the blade tested at 17,000 mg C1- kg-l (P. E. Fixen, unpublished data). Thus in any use of C1- plant analysis, plant growth stage and plant part sampled are critical considerations. In addition to soil CI- level, the amount of CI- applied in fertilizer or irrigation water, plant age, plant part, and several other factors can influence plant C1- concentrations. Interactions between CI- and N and be-

CROP RESPONSES TO ClILORIDE

137

Table I1 Suggested Chloride Critical Levels for Several Crops

Crop

Growth stage and plant part

Critical level (mg C1- kg-I)

General Potato Spring wheat Spring wheat Wheat and barley Coconut palm Coconut palm Coconut palm Oil palm

General Mature shoot Heading shoot Heading shoot Heading shoot Frond 14 Leaves Frond 14 Leaves

100 21310

I500 I500 1200-4000 2500 6000-7oooo 2500 - 45Wb 6 m

Ref. Johnson ef a/. (1957) Corbett and Gausman ( 1960) Fixen ef al. ( 1 986a) Fixen ef al. ( 1 987) Engel ef al. ( 1 992) von Uexkull ( 1972) Ollagnier and Wahyuni (1984) von Uexkull ( 1992) Ollagnier and Olivin (1984)

'Optimum levels. Critical level is defined as 2500; optimum is 4500.

tween CI- and P were discussed earlier. Relatively small differences between cultivars in C1- concentrations have been detected (Buchenau et al., I988a). Logically, one might expect differences in transpiration ratios to influence C1- uptake. Root distribution in the soil profile relative to C1distribution could also impact uptake. In spite of the many factors that influence CI- concentration in plants, it appears to be a useful predictor of the potential for response to C1- fertilization. Critical concentrations for several crops have been suggested and are summarized in Table 11. The most comprehensive summary of the relationship between plant C1concentration and yield response to C1- application was done by Engel et al. (1992). They combined data on spring wheat reported by Fixen et al. ( 1987) and on winter wheat reported by Bonczkowski ( 1 989) with unpublished data on winter wheat from R. E. Engel and on spring barley from R. J. Goos to generate Fig. 5 . Shoots were sampled from early heading to flowering and cultivars known to be nonresponsive were omitted from the summary. Three zones of differing C1- status were distinguished: low, 5 1200 mg kg-', significant response in 78% of the episodes; transition, 2 1200-4000, response in approximately 50% of episodes; and adequate, > 4000 mg kg-l, few significant responses to C1-. It is quite apparent that C1- levels needed for near maximum yield under field conditions are often considerably higher than the greenhouse value of 100 mg kg-' reported by Johnson et al. (1957) to prevent the onset of deficiency symptoms. They also are higher than the levels required for

PAUL E. FIXEN

138

110

0

2

4

6

8

10

12

Plant CI, g/kg Figure 5. Relationship between relative grain yield of cultivars of wheat and barley known to be c1- responsive and whole plant c1 concentrations in control plots of Great Plains studies. 1982- 1990 (Engel et at.. 1992).

photosynthesis (Robinson and Downton, 1984). Field requirements are likely tied to osmoregulatory functions, interactions with other nutrients, or plant reactions to various environmental stresses (discussed earlier).

B. SOILTESTING Plant CI- concentration in some cases appears to be related to the potential for response to applied CI-. Soil C1- levels have been highly correlated with plant C1- levels in several studies. Therefore, it seems logical to evaluate the potential of soil C1- level for predicting response to CI- application. Ozanne (1958) grew subterranean clover (Trqoliurn suhterruneun L.) in greenhouse pots containing two sandy loam soils that had C1- levels equivalent to 18 kg ha-' to 60 cm. After 3.5 months, acute symptoms of CIdeficiency were present for both soils and yields were reduced compared to pots in which C1- had been supplemented. Such data can serve as a reference for the situation where essentially no CI- is added in precipitation or imgation. One of the first applications of soil testing for C1- was made on potatoes by James el a/. (1970b) in the state of Washington. They observed that

CROP RESPONSES TO CHLORIDE

139

Table 111 Effect of Sampling Depth on Correlation between Soil a-Level and Plant Cl- Concentration r2 at sampling depth Crop

30 cm

60 cm

90 cm

120 crn

Ref.

Spring wheat Spring wheat Barley Sugar beets

0.6 1 0.6 I 0.58 0.59

0.69 0.95 0.80 0.53

0.39

0.25 0.20 0.60 0.79

Fixen et al. (1987) Mohr(1992) Mohr (1992) Moraghan (1987)

-

0.63

water-soluble CI- in the soil to a depth of 60 cm was a good indicator of CI- availability. The major application suggested was to aid in the interpretation of soil and tissue tests for NO,-. Levels of soil CI- measured were as low as 26 kg ha-', a level approaching what the authors speculated could result in C1- deficiency, although none was detected in these studies. Sampling depth for CI- has been evaluated by several investigators with rather predictable results when crop rooting characteristics are considered (Table 111). A depth of 60 cm produced the highest coefficient of determination ( r 2 )for spring wheat and barley in the northern Great Plains. This is consistent with results of sampling depth studies for soil nitrate in the same region and with common soil testing practices for nutrients existing in water-soluble forms in soils. Sugar beets required deeper sampling, again consistent with accepted soil sampling practice for nitrate. No sampling depth data have been located for winter wheat. However, it is possible that the optimum sampling depth for winter wheat is greater than for the spring cereals, as suggested by Engel and Grey (1 99 1). The somewhat deeper rooting patterns of winter wheat and the likelihood of significant CI- below 60 cm, especially in fallow rotations, may increase the importance of deeper sampling. Use of soil CI- as a means of monitoring the residual effects of CIfertilization was evaluated in eastern South Dakota on a loam-textured glacial till soil (Schumacher and Fixen, 1989). Both yield response to CIfertilization and residual soil C1 persisted for 2 years following application. The most extensive evaluation of soil CI- levels for prediction of response to CI- fertilization was done for spring wheat in South Dakota (Fixen et al., 1987). Trials were conducted at 36 locations across 5 years using cultivars known to be CI- responsive. A critical level of 43 kg CIha-' divided 83% of the sites into either low Cl--responsive or high CI-nonresponsive quadrants (Fig. 6). Such field performance is typical of commonly accepted soil test methods for other nutrients. However, the

PAUL E. FIXEN

w

c d

Partitioning = 83%in positive quadrants

I

0

Slgnlflcanl(O.10) w Not61 nlflcant

0

B

I

0

m 25

50

75

100

125

Soil CI to 60 cm,kg/ha Figure 6. Influence of soil C1- content on relative grain yield of spring wheat (Fixen ef al.. 1987).

calibration differs from typical data sets for N or P in that the magnitude of response is on the average more limited. As with other soil tests, soil CIlevel primarily indicates the long-term probability or frequency of response and guarantees nothing relative to individual events. Low, medium, and high categories were established as defined in Table IV. These results were used to develop a CI- fertilizer recommendation equation: CI- to apply = 67 - soil C1- to 60 cm in kg ha-’. In this work it was advised that care should be taken in extrapolating these results to other geographic areas and that local evaluation under the climatic, soil, and cultural conditions of the area should be performed. Chloride response of “Marshall” spring wheat was evaluated at five sites varying in soil CI- levels in northwestern Minnesota ( Windels ef al., 1992). No response was measured at three of the sites testing above the 43 kg ha-’ critical level shown in Fig. 6. The other two sites tested 30 and 3 1 kg ha-’ but also failed to respond significantly to Cl-. One of the two sites had very low yield levels for the area due to drought and thus likely had very limited potential to respond to any nutrient application. Spring wheat and barley CI- experiments conducted across 2 years in Manitoba resulted in 0 out of 8 wheat responses and 2 out of 8 barley

141

CROP RESPONSES TO CHLORIDE Table N

Summary of CI- Calibration Data for Responsive South Dakota Spring Wheat CultivarsD Response

Category LOW

Medium High Total a

Soil chlorideb (kg ha-‘) 5 34 35-67 >67

No. of sites

Frequency

CI-

(96)

Average‘ (kg ha-’)

requiredd (kg ha-’)

16 13 7

69 31 0

270 175 20

65 75

-

36

42

189

67

After Fixen ef al. (1987). To 60 cm depth. Average of all sites in category. Soil plus fertilizerCI- for responsive sites

‘

responses, even though many of the sites tested low in soil C1- (Mohr, 1992). However, followup studies comparing four cultivars of each crop revealed that the “Bedford” barley and “Katepwa” wheat used in the earlier studies, though very common in Manitoba, were in both cases the least responsive of the four cultivars tested.

C. OTHER FACTORSINFLUENCINGRESPONSE Response at any given level of soil CI- in a specific year Will potentially vary with several factors, including cultivar, soil C1- distribution relative to root distribution, foliar and root disease pressure, and the timing of moisture or temperature stress relative to the effect of C1- on plant develop ment. As observed for winter wheat by Engel and Grey (199 I), a given field may respond to C1- in one year and not in the next. Soil testing and plant analysis are perhaps most useful for identifying high-Cl- environments that will not respond to Cl- fertilization. When levels are low, actual response will depend on the factors mentioned earlier.

VI. SUMMARY AND FUTURE RESEARCH NEEDS Considerable progress has been made in our understanding of the role of C1- in crop production since CI- was first established as a plant essential nutrient. The potential for yield response in the field to CI- application has

142

PAUL E. FlXEN

now been well established. These responses likely involve the osmoregulatory functions of CI-, indirect effects on crop development, and interactions with diseases and other nutrients. Wheat and barley have been generally more responsive to C1- in the field than other major temperate-region crops. Corn has shown response in some situations but the frequency and magnitude of response have been limited. Both plant and soil analysis appear to be helpful in predicting CIresponse. They are most effective at identiFying situations in which the probability of C1- response is very low. It is clear that several other factors, in addition to CI- supply, influence response. These factors include crop species, cultivar, disease pressure, and the timing of crop stress relative to effects of CI- on plant development. Our ability to capitalize on the potential benefits of optimum C1- levels in crop production would be improved if several knowledge gaps could be eliminated. A few of these gaps are discussed below. Significant cultivar differences in C1- response potential appear to exist for several crops. The dynamic nature of dominant cultivars in specific regions and the multitude of cultivars used in agriculture make cultivar interactions exceedingly difficult to manage unless the fundamental cause of the interaction is understood. The fundamental genetic cause of lack of C1- response by some cultivars needs greater study to allow categorization of cultivar response type without extensive field testing of each cultivar. Recent studies indicate that increased host tolerance is an important component of at least some CI- disease interactions. The mechanism by which this is accomplished is critical information for predicting when such an effect will occur, and deserves research attention. Several studies have demonstrated that CI- can influence plant water relationships and plant development rates. A host of unresolved questions remain about the real importance of these effects in modifying the impact of drought and temperature stress on crop yield. More needs to be known concerning the effects of C1- on the foliar disease infection process. For example, uredospore germ tubes of rust must grow across the leaf surface and locate stomata in order to infect a host. Studies by Edwards and Bowling (1986) have strongly suggested that the germ tubes use pH gradients as a means of locating stomata. Because CIserves as a counterion in the transport of ions across membranes and may compete with HC0,-, OH-, or organic acids in that role, it is possible that foliar CI- concentration could impact pH gradients and thus the ease with which germ tubes locate stomata and infect the host. Additional data are needed for soil test calibration and plant analysis interpretation that are appropriate for specific geographic areas, crop species, and cultivars. The C1- responsiveness of certain major crops, such as

CROP RESPONSES TO CHLORIDE

143

alfalfa, has received little or no direct research attention. For some crops, such as winter wheat, the optimum sampling depth is uncertain. We know little about sampling requirements in fields with unusually high spatial variability in soil C1-. The potential for improving response prediction accuracy by considering interactions with N, P, and other nutrients also needs further exploration.

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REDOXCHEMISTRY OF SOILS Richmond J. Bartlett' and Bruce R. James2

' Department of Plant and Soil Science, University of Vermont, Burlington, Vermont 05405

' Department of Agronomy, University of Maryland, College Park, Maryland 20742

1. 11. 111. IV. V. V1.

VI1.

VIII.

1X.

X. XI. XII.

Introduction N a m e of the Electron Derivation of Thermodynamic Relationships for Electron Activity in Soils Kinetic Derivation of Thermodynamic Parameters for Redox Uses of pc-pH Thermodynamic Information Uses of pc-pH Diagrams A. Oxygen Species B. Nitrogen Species C. Manganese Oxide Specie; D. Iron Species E. Carbon and Sulfur Species Measurement of Oxidation- Reduction Starus of Soils A. Construction and Use of Platinum Electrodes B. Inadequacies of Platinum Electrode Potentials C. Alternative Strategies for More Accurate Measurement of Soil Redox Status Free Radicals in Redox Processes A. Formation of Free Radicals in Soils B. Behavior of Soil Free Radicals Manganese and Iron A. Living Earth Redox Scheme B. Catalytic Oxidation of Organics by Iron C. Manganese, the Transcendental Transition Metal D. Manganese and Nitrogen Transformations Soil Chromium Cycle Photochemical Redox Transformations in Soil and Water Iiumic Substances A. Involvement of Manganese in Forming Humus B. E Horizon Development C. Mechanisms and Processes in Formation of A Horizons Advance, m Apvnmny, Vol. I0 Copyright 0 I993 by Academic Press, Inc. AU rights of reproduction in any form reserved.

151

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R I C H M O N D J. BARTLETT AND BRUCE R. JAMES

X111. Wetland and Paddy Properties and Processes A. Paddies, Beaver Ponds, Bogs, Marshes, Swampland, and Poorly Drained Soils B. Redox-Related Reasons for Wetland Preservation C. Redox Interfaces XIV. Empirical Methods for Characterizing Soil Redox A. Soil Handling B. Lab Incubations C. Determination of Empirical p' D. Field Tests for Mn and Fe Oxides and Oxidizing Free Radicals E. Preparing Synthetic Amorphous Mn(1V) F. Standard Chromium Net Oxidation Test G . Soil Reducing Intensity H. Available Reducing Capacity I. Electron Demand by Iodide Oxidation J. Disrnutation of H,O, K. Interferences References

I. INTRODUCTION Why, on the hill at its crest Is the soil pale yellow or red But then, farther down, its essence is brown And why, when it's black, is it best? (Anonymous) These lines pose an oxidation -reduction (redox) question. Would that we were as adept in answering the question as the poet in asking it. Redox answers seem to beget questions more effectively than questions beget answers. Energy flow in the universe toward thermodynamic equilibrium would seem to be its natural course as surely as water flows down to the sea. Natural laws are not violated. Therefore the existences of soils and people, and sugar in the bowl on the kitchen table, are thermodynamically extremely precarious. Yet, they exist. How? They exist in the face of thermodynamic disequilibrium because progress toward equilibrium is extremely slow. Probably this is telling us that the existences of soils and people, and sugar, all are temporary, and much shorter, on the grand scale of time, than the tiniest fraction of a nanosecond. But to us, it is eternity, because we do not have the ability to see the grand scale. Perhaps our efforts in trying to understand the redox chemistry of soils will put us at peace with our thermodynamic disequilibrium. The thermodynamic data

REDOX CHEMISTRY OF SOILS

153

in Table I, in Figs. 1-6, and in Section VI help us to predict and understand some of the phenomena that are important to us in our immediate relationship to the earth and are at least in part under our management and responsibility. This article is organized so that the first sections are based on interpretations of thermodynamic data and theoretical concepts relating to soil redox characteristics, processes, and mechanisms. The discussion moves progressively from theory toward the empirical, with application of theory grounded less on theory and more on experience and experimental data. Explanation and discussion are based partly on factual results and partly on conclusions that are at the same time hypotheses that will require future testing by experimentation. The unifying goal is the understanding and prediction of soil chemical redox processes.

11. NATURE OF THE ELECTRON

I wish to give an account of some investigations which have led to the conclusion that the carriers of negative electricity are bodies, which I have called corpuscles, having a mass very much smaller than that of the atom of any known element, and are of the same character from whatever source the negative electricity may be derived. (Joseph John Thompson, discoverer of the electron, in Weaver, 1987) These words began the Physics Award Address when J. J. Thompson received the Nobel Prize in Physics in 1906 for his 1897 discovery of “corpuscles” in atoms, the first hint of the existence of particles smaller than atoms (Castellan, 1983). Later to be called electrons, these subatomic particles have defied exact characterization since their discovery, despite their central role in chemical reactions. To understand and characterize the vague nature of electron activity in soils and soil solutions, appreciation of the characteristics of electrons and closely allied protons is needed. An examination of the complementary nature of electrons and protons affirms the importance of hydrogen ion and electron activities as master variables in soils (Sillen, 1967). The hydrogen atom, composed of one proton and one electron, may be visualized and modeled as a spherical puff of cotton candy with a radius of approximately 10 cm and a proton nucleus having a diameter of 0.005 mm; an invisible fleck of unspun sugar in the center! The remaining volume of the atom is occupied by the electron: the density of the spun sugar represents the probability of finding the electron in any one location,

154

RICHMOND J. BARTLETT AND BRUCE R. JAMES

and it becomes increasingly thinner with distance away from the positively charged proton (Castellan, 1983). The radius of the H atom (0.3 A), therefore, is approximately 20,000 times that of the proton (approximately 1.5 X A). The size of a proton compared to that of an H atom also may be visualized as the size of a 0. I-pm colloidal clay particle, compared to a 2000-pm sand grain in a soil. In contrast to the large proportion of the volume of the H atom occupied by the electron, the negatively charged, wavelike “corpuscle” has only neglible mass, equal to approximately 550 pg/mol, I / I836 of the mass of the hydrogen atom ( lo6pg/mol). The tiny proton has substantial mass and persists in hydrated form in aqueous media as H30+, the hydroxonium or hydronium ion. The hydrogen atom can form the hydrogen ion only when its compounds are dissolved in media that solvate protons, so the H+ cannot exist in solid phases (Cotton and Wilkinson, 1980). The solvation enthalpy of - 26 1 kcal/mol provides the energy for bond rupture. Protons migrate rapidly between water molecules, and an individual H,O+ ion has a lifetime of approximately sec. Its concentration and activity can, therefore, be measured and understood in terms of the hydration of a cation in solution. The activity coefficient can be calculated based on a knowledge of ionic strength, temperature, and solvent characteristics, and H+ activity can be calculated or measured ( Westcott, 1978). The relatively large charge-to-size ratio of the electron prevents it from persisting in free form in aqueous systems, as does the solvated H+. The ephemeral “hydrated electron” is a powerful reducing agent with a potential of -2.7 V relative to the H+/H, reference potential of 0.0 V, and it has a half-life of < 1 msec. It reacts rapidly with second-order rate constants of lo*to loLo,near the diffusion-controlled limit (Sullivan ef al., 1976). In soil chemical calculations and theory, we consider the electron as a species, designated e-, with neglible mass.We treat it thermodynamically as a ligand, reactant, and product (Sposito, 1981). The electron is not ionic, but it is negatively charged as the camer of negative electricity (Thompson, 1923). Its activity is conceptually analogous to that of H+, but its concentration (moles/liter) is undefined. All these caveats about the electron require that we understand that electron activity in soils and natural waters be regarded as related strictly to energy functions and can be described simply as “the ability to do work,” “electrochemical potential,” and, more colloquially, “electron pressure.” The concept of H+ activity and pH also can be described in terms of thermodynamic work, dw, defined as the product of an intensive property and extensive variation, such as P d V, where P i s pressure and d V is change in volume (Stumm and Morgan, 1981 ). Similarly, electron activity may be defined as % de, where % is potential and de is change in charge of the

REDOX CHEMISTRY OF SOILS

155

system. Proton and electron activities also may be defined as chemical work, p dn, where p is chemical potential and dn is charge in moles (Sposito, 1981). In considering the sibling concepts of proton activity and “electron activity” in soils, they must not be considered twins. Recognition must be given to similarities and differences in the formulation of conceptual and operational definitions for these key variables, and such comparisons are based on the differences in the nature of the proton and electron, as described above.

111. DERIVATION OF THERMODYNAMIC RELATIONSHIPS FOR ELECTRON ACTIVITY IN SOILS Because electrons are transferred from reductants (e- donors or reducing agents) to oxidants (e- acceptors or oxidizing agents) and do not exist free in soil solution, reduction reactions must be coupled to oxidation reactions to describe complete oxidation - reduction processes (redox reactions). By convention, reduction half-reactionsare written, thermodynamic relationships are derived from them, and the log K values (where K is the equilibrium constant for the half-reaction) for the coupled reactions may be compared to predict the likelihood of a reaction occurring spontaneously as written. For example, development of reduction half-reactions to predict whether H,S could reduce FeOOH may be completed in the following manner: I . Write the oxidized and reduced species on the left and right sides of the equation, respectively, with the appropriate number of electrons per mole of oxidant written on the left side:

+ e- = Fez+ + 8e- = H2S

FeOOH

S@-

(1)

(2) The number of electrons needed for reduction is calculated from the oxidation numbers of the elements in the oxidized and reduced species (Vincent, 1985). 2. Add H,O to balance the moles of 0, usually to the right side of the equation: FeOOH S@-

+ e-

= Fez+

+ 2H20

+ 8e- = H2S + 4 H 2 0

(3) (4)

156

R I C H M O N D J. B A R T L E T T A N D BRUCE R. JAMES

3. Then add H+ to balance the moles of H, usually on the left side of the equation:

+ e- + 3H+ SO:- + 8e- + lOH+

FeOOH

+ 2H,O = H,S + 4H2O = Fez+

(5)

(6)

4. Check charge and mass balances for the equations. These equations can now be used to develop expressions for electron activity, based on equilibrium expressions and free energy data. The principles for doing this can be understood by starting with a generic reduction half-reaction: A(ox)

+ B(e-) + C(H+) = D(red) + E( H,O)

(7)

where A, B, C, D, and E are reaction coefficients for the oxidized species (ox), electron (e-), proton (H+), reduced species (red), and water (H,O), respectively. The expression for the equilibrium constant, K, is equal to

K = (red)D( H Z O ) E / ( ~ ~ (e-)B ) A ( H+)c

(8)

Taking the log of both sides of the equation, log K

+ log I / ( c ) ~ + log l/(H+)c

= log(red)D/log(ox)A

(9)

And, log K = D log(red) - A log(ox)

+ B(pe) + C(pH)

(10) where pe and pH are defined as -log of electron activity and hydrogen ion activity, respectively. The “p” notation means “power” and denotes the exponent for the mol H+/kg water. hydrogen ion activity, e.g., pH 4 is equivalent to Therefore, H+ activity equals 10-PH. In contrast, the exponent for eactivity is not lO-w, because the electron activity is not defined in terms of moles of e- per liter. Both pe and pH are analogous, though, if viewed as related to the ability of e- and H+ to do thermodynamic work. Further rearrangement of Eq. (10) yields a pe-pH relationship of the following form: [ 1/B log K - D/B log(red)

+ A/B log(ox)] - C/B(pH) = pe

( 1 1)

Equation ( 1 1 ) is the equation for a straight line with slope C/B and the intercept is the terms in square brackets. It is clear that the intercept is a function of log K for the half-reaction and the activities of the oxidized and reduced species.

REDOX CHEMISTRY OF SOILS

157

For a one-electron transfer ( B = 1) coupled with one-proton consump tion (C = I ) , and when D = A and (red) = (ox), pe

+ pH = log K

(12)

And at pH = 0 ( 1 M H+ activity), (13) pe = log K Knowledge of pe and pH is pertinent to describing the equilibrium condition of a soil as defined by the master variables, pe and pH (Lindsay, 1979). The concepts of electron activity and hydrogen ion activity are closely coupled and cannot be separated in assessing the oxidationreduction status of a soil system. To relate the concept of log K to the Gibbs free energy change (AG,") for a given half-reaction in the soil, the following expressions are pertinent: AG,"= -RTln K (13) where AGIO is Gibbs free energy of reaction under standard conditions (298.15 K and 100 kPa), R is the universal gas law constant (0.001987 kcal/mol/K), and T is absolute temperature (298. I5 K). Converting to log,, (In K = 2.303 log K ) yields AG,"/- I .364 = log K

(14) Therefore, log K may be estimated simply from knowledge of free energies of formation of H,O, red, and ox, because those of H+ and e- are zero, by convention. To relate log K directly to pe as defined by Eq. ( I I ) for a one-electron transfer, log K values must be divided by B. Log K values also are related to measured electrochemical potentials, 8,, according to the following expressions: AGIO= - n98,

(15)

where n is the number of e- transferred/mole and 9 is the Faraday constant (23.1 kcal/V/equivalent). Because both Eqs. ( 1 5) and ( 1 3) are expressions for AG,", -n98,

= -RTln

K

And,

RT 2.303 log K = 0.059 log K n9

ifh =

If n

=

1 and (red) = (ox), pe

+ pH = log K [Eq. (12)], and

(16)

158

R I C H M O N D J. BARTLETT A N D BRUCE R. JAMES

or, (8,/0.059) - pH = pe

(19)

Therefore, calculating and interpreting 8, values rigorously requires a knowledge of pH of the soil/water system. Equation ( 19)also demonstrates the coupling of H+ and e- reactions in redox processes. Applying these principles to the H,S-FeOOH problem above [Eqs. ( 5 ) and (6)], the following pe-pH expressions can be derived: For FeOOH - Fez+, pe = log KFe- log(Fs+)- 3pH

(20)

where activity of FeOOH is assumed to be 1, an assumption that may not be valid for redox processes. For SO, - H,S, pe = (1/8)log Ks- (1/8)log pHS/(so4)- (5/4)pH

(21)

where KFeand Ks are the equilibrium constants for the FeOOH and SO, reduction half-reactions. Calculating log K values and substituting into Eqs. (20) and (21) yields pe = 13.0 - log(Fe2+)- 3pH

(22)

pe = 5.2 I - (1/8)log PHS/(SO,) - (5/4)pH

(23)

and where PHSis partial pressure of H,S in the soil atmosphere. At defined activities for the ions and H,S, pe may be calculated, and if pe for Fe reduction is greater than pe for SO, reduction, then FeOOH will be reduced and H,S will be oxidized at equilibrium. The same result will be obtained if, after subtracting the log K value for S from that for Fe, a positive answer is obtained.

IV. KINETIC DERIVATION OF THERMODYNAMIC PARAMETERS FOR REDOX This thermodynamic derivation of log K and its relationship to pe and pH of soils is based on free energy of formation data for oxidants and reductants, and it is not related to reaction mechanisms or rates. An alternate procedure to obtain log K values is to use a kinetic approach

REDOX CHEMISTRY OF SOILS

1 S9

suggested by Sparks (1985) for cation exchange and by Harter and Smith ( 1981) for adsorption processes. This approach has been little used in redox soil chemistry, probably due to difficulties in obtaining rate coefficients for many electron transfer processes in soils. The application of such a p proaches should, however, be appropriate for certain, reversible redox reactions in soils, especially in situations where metastability and lack of chemical equilibrium prevail, or when inaccurate free energy of formation data are used for oxidants and reductants. The equilibrium constant, K, for redox equilibria can be estimated from the ratio of forward and reverse rate coefficients for a given reversible reaction in soils:

K = kflk (24) where k, and k, are the rate constants for forward and reverse reactions, respectively. The reliability of such an approach depends on the true reversibility of the redox reactions, an assumption that is not true for many redox couples, especially those mediated or catalyzed by microbial enzymes. The Gibbs free energy for the reaction then could be calculated by substituting In K into Eq. ( 13). Preparation of Arrhenius plots of the rate coefficientsas a function of 1/T permits estimation of activation energies for the forward and reverse reactions, and enthalpies for the reactions can be calculated as AH a = E:- E:

(25)

where AH' is enthalpy for the redox reaction and E: and E: are the activation energies for the forward and reverse reactions. With a knowledge of AG and AH ', entropy of the reaction can be calculated as

AS'

= AHo - AG"/T

(26) Because the e- and H+ are key reactants, products, and ligands in the thermodynamic sense, a refined knowledge of mechanisms for particular redox reactions is needed because of our current limitations to understanding how similar or different e- and H+ reactions are in soils and natural waters. A knowledge of thermodynamic stability for a redox system does not necessarily predict kinetic lability, a concept directly pertinent to reactivity of different types of complexes (Cotton and Wilkinson, 1980). The kinetic lability of redox processes in soils has not been compared systematically with predictions of stability based on thermodynamic data. Such information could affirm the reliability of using thermodynamic data to predict bioavailability of nutrients and pollutants, and to estimate true reactivity of electron donors and acceptors in soil environments. The

160

R I C H M O N D J. BARTLETT AND BRUCE R. JAMES

relative rate of organic ligand exchange and electron transfer is an example of a problem pertinent to plant nutrition and to environmental quality concerns, and one that is little-studied in soils.

V. USES OF P-PH THERMODYNAMIC INFORMATION Values of log K derived from Gibbs free energy data or kinetic evaluations of redox reactions provide powerful tools for predicting the likelihood of a reduction reaction coupling with an oxidation reaction to allow the spontaneous transfer of electrons from reductant to oxidant. Because soils are only metastable and highly heterogeneous in nature, such predictive capability is necessary to formulate hypotheses for many processes that may occur in the field, even if they require catalysis or other coupled reactions to occur at ambient temperatures and pressures of soil- waterplant systems. Table I provides a listing of reduction half-reactions for species of N, 0, Mn, Fe, S,and C, various pollutants sensitive to redox conditions in soils, and several reactions pertinent to the analysis or characterization of redox conditions. Within groups, the half-reactions are arranged in descending order of log K values, calculated as described above. These values are pe values at pH 0, when activities of oxidant and reductant are 1, and may be considered standard reference pe values for the reactions. The pe values listed at pH 5 and 7 are calculated to represent typical activities and partial pressures of gases in soil environments. Higher log K or pe values indicate greater “ease of reduction” of an oxidant (left side of equation) to its reduced form than do lower values. This means that for predictive purposes, an oxidant in a particular reduction half-reaction is able to oxidize the reductant in another half-reaction with a lower pe, at a specified pH. For example, oxides of Mn (111, IV) would be expected to oxidize Cr(OH), to Cr(V1) at pH 5 because the range of pe values for reduction of Mn ( 12.8 - 16.7) is greater than that for Cr( VI) reduction (10.9). This has been demonstrated to occur in the pH range of 4-7 in most field-moist soils containing oxides of Mn(II1, IV) (Bartlett and James, 1979; James and Bartlett, 1983). Conversely, these oxides would not be expected to oxidize N, to N,O (pe at pH 5 = 22.9). Even though the log K or pe for one reduction half-reaction is less than that for a second half-reaction, the reduced form in the first reaction may still be oxidized by the oxidized species in the second half-reaction. For example, the pe at pH 5 for reduction of CO, to C,H,,O, is - 5.9 and that for reduction of 0, to H,O is 15.6. Based on this difference, one would

REDOX CHEMISTRY OF SOILS

161

predict that reduction of 0, to H,O would be coupled to oxidation of C6H1,06 to CO,, and coupling oxidation of H,O to 0, with reduction of CO, to C6H1206would not be thermodynamically possible. In fact, both respiration (the predicted reaction) and photosynthesis (the second, “impossible” reaction) occur together, and the balance of the two is responsible for the existence of the aerobic lifestyle and the persistence of organic matter in soils. Photosynthesis is made possible via a complex series of coupled reactions that make an overall thermodynamically impossible reaction occur rapidly in sunlight. Similarly, a reaction predicted to be thermodynamically possible may not occur under natural conditions. The pe for NO, reduction to N, at pH 5 (14.3) is greater than that for HCrO,, reduction to Cr(OH), (10.9), but this NO, oxidation of Cr( 111) has not been demonstrated in soils or plants, probably because the reduction of NO, requires enzymatic processes to lower the energy of activation at such a high pH. The order of log K values for reduction-half reactions also has been used to predict the sequence of reduction reactions camed out by respiring soil microorganisms following saturation of a soil (Ponnamperuma, 1972). The descending order of preference (pe) for the electron acceptors at pH 7 (proportional to free energy derived from the reduction) is 02/H,0 ( 13.6), NOJN, (1 1.9), MnO,/MnZ+ (8.8), Fe(OH),/Fe2+(- 1.2), SO,/H,S (- 3 . 9 , and C02/CH,, (-4.1). Heterotrophic bacteria are using organic compounds as the electron donors (energy source) in their respiration to produce CO, or organic acids (pe range at pH 7 of - 8.7 to - 3.1 ), so most of the organic compounds can be used throughout the reduction sequence following depletion of atmospheric 0,. The predicted pe values at a given pH are determined by chosen activities of reductant and oxidant, and by the values of Gibbs free energy of formation (AGa used to calculate log K values. As shown in Table 11, the sensitivity of calculated pe due to changes or error in log K and activities varies considerably among reduction half-reactions. Predicted pe values for O,, NO,, CO, , and SO,, reduction changed less than 0.5 units in response M . In contrast, an error of only 10 to decreasing activity from lo-‘ to kcal/mol in the AG,“resultedin changes in the calculated pe values of 3.6, 1.5, 0.3, and 0.9, respectively, for the above half-reactions. This result indicates that wide variation and error in estimates of activities will have a smaller effect on pe than will errors in free energy of formation data, especially for 0, and NO, reduction reactions. In contrast to these half-reactions involving the reduction or oxidation of gases, those involving oxides and oxyhydroxides of Mn and Fe are subject to greater error ( 1 .O to 3.0 units) in estimation of pe due to variation in and M. Similar to the gas activity of Fe2+ or Mn2+ between

Table I Selected Reduction Half-Reactions Pertinent to Soil, Natural Water, Plant,and Microbial Systems

Reaction

-

Nitrogen species iN,O e H+ jN, jH,) NO e- H+ = j N 2 0 tH,O +NOT C fH+ = tN20 fHtO fNO; e- tH+ = &N, fH,O N q e 2H+ = NO HzO +NO; e- fH+= tN,O )H,O &NOT C 3H+ = fNH: jHiO +NOT h fH+ +NH: + H i 0 tNOT e H+ = 4NOT +HzO &NO; C )H+ iNH2OH +H20 &N2 e- fH+= JNH:

+ + + + + + + + + + + + + + + + + + + + + +

+ +

+ + + + + + +

-

+

Oxygen species j O 3 C H+=jO2 +HZ0 OH. e -OHOr e 2H+ Hz02 +HzOi C H+ = H i 0 C H+-+H,O j O 2 C H + = jH202 0 2

+ + + + + + + + + + + + e- - 0;

-

+

Sulfur species t S 0 - e fH+= tH,S jH,O +so,- Q 2H+ = jS02+ HzO

+ + + +

+

Iron lad mpa%lwse compollllds jMn,O, e 4H+ = fMn2+ 2H20 jMn203 e 3H+ = M d + f H 2 0 Mn3+ e = Mn2+ yMnOOH e 3H+ = M d + 2H,O 0.62Mn0,,1 e- 2.2H+ = 0.62Mn2+ 1.1H20 jFe,(OH), e 4H+ = jFG+ 4H20 jMn02 e 2H+- jMnz+ H,O [Mn3+(PO,),I3- e- = (Mn'+(FQ,)l14Fe(OH)Z h 2H+ = Fe'+ 2H20 +Fe,04 e 4H+ = f F P 2HI0 Mn02 e 4H+ = Mn3+ 2H20 Fe(OH), C 3H+ = F g + 3Hi0 Fe(OHP+ C H+ = FS+ HzO iFe20, e-+ 3H+ = FG+ jH,O FeOOH e 3H+ = FZ+ 2HzO Fe3+ e = Fe'+ phcnanthroline Fe'+ e- = FG+ Fe3+ e- = Fe'+acetate Fe'+ e = F$+ malonate Fe'+ e- = F P salicylate Fe)+ e- = FG+ hemoglobin Fe'+ e- = Fe2+cyl b, (plants) Fe'+ e- = F P oxalate

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ +

+

+ + + +

+

+

+

+

+

162

29.8 26.8 23.6 21.1 19.8 18.9 15.1 14.9 14.1 11.3 4.6

22.9 19.8 15.1 14.3 9.8 12.1 8.4 8.6 9. I 5.4 -0.7

20.9 17.8 12.1 11.9 5.8 9.6 5.7 6. I 7. I 3. I -3.3

35. I 33.6 32.6 30.0 20.8 11.6 -9.5

28.4 33.6 22.6 23.0 15.6 8.2 -6.2

26.4 33.6 18.6 21.0 13.6 6.2 -6.2

5.2 2.9

- 1.0

- 3.5 -11.1

30.7 25.7 25.5 25.4 22. I 21.9 20.8 20.7 20.2 17.8 16.5 15.8 15.2 13.4 13.0 18.0 13.0

-

-7.1 16.7 14.7 25.5 14.4 13.4 7.9 12.8 20.7 10.2 3.9 0.54 4.8 10.2 2.4 2.0

-E

13.0 5.8 4.4 (pH 4) 4.4 (pH 4)

-

-

8.7 8.7 25.5 8.4 8.9 -0.1 8.8 20.7 6.2 -4.1 -7.5 - 1.2 8.2 -3.6 -4.0

-

13.0

-

2.4 0.68 0.034 (conrinued)

Table I (Continued) peb

Reaction

+ +

FZ+ e- = FP+ pyrophosphatt Fe3+ e- = FP+peroxidase Fe3++ e- = Fe? ferredoxin (spinach) fKFe,(SO,h(OH), Q 2H+ F 9 + + 2 H 2 0+ fS@[Fe(CN),]’- + F = [Fe(CN),]‘-

-

+ +

Carbon species JCHJOH C H+ = JCH, JH20 toquinone F H+ = *phenol tpquinone F H+ = thydroquinone &H,,O6 c H+ fC2HSOH iH2O Pyruvate + F H+ = lactate KO, e- H+ = K H , f H 2 0 jCH,O F + H+ = jCH,OH tHCOOH c H+ KH2O + jH2O KO, c H+ = hC,jH,,O6 fH2O +deasc + e- H+ = jasc KO2 + C + H+= jCH2O + tH2O KO2 c + H+ = tHCOOH

+ + + + + + + + + + + + + + + + + +

+

-

+

+

+

Pollutant/nutrient group CO)+ e- = cd+ JNiO, F 2H+ = /Ni2+ HzO Puo; e- = Puo2 2H+= tPff + H I 0 jPbOz PuO2 6 4H+ = Pu3+ 2H20 +HCrO; C /H+ = KNOH), + jH,O j A ~ ( 3 - F 2H+ tAsOi H2O Hg2+ e- = fH& +MOO- F 2H+ = J M d Z H 2 0 jS&6 H+ jw- 4H2O f S e 0 - e- fH+ = iSe fH2O f S e 0 - tH+ = tH2& tH,O tV0; e- /H,O+ = fV(OH), Cu’+ e- = c u + PuO, F 3H+ = PuOH2+ H I 0

+ + + + + + + + + + + + + + + + + + + + + + + + +

+ + + +

-

+

+

+

+

+

M y t i c a l couples Ce02 e- 4H+ = Ce3+ 2H20 jCI0- 6 H+ = jC- + H i 0 H a 0 + C + j C l 2 + H2O

+ + + + + + Kl, + F = a&lor + e- + H+ = il- + t H 2 0 )F?(OH), + e- + H+ = tPt + H 2 0 +I2 + e- = ItHg2C12+ F = Hg + C T C + H+ = +H2 jPts + e - + H + = +Pt + t H 2 0

iog~4

-2.4

-

+ +K+

8.9

-

9.9

-

4.4

-

2.9 2. I I.5 -0.21

I .o

-1.2 - 1.9 30.6 29.8 26.0 24.8 9.9 18.9 16.5 15.4 15.0 14.9 14.8 7.62 6.9 2.6 2.9 47.6 29.0 27.6 23.0 18.6 16.6 9. I 4.5 0 -5.0

~ H S

6.9

-

4.9

-

0.I -2.1 -2.9 - 3.5 -5.9 -3.5 -6.1 -6.7 30.6 21.8 22.0 16.8 -6.1 10.9 6.5 13.4 3.0 9.9 6.3

p ~ 7

-4.6 -7.3 2.9 6. I 2.9 5.9 4.7 - 1.9 -3.1 -4.1 -4.9 - 5.5 -7.9 - 5.5 -8.1 -8.7

2.4 2.6 -8.1

30.6 17.8 22.0 12.8 -14.1 8.2 2.5 13.4 - 1.0 7.9 3.3 -1.7 I .4 2.6 -14.1

31.6 24.0 20.6 25.0 13.6 11.6

23.6 22.0 18.6 25.0 11.6 9.6

11.1

11.1

3.9

3.9 -7 - 12.0

1.o

-5 - 10.0

Calculated for reaction as written according to Eq. (14). Free energy of formation data were taken from Lindsay (1979) as a primary source, and when not available from that sou-, from Gamls and Christ ( I 965) and Loach ( I 976). *Calculated Using tabulated log K values, reductant and oxidant = IO-‘ Msoluble ions and molecules, activities of solid phases = 1; partial pressures for gases that are pertinent to soils: IO-, atrn for trace gases, 0.21 atm for 0,. 0.78 for N,, and 0.00032 for CO,. Values not listed by h h ( I 976). 163

164

R I C H M O N D J. B A R T L E T T A N D BRUCE R. JAMES Table I1

Sensitivity of Calculated pe at pH 0 to Variation in ACj nod Activities of Reductant or Oxidant in Selected Reduetion HIII-ReactionsPertinent to Soils

Column

-Log activity

Couple

AGj

ox

Red

ox

Red

Pe

0.68 2.68 0.68 4 6 4 0 0 0 0 0 0 0 0 0 3.5 3.5 3.5 0 0 0 0 0 0 4 6 4

0 0 0 0.1 1 0.11 0.11 4 6 4 4 6 4 4 6 4 4 6 4 4 6 4 4 6 4 4 4 4

0 0 0

- 56.70 - 56.70

- 26.43

0 0 0

20.6 1 20.11 24.20 20.30 19.90

-26.43 - 36.43 - 306.20 - 306.20 -3 16.20 -111.10 -111.10 - 121.10 - 133.10 - 133.10 - 143.10 -94.26 -94.26 -94.26 - 170.40 - 170.40 - 180.40 - 177.10 - 177.10 - 187.10 - 177.95 - 177.95 - 167.95

-66.70

- 54.40 - 54.40 - 54.40 - 54.40

- 54.40 - 54.40 - 54.40 - 54.40 - 54.40

-2 18.58 - 2 18.58 -228.58 -21.80 -21.80 -21.80 -21.80 -21.80 -21.80 -8.02 - 8.02 - 8.02

18.80

36.70 39.70 33.00 22.80 23.80 19.10 29.40 3 I .40 22.10 -0.91 -0.83 -0.60 19.80 2 I .80 12.40 17.40 19.40 13.70 5.20 5.50 6.10

Ape‘ -0.50 3.59 -0.40 - 1.50

3.00 -3.70 1 .00 -3.70

2 .OO -7.30 0.08 0.3 1

2.00 -7.40 2.00 -3.70 0.30 0.90

‘Change in calculated pe resulting from change in activity of oxidant or reductant (column I or 2) or resulting from use of A G j 10 kcal/mol different from the published value (first row, column 3 or 4).

group, pe values for the oxide group showed considerable change due to error in free energy of formation, ranging from 3.7 to 7.4 pe units. Differences between MnOOH, Mn,O,, and MnO, also were large, and similar differenceswere found for the Fe oxides. This indicates that correct identification of Mn oxide mineralogy significantly affects pe- pH relationships

REDOX CHEMISTRY OF SOILS

16.5

and predicted energy changes associated with these reductions in soils at certain pH values. These observations of the sensitivity of predicted pe values due to variation in activities and due to error in free energy of formation indicate that exact pe values for the reduction of a particular oxidant are difficult to obtain, and ranges of values may be more accurate. The use of such ranges also recognizes the heterogeneity of soii minerals, gaseous composition, and ion activity in space and time. Values for log K for reduction half-reactions are especially sensitive to changes in activity of oxidant or reductant if oxygen atoms are transferred from the oxidant to H20, e.g., in the reduction of MnOOH to Ma”, in contrast to the reduction of Mn3+ to Mn2+. The formation of H20 is favorable because it increases entropy of aqueous systems (Cotton and Wilkinson, 1980)and thereby tends to lower calculated AGro.Water molecules formed as a product of a reduction half-reaction are balanced by H+ on the left side of the equation, resulting in a larger ratio of H+ to econsumed. This increases the slope of the pe-pH relationship, resulting in a greater error in the estimation of pe due to an error in pH measurement.

VI. USES OF p - p H DIAGRAMS Individual pe-pH relationships can be defined by the equation for a straight line [Eq. ( 1 l)] in which log A’ and the activities of the oxidant and reductant determine the y intercept, and the ratio of H+ to e- consumed determines the slope. When such lines are plotted together, they predict whether the oxidation - reduction reaction can occur “spontaneously,” that is, with AG;
A. OXYGEN SPECIES Whereas the pe for reduction of 0, to H,O ranges from 20.8 at pH 0 to 13.6 at pH 7 (Fig. 1 and Table I), the intermediates associated with one-electron transfers show a wide fluctuation in their oxidizing power, a property of 0, that is pertinent to understanding the transition in soils from “aerobic” to “anaerobic” conditions. Anaerobic respiratory enzymes are produced when Po, reaches approximately 1% of atmospheric levels (0.2 kPa or 0.002 1 atm). The data in Table I1 also indicate that the pe for

166

R I C H M O N D J. BARTLETT AND BRUCE R. JAMES

a,

a

PH Figure 1. A pe-pH diagram for 0, reduction reactions, including partially reduced intermediates:superoxide (Oi), hydroxyl free radical (OH .), and hydrogen peroxide (H202). Reduction of 0,to O2is also included for comparison with 0, reduction reactions. Activity of oxygencontaining ionic or molecular species is 1 6 ‘ M.except that Pa is 2 1 Wa.

reduction of 0, is relatively insensitive to the 0, partial pressure in this range. and the hydroxyl free radical (OH-)are the most powerful Ozone (0,) oxidants among the oxygen species (Table I), and the latter may be formed during stepwise, four-electron reduction of 0, to Oy, H,O,, and H,O (Fridovich, 1978). The low and high positions of the pe-pH lines for superoxide (0;) reduction to H,O, and for superoxide oxidation to 0, indicate that both a powerful oxidlzing and reducing agent is formed in the first step of reduction of 0,. The enzyme superoxide dismutase scavenges 0; in living cells using 0, as the terminal electron acceptor, but relatively little is known about its reactivity in biological and chemical processes in soils that may be pertinent to our understanding of the formation of highly reduced components (e.g., soil organic matter) and highly oxidized species (e.g., NO;) that coexist in soil at chemical equilibrium or quasi-equilibrium.

B. NITROGEN SPECIES Most reduction reactions of N species (Fig. 2 and Table I) are not reversible, and therefore are not well defined by thermodynamic pe-pH

REDOX CHEMISTRY OF SOILS

167

a,

a

0

1

2

3

4

5

6

7

8

9 10

PH Figure 2. A pe-pH diagram for nitrogen species half-reactions, including intermediates formed or consumed in denitrification and dinitrogen fixation. Partial pressures of gaseous intermediates are 0.01 kPa, except N, is 78 kPa; activity for NO;, NO:, and NH:is lo-' M.

relationships. The series of half reactions composing the process of denitrification, though, is instructive in that it identifies the wide range of pe for reduction of each of the intermediates believed to form in the sequence of electron acceptors used by microbes:

NO; Step

- NO?

(1)

(2)

NO

+

(3)

N,O

-

N,

(4)

Step 1 of the sequence occurs at pe values less than those for reduction to 0, to H,O, whereas those for steps 2, 3, and 4 are increasingly higher. The overall reduction of NO; to N,, though, is almost identical to that for the 0 2 / H , 0 couple. The overlap of pe values for the 0, and NO; reduction intermediates indicates that denitrification and aerobic respiration may occur at the same time under certain conditions when organic C is used as the electron donor. They may not be mutually exclusive, as predicted from log K values for the overall reactions, 0,to H,O and NO; to N, .

C. MANGANESE OXIDE SPECIES Manganese exists in soils in the 11, 111, and IV valence states, and the latter two are most stable as oxides or oxyhydroxides. Trivalent Mn may

168

R I C H M O N D J. B A R T L E T T A N D BRUCE R. JAMES

-15

0

1

2

3

4

5

6

7

8

9 10

PH Figure 3. A pe-pH diagram for Mn oxides, trivalent Mn ions, and superoxide. Ion activities and H 2 0 2concentrations are lo-' M,Pa is 21 Wa.

exist as a cation, especially if stabilized by ligands, such as pyrophosphate or citrate. The pe-pH relationships of Fig. 3 predict that different valences of Mn in Mn,O,, MnOOH, and Mn02 affect the pe at which Mn2+would be expected to form at pH < 7, but they are all similar at pH values near 7. The Mn3+/Mn2+line indicates that at approximately pH 4, Mnw is a powerful oxidant similar to 0;and Mn,O, (Fig. 3) if in equilibrium with Mn2+.At pH values near 6.5, Mnw in equilibrium with MnO, is a powerful reductant, similar to H2and 0;. This powerful oxidizing ability of Mn3+ in equilibrium with Mn2+ may be pertinent to anaerobic soils that are exposed to 0,, and in which Mn2+ is oxidizing to form Mn(II1, IV) oxides via Mn3+. In oxidized soils containing MnO, ,flooding and the process of becoming reduced may produce Mn3+,which is a powerful reducing agent. The trivalent Mn species may be ephemeral intermediates in such processes at redox interfaces, such as in the rhizosphere of plant roots or between soil water and groundwater. As the metal analog of superoxide in its oxidizing/reducing power and as a free radical, Mn3+is appropriately referred to as the supermanganese ion. Because many Mn(II1, IV) oxides are nonstoichiometric and no compound with the exact composition of MnO, is known (Arndt, 1981), predictions of their redox properties as a function of mineralogy or valence

REDOX CHEMISTRY OF SOILS

169

PH Figure 4. A pe-pH diagram for Mn3+, MnO,, and Mn,O,; as compared with reduction values between pH 5 and 7 for Co, Cr, Se, As, V, and Pu.Activity for ionic species is lo-' M.

in heterogeneous soils may be hard to formulate. Despite the uncertainty of thermodynamic predictions for the redox behavior of Mn, the chemistry of this element is pertinent to a number of processes governing speciation and valence state of trace elements and pollutants found in soils. The pe-pH data indicate that oxides of Mn may oxidize Pu(1II) to Pu(IV), V(II1) to V(V), As(II1) to As(V), Se(IV) to Se(VI), N(II1) to N( V), and Cr( 111) to Cr( VI), because the pe for each of these couples falls below that for Mn oxides (Fig. 4 and Table I). The oxidations of Pu(III), As( 111), Se(IV), N( 111), and Cr( 111) all have been demonstrated to occur in soils containing Mn oxides or by synthetic Mn oxides (Amacher and Baker, 1982; Bartlett and James, 1979; Bartlett, 1981b; Blaylock and James, 1992; Moore ef af.,1990). The instability of Mn3+and its ability to dismutate, as do H202and OF, mean that kinetic constraints may be particularly important in understanding the redox behavior of Mn in soils undergoing transitions between anaerobic and aerobic conditions. The kinetic lability of these species is poorly understood and new knowledge could contribute significantly to predictions of bioavailability and toxicity of numerous plant nutrients and pollutants in a range of types of soils from rice paddies and wetlands to well-drained agricultural and forest soils.

I70

RICHMOND J. BARTLETT AND BRUCE R. JAMES

aJ

Q

0

1

2

3

4

5

6

7

8

9 10

PH Figure 5. A pe-pH diagram for Fe(II1) oxides and dihydroxy species, Few/Fe2+in the presence or absence of five organic complexing Ligands. and the HCrO;/Cr(OH), redox couple. Activity of ionic species is IO-‘ M.

D. IRON SPECIES Predictions of the redox behavior of Fe( 11) and Fe( 111) species indicate that it falls below most Mn oxides species (lower pe values and less free energy released per equivalent upon reduction), but intermediate hydrolysis products, such as Fe(OH)l, theoretically can oxidize Cr(111) to Cr( VI) at pH <4 (Fig. 5 and Table I). In addition, complexation of Fe2+ by organic ligands lowers the pe values at which Fe3+is converted to Fe2+and the redox couples are similar to those of Fe( 111) oxides in the pH range of 5 to 7. This phenomenon suggests that Fe2+ becomes a more powerful reducing agent when complexed, and may explain the ability of Fe to act as a cofactor in enzymes involved in redox processes, such as peroxidases and superoxide dismutases. These enzymes reduce or dismutate H20, and Oy. The application of such concepts to abiotic redox processes in soils remains a key area for future research.

E. CARBON AND SULFUR SPECIES Reduced forms of C and S are normally viewed as reductants in soils, either in chemical or biological processes. Thermodynamic predictions

171

REDOX CHEMISTRY OF SOILS

pyruvate/lactate

A dehydroascorbate/ascorbate

25

- - _- _- _

- _- - _- - _

-5 1-

0

1

2

3

4

5

6

7

8

9 10

PH Figure 6. A pe-pH diagram for S, C, and Se species. Ion activity and molecular concentrations are lo-' M and Pa is 0.032 kPa.

support this idea for carbohydrates produced in photosynthesis, methane from methanogenesis, and hydrogen sulfide from reduction of SO, (Fig. 6 and Table I). The reduction reaction of 0- and pquinone suggest that these compounds may be reduced at higher pe values than are C02and SO,. The low position of these lines, however, coincides with the Mn02/Mn3+couple at pH 7, suggesting that Mn3+ may act as a reducing agent for certain organic species in near-neutral soils. Coupling of reduction of the organic with oxidation of Mn may result in formation of free radical species. This is pertinent to understanding the formation and persistence of organic matter in high-pH soils that may contain reactive forms of Mn oxides. Reactions of H2S and H,Se are predicted to be similar with respect to SO, and S e O , formation (Fig. 6). Sulfidization has been studied as a mechanism for precipitation of Fe and other heavy metals in tidal marshes and natural or constructed wetlands (Rabenhorst and James, 1992; Rabenhorst et al., 1992; Hines et al., 1989; Kittrick et al., 1982), and selenide formation may result in analogous products in sulfidic soils (Masscheleyn et al., 1991). Although SeO, and SO, are similar chemically, the oxidation of SeO, to SeO, is predicted to occur at higher pe values than is the oxidation of H2S to SO, (Fig. 6 and Table I). Blaylock and James (1992) observed that Mn oxides in soils or in pure form will oxidize SeO, to SeO,, as predicted by thermodynamics (Fig. 4). They also observed that adding reducing, phe-

172

RICHMOND J. BARTLETT AND BRUCE R. JAMES

nolic acids, such gallic and ascorbic acids, actually enhanced this oxidation. They hypothesized that partial reduction of MnO, in soils converted the Mn oxide into a Mn(II1) form that was a more powerful oxidant for SeO, than was MnO,. Such a hypothesis is supported by the relative oxidizing power of MnO,, MnOOH, and Mn,O,, where the latter two oxides contain Mn( 111) (Fig. 3).

VII. MEASUREMENT OF OXIDATION- REDUCTION STATUS OF SOILS The most common method for quantifying electron activity of soils and natural waters is to measure the potential difference between a Pt indicator electrode and a calomel or Ag/AgCl reference electrode, both connected to a voltmeter of pH meter (Rowell, 1981 ;Bricker, 1982). In this method, the Pt electrode is presumed to be inert and to not react chemically while coming into equilibrium with electroactive species in soil solution and on soil colloids. Recent advances in evaluations of the reliability of this potentiometric measurement have generally resulted in it being considered unreliable for accurate assessments of redox status of soils, especially aerobic ones (Bartlett, 1981a). Other methods that employ analyses of soil solution analytes indicative of redox status, along with thermodynamic half-reactions, as discussed above, may prove more reliable for calculating pe ranges for aerobic and anaerobic soil systems.

A. CONSTRUCTION AND USEOF PLATINUM ELECTRODES Platinum and suitable reference electrodes are relatively easy and inexpensive to construct (Mueller et al., 1985; Farrell er al., 1991), but the measurement technique may significantly alter measured voltages; several aspects of electrode use and misuse with respect to the reliability of recorded voltages for natural systems have been described (Bartlett, 198la; Bricker, 1982; Matia et al., 1991).

B. INADEQUACIESOF PLATINUM ELECTRODE POTENTIALS Assessing “electron activity” in soils relates strictly to an evaluation of the ability of the electron to be transferred, to do thermodynamic work, and not to its concentration in soil solution, as can be defined for H+. Because of the nature of the electron and its differences from the H+, a

REDOX CHEMISTRY OF SOILS

173

number of caveats must be described and recognized when evaluating Pt electrode potentials. 1. Dissolved Oxygen Status

A stable potential can be obtained for a Pt reference electrode pair immersed in an oxygenated soil suspension, but it is unreliable as a measure of dissolved oxygen status (Bricker, 1982; Stumm and Morgan, 198l ). The Pt surface may react with 0, to form ROH, which develops a potential with elemental Pt with a pe of 9.6 at pH 7 (Table I). In addition, the measurement may not be that of the 0 2 - H 2 0 couple, but may be responding to 0, reduction intermediates, such as H,02and 0;-(Bricker, 1982). In addition, predicted pe values are relatively insensitive to changes in dissolved O2 between 0.21 and 0.0021 atm (Table 11), the range of 0, partial pressures in which aerobic respiration occurs (Russell, 1973). For these reasons, Pt electrode potentials cannot be used reliably as a measure of redox status for aerobic soils, but empirical values for pe (EMpe) may be obtained for comparison purposes (Bartlett, 198la). Although more faith is placed in measurements of soil pH, it also should be considered an empirical measurement because of uncertainty about the form of the hydrogen ion in colloidal environments and about the behavior of the glass electrode in such systems. For these reasons, both pe and pH measured with electrodes in soils may be very uncertain for accurate descriptions of the redox status of soil environments containing air-filled pores. 2. Irreversibility of Redox Couples

Many of the important redox processes involving C, H, N, 0, and S (the “light” elements, relative to the “heavy” metals) are irreversible in the thermodynamic sense, and nonelectroactive gases and molecules may be consumed or formed. As a result, potentials generated by redox couples for these elements are difficult to obtain and interpret using a Pt electrode. In addition, many of these reactions do not reach true chemical equilibrium, and activities measured in soil solution may be kinetically constrained (Liu and Narasimhan, 1989). Because the redox status of soils is often set by “microbial potentials,” consuming or producing compounds or ions containing one or more of these elements may render Pt electrode measurements inaccurate. 3. Mixed Potentials

The goal of relating measured redox potentials to species and valence states of various elements in soils requires that a given, singular redox

174

R I C H M O N D J. BARTLETT AND BRUCE R. JAMES

couple is responsible for the equal cathodic and anodic currents present when the applied potential is zero at the Pt electrode (equilibrium is achieved). For example, a Fe2+-Fe3+ couple at activities greater than M generates sufficient currents to obtain a measurable voltage for the system. In heterogeneous soils, however, a mixed potential may be observed where anodic and cathodic currents are equal, but the redox partners may not be in equilibrium with each other. Such a situation disallows quantitative interpretation. An example would be the coupling of an Fez+-Fe” couple with an 0 2 - H 2 0 couple (Stumm and Morgan,

1981). The establishment of a measureable balance between the cathodic and anodic current at the Pt electrode at the point of zero-applied voltage Mya condition that may not exist for many requires activities of > may be common, redox-active species of interest in soils. Activities < especially for certain plant nutrients and soil pollutants. For this reason, application of measured Pt electrode potentials to predicting soil composition may not be possible if activities of oxidants and reductants are low. 4. Coupling of pH and pe

Based on the complementary nature of pH and pe (Stumm and Morgan,

+

198I), summing “pe pH” to describe log K for soils is possible theoretically. Because the Pt electrode is an “all-purpose” electrode that responds to pH as well as electron activity, pH should always be measured and reported with pe. The negative slopes of the pe-pH (Figs. 1 -6) relationships of many of the reduction half-reactions also indicate that the energy change associated with a particular reduction decreases with increasing pH. Therefore, an 8, measurement cannot be used to predict the presence of a particular redox couple unless pH is known. Because higher pe values at lower pH values correspond to larger releases of free energy, reduction reactions are expected to be favored by lower pH. That is, such systems are ones in which “reduction is favored.” In contrast, loss of electrons from reductants of a particular couple is favored at higher pH, or the system is more “prone to oxidation.”

C. ALTERNATIVE STRATEGIES FOR MOREACCURATE MEASUREMENT OF SOILREDOXSTATUS 1.

Using Electrochemical Relations in Reverse

Given the uncertainty associated with measured Pt electrode potentials in soils to quantify pe, actual measurements of reductant and oxidant

REDOX CHEMISTRY OF SOILS

175

activities, along with a reliable pH measurement, may be a better approach (Stumm and Morgan, 1981). The activities are substituted into appropriate half-reactions relating pe and pH, and pe is thereby obtained by calculation. As shown in Table 11, the predicted pe by such a technique will be inaccurate to different degrees in different half-reactions. For example, an error of two log units for H,S or 0, partial pressures will only produce errors of 0.3 to 0.5 pe units. In contrast, similar errors in measurement of Mn2+or Fe2+will result in pe errors of 1.O to 3.0 pe units. Because Mn and Fe are relatively easy to measure accurately by atomic absorption or colorimetric methods, and mineralogy of associated oxides can be made with infrared or X-ray techniques, assessing pe values for Mn and Fe oxide-dominated systems could be reliable. Detailed research is needed to prove this hypothesis. In contrast, dissolved gases are harder to measure accurately, and qualitative estimates may be sufficient to obtain accurate evaluations of pe. If calculated pe values obtained with this “reverse electrochemical technique” are equal for two different reduction half-reactions, then chemical equilibrium may be assumed to exist. If the pe values are unequal, then disequilibrium and a metastable, kinetically limited soil system probably exist. This latter condition is common in soils due to spatial heterogeneity of soil solution, oxide mineralogy, and organic matter reactivity. New thinking and hands-on research are needed to provide new ideas for evaluation of the “electron activity” for such soils. 2. Redox Ranges for Empirical pe Values

These limitations to assessing soil pe based on R electrode or reverse electrochemical methods indicate that our sense of accuracy for soil redox status must be modified. If we surrender in our efforts to conceptualize and operationally define soil redox status, we will have lost a challenging scientific crusade. Rather, we should retreat temporarily. This means that while new ideas are being developed, we accept a lack of knowledge of pe values more accurate than ranges bracketed by whole numbers. Liu and Narasimhan (1989) have described “redox zones” in which a range in gh or pe defines an electron activity condition. The oxygennitrogen range is defined by 8, values of 250 to 100 mV, the iron range is 100 to 0.0 mV, the sulJhte range is 0.0 to -200 mV, and that for methane-hydrogen is defined at 8 h <-200 mV. Sposito ( 1989) proposed oxic soils as those with pe > 7, suboxic ones in the range of pe between 2 and 7, and anoxic soils with pe < 2, all at pH 7. These ranges correspond roughly to redox control by oxygennitrogen, manganese - iron, and sulfur couples.

+

+

+

+

+

+

176

R I C H M O N D J. BARTLETT AND BRUCE R. JAMES

Berner (198 1) proposed categories for redox named oxic, postoxic, suljidic, and methanic controlled by transformations of oxygen - nitrogen, iron, sulfur, and methane- hydrogen, respectively. James ( 1989) has proposed these names be assigned to ranges in EMpe (Bartlett, 198 1 a) of 7 to 1 3, + 2 to +7, - 2 to +2, and -6 to -2. The appropriateness of these categories and names will require further evaluation of new operational definitions for the concept “redox status” in soils.

+ +

VIII. FREE RADICALS IN REDOX PROCESSES

A. FORMATION OF FREERADICALSIN SOILS A free radical is an odd electron species, that is, an atom or group of atoms either donating or accepting a single electron and ending up with an unpaired electron. Because the spin of an odd electron is not cancelled by an electron of opposite spin, the free radical generates a magnetic moment that makes it paramagnetic. This means that it is attracted to an external magnetic field and can be detected and studied by means of electron spin resonance (ESR) spectrophotometry. Electron cleavage in its formation results in a high-energy free radical with a strong tendency to lose some of its energy by forming new chemical bonds. It acts either as a powerful reducing agent by donating its unpaired electron or as a powerful oxidizing agent by accepting a mate for its odd electron. Equation (30) shows a simple straightforward reduction half-reaction in which one molecule of dioxygen (0,)accepts four hydrogen atoms, consisting of four protons and four electrons, and forms two molecules of water. Restricting conditions of interaction between the availabilities of soil 0, and electron donors, for example, at an interface between oxygenated water and anaerobic soil in a rice paddy, or between a living root and the rhizosphere soil surrounding it, tend to favor electron transfers in single steps, one electron at a time. Whenever the transferred electrons are not in pairs, free radicals will be formed. Equations (27) and (29) show formation of oxygen free radicals. The products in Eqs. (28) and (30) are not free radicals. The following reactions are half-reactions showing odd and even electron additions to O,, and reactions showing transfer of electrons among oxygen species:

+ + H+ 0, + 2e- + 2H+

O2 e-

-

__+

H Q (protonated superoxide) H,O,

(hydrogen peroxide)

(27) (28)

REDOX CHEMISTRY OF SOILS

0,

+ 3e- + 3H+ H,O + OH (hydroxyl free radical) 0, + 4e- + 4H+ 2H,O (water) HO; + H,O, H,O + 0, + OH. -+

-

*

OH. + H 2 0 2 - H 2 0 + H Q

+

(30) (31) (32) (33)

2H202 a 2 H 2 0 + 0, 0.

(34)

MnO,

OH

(29)

2HO; x H Z O Z 0,

o+ OH

177

+ 3H+

-0

+ Mn3? + 2H,O

(35)

OH

B. BEHAVIOR OF SOILFREERADICALS With one electron, a hydrogen atom is a free radical, a single protonated electron in frantic search for an electron mate. It is the simplest and most reactive (least stable) free radical. As H,, however, it is quite stable, because the electrons are paired. Catalases or fresh recently oxidized Mn oxides at pH > 6 (Bartlett, I98 la) will catalyze the oxidation of one of the oxygens in H,Oz by the other and thereby will destroy the peroxide by dismutation [Eq. (34)]. In acid media, oxidized Mn will oxidize H,O,. The dismutation of HO; to H,O, and 0, by superoxide dismutase (SOD) enzymes, Eq. (33), followed by H,O, dismutation to 0, and H,O or oxidation of H,O, to O,, make aerobic life possible (Fridovich, 1975; Halliwell, 1974) by preventing the formation of the biodestructive hydroxyl free radical by Eq. (3 1). Reforming HO; by Eq. (32) is prevented. Oxygen free radicals are much more reactive than 0,. Probably free radical mechanisms explain why kinetically very slow and seemingly unlikely redox transformations sometimes occur readily. The hydroxyl free radical, (OH the superoxide free radical, -OF, and the supermanganese free radical ( Mn33are close to being the most powerful oxidizing agents in soil systems, and superoxide and supermanganese also are the most powerful reducing agents (Table I, Fig. 3, and see Sections VI,A and VI,C) (Bartlett, 198 I a). Oxygen free radicals are among the few species having the thermodynamic capability for oxidizing Mn(I1). This means that Mn is one of the few elements that is capable of scavenging these radicals and protecting life a),

178

RICHMOND J. BARTLETT AND BRUCE R. JAMES

forms from their biotoxicity. By scavenging free radicals, Mn disrupts the tendency toward thermodynamic equilibrium between 0, and soil organic matter (or living roots), allowing the persistence of metastable humus and roots in an oxidative environment. Equation (35) shows the MnO, oxidation of hydroquinone by a single electron step to form two free radicals, the semiquinone free radical and the supermanganese free radical, Mn? It seems that free radicals could be the latchkeys to the linking together of reducing phenolic compounds into organic polymers that are stable in the presence of 0,. For example, a single OH free radical initiates the linking process in the formation of a polyethylene chain. The free radical attacks and breaks the 71 bond of an ethylene molecule, forming a new free radical, which then attacks another ethylene molecule and forms another new free radical, and so on, until thousands of molecules are joined (Zumdahl, 1986).

-

IX. MANGANESE A N D IRON A. LIVINGEARTHR E D ~ X SCHEME The redox elements, oxygen and carbon, are basic to the building and functioning of soils and all living systems, and basic to the functioning of oxygen and carbon in life’s overall redox scheme are Mn and Fe (Figs. 7 and 8). As the key that unlocks oxygen from water in the process of photosynthesis, Mn is responsible for the presence of the oxygen in the atmosphere of the planet Earth. Soil Mn also is a protector of life, the scavenger of death-dealing oxygen free radicals. Manganese and iron together provide the key to the establishment of the organic mantle, the humified soil top layer that covers the surface of the earth and serves as the nurturing home for the roots of all plants and carbon-recycling microorganisms. It is the sole provider of food for a variety of Earth’s creatures, including people. Iron is vital in the life systems of all plants, animals, microbes, and soils. In biological systems, Fe appears to play many roles similar to those of Mn. In animals, on the other hand, Fe is supreme, and Mn, although essential, displays some toxicity tendencies that may prevent its predomination of Fe. Iron is the chief camer of oxygen in blood, and it is a camer of electrons to oxygen in both plants and animals. Except in the soil redox scheme, Fe appears to have a bigger role than Mn in many living redox systems. In redox of soils, Fe plays second fiddle to Mn, but the melody of soil processes requires both metals.

REDOX CHEMISTRY OF SOILS

179

H20 /02

0 f r e e radical

,

\

/

4

everse dismutatio e-

Mn(II1)-organic

acid]

Mn3'.Fe3*

0 free radicals oxidative polymerization

\

lU,,mir

-,,h.-C---,.-l

Figure 7. The manganese redox system: rather than a simple redox cycle or even a series of cycles, the Mn system appears to be more a complex web of interacting redox transformations.

Fe(I1) in Primary Minerals

I

::athering

[Low Solubility Fe(lI1) Minerals]

I

chelat ion acid dissolution

02-Carrying Proteins

Fe(ll) 0 free radicals Organic f r e e radicals

Hemoglobin- Fe ( II) Met hemoglo bin- Fe( III)

Reduced N Cr(II1. VI)

Cytochromes-Fe( 11,111)

co,7 027

Increases in: pe, pH, darkness, H O ,,

1

Catalases-Fe( 111) Per o x i d a s e s - Fe( III)

Figure 8. Iron redox cycling: though not depicted here as a circular scheme, the reversibility of the various Fe reactions shown here indicates that Fe is involved in cycling, once it moves out of its primary mineral origins.

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RICHMOND J. BARTLE1-T AND BRUCE R. JAMES

B. CATALYTIC OXIDATION OF ORGANICS BY IRON Direct oxidation of soil organic compounds by atmospheric 0, is a rare occurrence. The majority of apparently spontaneous oxidation reactions are catalyzed by microbial enzymes, metal oxides, peroxides, or free radicals. Such species serve as electron camers, that is, substances that will oxidize reduced substances and will in turn be oxidized by more highly oxidized species. Proteins containing metals-usually Fe, Cu, or Mnserve as redox enzymes in microbial systems carrying electrons from reduced carbon to 0,. These enzymes are essential links in the respiration of all aerobic organisms. Flavoproteins without metals can transfer electrons to O,, but the rate is too slow to account for aerobic respiration. The Fe in hemoglobin must be in the ferrous form in order for it to combine reversibly with O2and act as the all important carrier of 0, in the blood of animals (Fruton and Simmonds, 1961). If the Fe is oxidized to Fe( III), methemoglobin is formed, which does not combine with 0,. The danger of nitrate in drinking water, spinach, or forage plants is that nitrite, formed from nitrate by reduction in an infant’s intestine, or in the rumen of a cow, can oxidize hemoglobin to methemoglobin, causing methemoglobinemia, the inability of the blood to carry oxygen. In the heme structure of cytochrome c, the Fe is reversibly oxidized and reduced, but in catalases and peroxidases, also heme compounds, the Fe remains trivalent. In the enzyme cytochrome oxidase, Fe(II1) is reduced by an organic compound that is oxidized in the process. The Fe(I1) formed then “cames” an electron to atmospheric 0,, which oxidizes it spontaneously back to Fe(II1). Names of redox enzymes are sometimes confusing. An enzyme carrying an electron from an electron donor to O2 is called an oxidase because the donor is oxidized. But an enzyme that oxidizes a carbon compound by transfemng an electron to an oxidized substance other than O2 or peroxide usually will be referred to as a reductase. An oxidase that removes both a proton and an electron from an electron-donating substance is called a dehydrogenase, because a proton and an electron comprise a hydrogen atom. Peroxidase is a peroxide reductase (pe = -4.6 at pH 7; Table I ) usually refined from horseradish. A molecule (about 40,000 g mol-’) contains one atom of Fe(I1) (Fruton and Simmonds, 196 I). To pass along an electron to H,O,, the Fe(II1) borrows an electron from a carbon atom to form, for an instant, an atom of Fe(I1). The Fe( 111) is restored during that same instant as the H 2 0 2is reduced. Because it is so readily oxidized by O,, Fe( 11) can catalyze the oxidation of a phenolic compound that complexes Fe( 111). The phenolic is oxidized to a quinone when it reduces the Fe( 111) to Fe( II), which is reoxidized by 0, (Stumm and Morgan, 1981). The Fe(II1) formed is complexed and

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reduced by the excess phenolic compound, and so on. Trace levels of Fe or other metals can catalyze the oxidation and spoilage of foods by similar mechanisms. Citric acid is added to prepared food to tie up the Fe( 111) and prevent its complexation and reduction by the easily oxidized electrondonating food. This reaction occurs commonly in soils in the absence of light. More difficult oxidations that apparently do not occur in darkness frequently do take place when the Fe and reduced carbon are exposed to energy from sunlight (see Section XI). Because its reoxidation is so much more difficult than that of Fe, Mn is less effective than Fe in catalyzing complete oxidation of organics. Manganese is more likely to oxidize organic residues partially (making free radicals), setting them up for further microbial breakdown. It is interesting to note that of the three trivalent redox cations, Cr(II1) can hydrolyze or it can oxidize, Mn( 111) can only oxidize, and Fe( 111) can only hydrolyze.

C. MANGANESE, THE TRANSCENDENTAL TRANSITION METAL 1 . Ultimate Electron Acceptor

The outstanding contribution of manganese to life on Earth appears as a simple entry in the upper right-hand comer of Fig. 7. Both Mn(II1) and Mn(IV) are powerful oxidants in the soil redox system, especially the free radical supermanganese Mnw ion, which has the thermodynamic capability of oxidizing 202- to 0, gas. It does this in the leaves of green plants exposed to radiation from the sun in the process of photosynthesis and thereby is responsible for creating the oxygen in the atmosphere. However, the mechanism for this profound redox transformation is only partially understood (Brudvig and Crabtree, 1989; Thorp and Brudvig, 1991). It is possible that a singlet chlorine free radical is the direct electron acceptor, and that the Mn role is one of accepting an electron from C1- to form (CIS). Marschner (1986) discusses the evidence that chloride acts as a cofactor in the Mn-containing 0,-evolving system. 2. Proportionation and Disproportionation

Mn2+ is a soluble or exchangeable cation. It forms by reduction of Mn(II1) or Mn(1V) by a multiplicity of easily oxidizable, reduced organics, often microbial by-products. The reductive dissolution of Mn oxides by microbial metabolites has been studied extensively by Stone and Morgan ( 1984). Simultaneous formation of Mn( 11) and Mn( IV) can take place by the thermodynamically spontaneous disproportionation, or dismutation,

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of two Mn( 111) ions. One Mn( 111) loses an electron to the other to become Mn(IV), while the electron-accepting Mn( 111) forms Mn( 11). In a reverse of this dismutation reaction, two molecules of free radical Mn( 111) are constituted when a Mn( 11) gives up an electron to Mn( IV) as follows:

+

Mn2+ MnO,

+ 3(COOH),

-

2Mn(COO),

+ 2H20 + 2H+

(36)

This reverse dismutation would not be thermodynamically spontaneous if it were not for the energy of formation of the organic acid-Mn(II1) complex. An organic acid that readily couples with Mn(II1) and also is easily oxidized by it can drive the reverse dismutation equation to the right. With low-molecular-weight organic acids, the Mn( 111) complexes are soluble and range in color from yellow to yellowish brown to red. To be readily oxidized by Mn( III), the acid must have an oxygen on a carbon adjacent to a carboxyl group. Oxalic, citric, and tartaric acids are examples, but succinic acid, which chelates Fe( 111) and Al( III), does not complex Mn( 111) and will not drive the redox. Pyrophosphate is another ligand that drives this redox reaction by binding strongly to Mn(II1) to form a violet-pink color (Dion and Mann, 1946). Loss of complex color accompanying the oxidation of dissolved organic carbon by the Mn(II1) serves as a simple colorimetric method for measuring dissolved organic carbon (Bartlett and Ross, 1988). Pyrophosphate-bound Mn(II1) is not as powerful an oxidizing agent as Mn3+ (Table I). 3.

Mn(111) -Organic Acids as Reductants

In solution, the gradual fading of the color complex indicates that the organic acid is being oxidized to CO, and H 2 0 by the bound Mn( 111) while Mn(111) is being reduced to Mn( 11). The rates of formation of the color complex and its fading both are inversely proportional to the pH. When redox decomposition such as this takes place in a soil, base-forming cations are released, causing the pH to rise and the rate of oxidation of the organic to be slowed or stopped. Mn(III)-citrate made from K,.,-citrate at pH 4.7 decomposes fairly rapidly for a few hours until the pH reaches 7.6, and then it remains stable, with no more fading or C02 loss for several months. It was pointed out in Section VI,C and Fig. 3 that the supermanganese Mn3+ion can function as a double agent, powerful not only as an oxidizer but also as a reducer. Mn(II1)-citrate will reduce methylene blue and tetrazolium blue and oxidize tetramethylbenzidine (see Section XIV,D). In solutions and in soil in the laboratory, under normal fluorescent lighting, we found that Mn(II1)-citrate or oxalate, formed by reverse dismutation, reduced Cr(V1) much more effectively at pH 4-6 than the organic acid

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Figure 9. The marked lowering of net Cr oxidized, shown when Mn(I1) and oxalate ware added together to soil samples containing different levels of oxidized Mn, demonstrates the reducing power of Mn(ll1) formed by reverse dismutation.

alone. We also showed that Mn(II1)-citrate reduced Cr(V1) faster than the organic alone. These effects were strongly borne out in treated soil samples in which the chromium net oxidation test (see Section XIV,F) was used to characterize the oxidative minus the reductive powers of the samples (Bartlett, 1988). Figure 9 shows that adding Mn2+to soil samples already containing MnO, decreased the net oxidation of Cr by the soil. Part of the reason may be the temporary increase in positive charge (Fig. 7) in repelling Cr( 111). But the most probable reason is the reducing effect of Mn( III), which will form by reverse dismutation (see Section IX,C,2) when Mn2+ is adsorbed onto MnO,. Adding citrate alone had less effect than Mn2+alone on reduction of Cr(VI), as shown by the net test. However, adding both Mn2+ and citrate together markedly increased reduction of Cr( VI), as indicated by the net Cr oxidation test. Reduction by Mn(II1) formed by reverse dismutation most surely is the explanation for the huge lowering of the Cr oxidation net test with the two added together.

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Manganese (111) also has the ability to oxidize an organic compound by single electron steps to form a reducing organic free radical. For example, a carboxyl free radical (R-COO.) has a strong disposition for giving up its remaining odd electron to act as a reducing agent. 4. The Oxymoron: Enhanced Oxidation by Oxygen Restriction

In a slightly reducing environment, the first electron step in the partial reduction of 0, is the formation of superoxide, the oxidizing/reducing free radical. Manganese(III), formed in the first electron step in the partial reduction of MnO, , is another extremely reactive oxidizing/reducing free radical. Its reoxidation produces highly reactive “fresh” MnO, . This effect is demonstrated by Mn behavior in oxidizing Cr in soils. For example, partially restricting aeration by stoppering a flask escalated the Mn oxidizing behavior. Ten times the concentration of Cr(VI) was produced in a soil suspension incubated 9 days with MnSO, and Cr(OH), in a stoppered flask, as compared with the same volume of suspension swirled in an open flask. Vigorous aeration of a high-organic-matter soil increased net Cr oxidation by Mn oxides, but the same aeration lowered oxidation by a low-organic-matter soil. Stoppering the low-organic-matter soil increased net Cr oxidation, whereas stoppering the high-organic-matter soil halted oxidation entirely. Thus, the redox poise between reactivities of oxidants and reductants is critical. (R. J. Bartlett, unpublished data). Changing the balance may reverse the direction of whatever is happening. Compaction of soil in wheel tracks in turf typically results in dark green stripes on the surface, not between areas of compaction, but at the site of compaction. Chemical analysis of the vegetation shows that the extra green is associated with higher nitrogen. The increased nitrogen is the result of increased oxidation in the root zones of plants where the soil has been somewhat compacted. Thus, it appears that partial exclusion of oxygen by compacting a soil can increase certain oxidative processes in that soil. The favorable oxidative effects resulting from restricting aeration seem to be mainly related to the reactivity of Mn oxides. Fresh Mn oxides provide better aeration than air in an oxidizing soil environment. The oxides are more ready electron acceptors than oxygen. In paper towels impregnated with high-Mn-oxide soil compared with those with only nutrient solution, white clover seeds germinated more quickly and had a higher percentage of germination (R. J. Bartlett, unpublished data). When aeration and respiration and synthesis of electron donors get out of balance in the rhizosphere, dangerous-to-life free radicals may form. The enzymes that scavenge such free radicals depend on metals, generally, Cu, Mn, and Fe, to effect electron transfers. An example is a superoxide

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dismutase described by Fridovich ( 1979, containing Cu2+and also Zn2+, as a stabilizer. Reduced Mn acts as a scavenger for oxidizing oxygen free radicals, and oxidized forms of Mn together with Fe are responsible for accepting electrons from highly reactive reduced toxic or allelopathic organic compounds and then using these compounds for the synthesis of benign, nurturing, and stable humic substances. Manganese does these things in the dark, where plant roots grow and develop in soil. Roots benefit from the humified materials surrounding them with the right balance between 0, and H,O,while, at the same time, they are being protected by redox metals from toxicity of oxygen free radicals. 5 . Mechanism for Oxidation of Manganese in Soils

Manganese(IV) usually occurs in the soil as a colloidal solid oxide. Often the negative charges on the oxide surface are occupied by adsorbed Mn(II), and this gives the overall surface a positive charge (Loganathan et al., I977), enabling its adsorption by negatively charged colloidal organic matter (Fig. 7). The change in surface charge of MnO, from negative to positive is easily observed by a reversal in direction of electrophoretic mobility (Bartlett, 1988). The positive charges also could arise from adsorbed Mn( 111) ions formed by reverse dismutation of Mn( 11) and MnO,. It is axiomatic that living plants, animals, and microorganisms supply all of the soil organic substances that are redox reactive. Soil microorganisms are indispensable in synthesizing and making available phenolic and aliphatic acids and in influencing pH near reactive surfaces by mineralizing organic matter and releasing base-forming cations. They also “graze” and metabolize selectively the most biologically available electron-rich substances, those that would tend to interfere the most with oxidation of Mn. Because the autooxidation of Mn(I1) by atmospheric 0, cannot be demonstrated unless the pH is above 8 (Diem and Stumm, 1984), it has been a common assumption that formation of Mn oxides in most soils requires specific microbial enzymes, and activities of soil microorganisms have been studied in this regard [e.g., Ehrlich ( 1 976), Silver et al. (1986), and Sparrow and Uren (1987)l. Unfortunately, lack of Mn oxide formation after use of a chemical microbial inhibitor has been incorrectly used as conclusive evidence for dismissing the importance of abiotic mechanisms of Mn oxidation. Chemical inhibitors (e.g., chloroform or sodium azide) will reduce MnO, in any soil containing organic acids and will destroy the soil’s Mn-oxidizing mechanism (Ross and Bartlett, 198I). Using Cr oxidation to evaluate Mn oxides, Ross and Bartlett ( 1 98 I ) , showed that oxidation of added Mn was proportional to existing oxides. Arrhenius plots of rates of Mn oxidation at different temperatures were indicative of nonbio-

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logical characteristics for the oxidation. Ross and Bartlett (198 1) hypothesized that the oxidation was autocatalytic and that fresh Mn oxides tended to form on old oxide surfaces. Chemical oxidation of Mn(I1) added to an acid (pH 4.4)soil and to a neutral soil was demonstrated directly in another study (Bartlett, 1988). All of the oxidation of added MnSO, that was to occur during a 36-hr period occurred the first 15 min. Obviously the oxidation was dependent on the biochemical status of electron acceptors already present in the soils at the moment of addition and not on microbial growth in response to the Mn(I1) additions. After 5 days, there were marked increases in oxidized Mn in both soils, suggesting that a biochemical readjustment had taken place, presumably in response to the newly formed Mn(IV) and/or excess added Mn(II), and to changes in microbial activities. It is not safe to assume that Mn not extractable by a neutral salt has been oxidized because strong inner sphere binding of Mn( 11)by soil organic matter above pH 5 - 6 can prevent its exchangeability (McBride, 1982). Probably the hypothetical “manganese oxidase” enzyme of Silver ef al. (1986) in reality is either the hydroxyl free radical (OH - ) or the protonated superoxide free radical (HO;). These are the most likely electron acceptors in the oxidation of Mn(I1) to Mn(1V). Fresh, newly formed Mn oxides usually are found in soil regions where oxygen free radicals are being formed, at redox interfaces, in rhizospheres, and in regions where atmospheric 0, is in somewhat short supply. Free radicals form, and Mn(I1) is oxidized in scavenging them. Even if there are not microbes that have specific roles as manganese oxidizers, microorganisms nevertheless are the ultimate setters of the scene, and, of course, the oxygen free radicals can be considered to be indirectly the result of microbial activity. A Mn( 11) ion, in reducing OH and becoming oxidized in the process, is destroying it and is preventing a microaerophile, busy setting the scene, from being poisoned in its own juice. Atmospheric 0, is the terminal electron acceptor when an oxygen free radical oxidizes Mn( 11) to Mn( IV). When the soil pH and pe are both high, as in the presence of free CaCO,, 0, may oxidize Mn(I1) to Mn(1V) directly. Microbial processes also favor direct oxidation in high-pH microsites as they increase pH by releasing base-forming cations during decomposition of organic residues. a ,

D. MANGANESEAND NITROGEN TRANSFORMATIONS There are many bits of circumstantial evidence indicating that Mn is involved in soil nitrogen redox transformations, but there is little under-

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standing of the processes involved. Manganese oxides are thermodynamically capable of oxidizing NH, and N, to nitrate, but there is no hard evidence that this happens. Circumstantial evidence consists of good correlations between nitrate production and content of Mn oxides in incubated soils. The good correlations may have resulted because soil Mn oxides retarded or prevented denitrification by oxidizing nitrite back to nitrate as fast as it formed, or else Mn oxides perhaps prevented denitrification by scavenging readily available organic reducing agents, oxygen free radicals, or Fe( 11). Both the oxidation of NH,OH to nitrate and of nitrite to nitrate by synthetic amorphous MnO, are easily demonstrated in the test tube (Bartlett, 1981b, 1988). Bartlett ( I 98 1b) showed that the amount of nitrate formed in soils from added nitrite at 0.5”C was directly related to the net Cr oxidized by the standard oxidation test (see Section XIV,F), that is, to the net oxidizing ability of the soil Mn oxides. Nitrate formation and MnO, reduction were stoichiometrically related in the presence or absence of atmospheric 0,. When MnO,/NO? ratios were high, reduction to Mn( 111) was mainly observed; when low, Mn( 11) was the reduced product accompanying nitrate formation.

X. SOIL CHROMIUM CYCLE The Cr cycle, Fig. 10, begins where it has ended, with Cr in its least mobile form, Cr( III), precipitated or tightly bound by a variety of ligands, such as hydroxyls, humates, and phosphates, or, in its most inert forms, substituting for two atoms of Fe in the magnetite structure, as FeCr204,or for small amounts of octohedral A1 in clay minerals. Like Al, Cr( 111) can be mobilized by low-molecular-weight organic acids such as citrate. The chelated Cr3+ may then interact with negatively charged MnO, and become oxidized to Cr(VI), the HCrO; ion in the diagram. Some of the citrate ligands are recycled. If there is a surplus of citrate, the Mn2+formed when the Cr was oxidized may react with surplus MnO, and reversely dismutate to two molecules of Mn(II1)-citrate, according to Eq. (36). Highly reducing Mn(II1)-organic, when it forms, will temporarily interfere with further Cr oxidation. The next step is “dechromification,” or the reduction of Cr( VI) by carbon reduced by the sun’s energy through photosynthesis. An intermediate species, such as Fe2+or S2-, reduced by carbon, can serve as the direct electron donor. Direct sunlight may hasten the process of Cr(V1) reduction. It is theoretically conceivable that dechromification, like denitrifica-

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Figure 10. Redox cycling of chromium in soils and water.

tion, could be vital to life in preserving atmospheric 02.Chromium( VI) is the thermodynamically stable Cr form in equilibrium with the air, and without Cr( VI) reduction, all of the atmospheric O2theoretically would end up as chromate, even if each Cr in the earth’s crust took on an average of only one more 0. Of course, all life would be poisoned by Cr(V1) long before this could happen. The Cr3+ion formed by reduction becomes an exchangeable cation only in very acid soils. Chromium(II1) behaves much like AI(II1) in being converted to organic, hydroxy-precipitated, and polymeric species. As pointed out, mobile low-molecular-weight organic species are most susceptible to oxidation in less than extremely acid soils. The Cr cycle is kinetically rather sluggish.

XI. PHOTOCHEMICAL REDOX TRANSFORMATIONS IN SOIL AND WATER Oxidation reactions that take place extremely slowly or not at all in the darkness below the soil surface may occur rapidly in direct sunlight. We

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have begun study of this phenomenon at the University of Vermont using polyethylene beakers containing salt solutions varying in concentration from 40 to 200pM, exposed to sunlight through a glass window or to a daylight fluorescent tube for periods of one or more hours. We measured changes in solutions in Fe2+,Cr(VI), organic carbon, nitrate, nitrite, and pH that occurred in the solutions as a result of their exposure to light. The equations that follow are based on hypotheses developed to explain the results of some of our experiments (R. J. Bartlett, unpublished data).

+ 2H,O1lghiHO; + 3H+ + 3Fe(OH), Cr3++ 2H20 + HO; ~ighiHCr0;+ 4H+ KHC20, + 4Fe(OH)f + 2H,O llghi HO; + 4H+ + 4Fe(OH), + KC20; KC,O; + HO; + HCrO; + 3H+ llghi Cr3++ 3H20 + 30, + 2C02 + KOH 3Fe(OH)f

(37) (38) (39)

(40)

Amazingly, a significant fraction of the Fe(II1) in a dilute solution of FeCI,, exposed for 6 hr to through-glass sunlight, was reduced to Fez+ [ Eq. (37)]. The original solution, consisting only of 200 p M FeCI, in distilled water, was assumed to be largely Fe(OH)f, based on the pH. We concluded ( 1 ) that the electron donor for the Fe reduction was water, because no other reduced species was known to be present, and (2) that the water oxidation product probably was the protonated superoxide free radical. The superoxide radical was identified as the likely product by its behavior as both an oxidant, in oxidizing Cr(II1) [Eq. (38)], and as a reductant, reducing tetrazolium blue or methylene blue (Afanas’ev, 1989). Both phenomena were prevented by pretreatment of the solution with superoxide dismutase, made from horseradish. The protonated superoxide product in Eq. (37) became the superoxide reactant in Eq. (38), and the protonated superoxide and the oxalate free radical, products in Eq. (39), were considered to be reactants in Eq. (40). The light-induced reducing activity of the solutions for Fe was greatly enhanced by the presence of oxalate [Eq. (39)], citrate, or phenolic compounds extracted by water from dried soil samples. The organics disap peared from solution by apparent oxidation in the presence of light. The presence of these organics was accompanied by light-induced reduction of Cr( VI) [ Eq. (40)], and also by reduction of both nitrate and nitrite (equations not shown). Although Fe(0H)t theoretically can oxidize Cr at pH < 4 (Fig. 5 and Table I), it did not do so in the absence of light. Chromium was not oxidized at all when organics were present. More easily oxidized than

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RICHMOND J. BARTLETT AND BRUCE R. JAMES

Cr( HI), the organics were oxidized first. The order of oxidation apparently was related to ease of oxidation. It appears probable that these photochemical phenomena are of significance in waters above the soil surfaces in wetlands or paddies as well as on unshaded moist surfaces of better drained soils. Toxic and allelopathic organic substances could be destroyed or rendered harmless or even be enhanced in reactivity by this process, and both CO, and 0, may be evolved. Redox metals may be oxidized or reduced. Preliminary results show denitrification greatly enhanced by light as well.

XII. HUMIC SUBSTANCES A. INVOLVEMENT OF MANGANESE IN FORMINGHUMUS Next to putting the O2 into the atmosphere, covering surfaces of the earth with humified soil has to be the second most astoundingly important contribution of Mn to life on our planet. Development of soil organic matter seems to be a process that takes place in the absence of light, beneath the soil surface, and although oxidized Fe is an essential link in the process of humus stabilization, the role of Mn redox is paramount in the domain of darkness. Manganese ushers atmospheric oxygen from the open-air soil pores to the poorly ventilated interior pore regions where Fe(I1) and organic building blocks of polymers would tend to remain reduced, were it not for Mn. In observing profiles of acid forest soils in the northeastern United States and southeastern Canada over a period of 20 years, we noticed a relationship between the Mn oxide content of the horizons of the mineral soil, as measured in the field using tetramethylbenzidine (see Section XIV,D), and the organic matter distribution in the profile. Spodosol profiles that had well-developed E horizons (leached, bleached, low-organic horizons underlying organic layers) generally were almost devoid of Mn oxides. Profiles that had A horizons, mull types, with stabilized intermixing of mineral and organic material, without E horizons beneath them, invariably displayed positive tests for Mn oxides throughout the profile. In a subsequent study by Bartlett (1990a), chemical analyses of 4 1 pedons with spodic horizons directly under E horizons and 37 showing spodic horizons directly beneath A horizons, including oxalate and pyrophosphate extractions, Mn electron demand (see Section XIVJ), H,02 dismutation (see Section XIV,J), leached columns, and flooded soil equili-

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brations (see Section XIV,B), resulted in the conclusion that Mn oxides are associated with absence of a developing E horizon and that Mn presence strongly favors the formation of a mull type A horizon as the uppermost mineral horizon of a Spodosol.

B. E HORIZON DEVELOPMENT In the first part of a simple demonstration to illustrate the formation of low-organic-matter E horizons in acid parent materials, dissolve a pinch of FeSO, in a water extract of partially decomposed leaves (ordinary black tea will do), and pour the solution through some light-colored fine sand from an E horizon. The water coming through is still tea-colored, and the pale E horizon retains its bleached E character. This could be considered an extreme example of the white sand - black water phenomenon described by Jenny (1980), i.e., a pale mineral E horizon disassociated from organic matter that has been mobilized from the soil profile, giving color to the leaching waters. In soils developed by the podzolization process in average or low-Mn parent materials, Mn(I1) and Fe(I1) both will tend to be reduced by organic residues in the acid environment, and both will be leached downward. Humus accumulation in the surface mineral soil layer will be minimal, and eventually it will develop into an E horizon. The organic matter distribution picture in acid soils would be skewed in the extreme without mention of Al. In very acid soils containing only trace amounts of Mn, the humified organic matter, mostly fulvic acids, is precipitated by Al, and is almost entirely in the B horizon, beneath the E. Spodosol criteria are based on the amounts of accumulated Al-bound organic matter in the B horizon. In sandy Ac;..ods, developed in poorly drained parent materials in warm climates, both Fe and Mn tend to be reduced and move completely out of the upper profile, and E horizons above the Al -organic accumulation frequently are more than a meter thick. Such profiles always will be practically devoid of Mn.

C. MECHANISMS AND PROCESSES INFORMATIONOF A HORIZONS 1. A Horizon Development

In the second part of our demonstration, add a few drops of fresh synthetic MnO, suspension to the FeSO,/tea solution, and almost instantly the Fe( 11) is oxidized, and inky black precipitated Fe( 111)-tannins cloud

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the once-clear tea. Pouring the suspension through the pale-colored sand causes the sand to turn black as it adsorbs the organic precipitate. The filtrate coming through the sand will be clear and colorless, and the fine sand is on its way to becoming an A horizon. Topsoil-as in “all that’s between us and starvation is 6 inches of it”-is the generic name for A horizon material of a generic soil. Evidence seems to point to Mn as the key to the formation of A horizon topsoil in both acid and nonacid soils. Iron and Mn are both important in formation of A horizons in acid soils and in poorly drained soils, but in soils that rarely reduce it (well-drained nonacid soils), Fe is much less important than Mn in A development. When atmospheric 0, oxidizes Fe( 11), protons are released by hydrolysis of the resulting Fe(III), and the pH is depressed, causing this oxidation process to be self-limiting [Eq. (4 l)]. When Mn oxidizes the Fe(11) instead, protons are consumed as the Mn is reduced, and half as many protons are released by the overall Fe oxidation/hydrolysis [Eq. (42)]. But when Mn oxidizes organic substances, the pH tends to rise because electrons are consumed during the Mn reduction [ Eq. (43)].

+

2Fe2+ 5H,O

--

+ 1/20,

+ + MnO, 4H+ + (COO-), + MnO,

2Fe2+ 4H,O

+ 4H+ 2Fe(OH), + MnZ++ 2H+ 2C0, + MnZ++ 2H,O 2Fe(OH),

(41) (42) (43)

Addition of Eqs. (42) and (43) will show that the net effect of oxidizing both Fe and the organic acid together by Mn is still one of H+ consumption and pH rise, despite hydrolysis of the Fe(II1). If the Fe(II1) has been complexed by fulvic acids as it formed, the pH effect, because of proton release, might have been similar to that of hydrolysis. Loss of organic matter and consumption of protons during the oxidiation would increase both pe and pH in the environment of the Mn(II), and this should favor reoxidation of the Mn( 11). The Mn oxide-mediated synthesis of the A horizon in an acid soil involves two roles for Mn: the oxidation of Fe(I1) and the oxidative polymerization of humus components. Iron catalyzes the oxidative decomposition of phenols and other ready electron donors in soils (Section IX,B). By oxidizing the Fe(II), Mn oxide interrupts the reaction of Fe( 11) and 0, with the easily oxidizable organics and prevents their degradation. The Fe(1II) and the phenolics form a black resistant precipitate that may become part of metastable humus. Besides oxidizing Fe, the Mn oxides may also partially oxidize some of the phenolics to free radicals, raw materials for instigation of oxidative polymerization.

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2. Oxidative Polymerization by Mn Oxides

Synthetic, amorphous MnO, causes the browning at room temperature of a glucose-glycine solution and results in the formation of a stable dark reddish brown gellike polymer similar to that obtained by boiling the same sugar-amino acid mixture (Bartlett, 1988). At neutral pH, Mn( IV) causes almost instant browning of phenolic compounds. Shindo and Huang ( 1982, 1984) showed that browning of hydroquinone by MnO, was related to the formation of colors with spectra suggesting polymerization, and Shindo (1990) investigated synthesis of humic acids from phenolic compounds by Mn( IV). Iron oxides produced little browning effect. Pohlman and McColl(I988) showed that polyhydroxyphenolic acids with para and ortho phenolic OH groups were rapidly oxidized by Mn oxides, with spectral evidence indicating that the reaction led to polymeric humic products by way of benzoquinone derivatives. The effects of Fe and Mn oxides, and other soil minerals, on abiotic catalysis of oxidation and browning and subsequent polymerization of phenolic compounds are discussed in a review by Wang et al. ( 1986). Equation (35) shows the induction by MnO, of a semiquinone free radical and also a Mn3+ free radical in the same reaction. By readily accepting from or donating electrons in single electron steps to organic species, Mn( 111) has the potential for creating semiquinone and organic acid free radicals from fulvic acids, tannins, and simpler phenolic acids (Bartlett, 1988). Like Mn( III), which may have helped form it, an organic free radical can act as either a reducing agent or an oxidizing agent by either donating its odd electron or by accepting a mate for it. Thus organic free radicals serve as connecting links in inducing polymerization of humic substances to form stable humus (Bartlett, 1986, 1988; Schnitzer and Khan, 1972; Senesi and Schnitzer, 1978). Schnitzer and Khan (1972) suggested that unstable free radicals induce humification, and the presence of stable free radicals denotes that humification already has taken place. Suflita ef al. ( I98 1) proposed a mechanism whereby a hydroxyl free radical dehydrogenates a phenolic substrate, forming phenoxy free radicals, which, they suggested, will spontaneously couple among themselves or attach to the polymeric structure of soil humus. 3. Stabilization of Organic Matter in Acid Soils

In acid soils developed from high-Mn parent materials, under podzolization perhaps less severe than in Spodosols that develop without A horizons, small amounts of Mn may resist reduction and leaching and will

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persist in the profile. Some of this Mn could eventually be oxidized (probably by scavenging free radicals in rhizospheres) and then catalytically oxidize Fe(I1) held in the reduced state by acid organic matter. Freshly oxidized Fe will be in highly reactive amorphous oxide forms that become attached to organic surface microsites. The result is precipitation and stabilization of the Fe - organic matter and subsequent development of an A horizon, perhaps over an E horizon or as an intermediate A/E horizon, although usually, if a well-expressed A horizon is present in a Spodosol, the E will be absent. In effect, the Mn acts as an oxygen pump, moving 0, from the soil pores and stabilizing that oxygen as oxides on the soil surfaces that would be highly reductive, were it not for the Mn. By preventing mobilization of organic matter in the surface mineral horizon, presence of Mn in the A horizon will prevent the formation of an E horizon beneath it. It is apparent that Mn in the parent material of a developing acid soil detracts from expression of spodic character in the soil profile, but the effects may not be enough to prevent the soil from qualifying as a Spodosol. 4. Stabilization of Organic Matter in Near-Neutral Soils

Although it may not be possible to say which is adsorbed by which, there appears to be an almost universal association between oxidized Mn and the organic matter in A horizons. Freshly formed Mn oxides have negative charges and measurable CECs and thus show strong tendencies to adsorb Mn( 11) cations. Electrophoretic mobility observations show that adsorption of Mn(I1) will reverse the charge on a Mn oxide from negative to positive (Bartlett, 1988). The positive charge induced by adsorption of Mn(11) seems to be a likely source of the attraction by oxides to negatively charged organic substances (Fig. 7). The adsorption onto organic matter sometimes causes the oxides of the heavy metal Mn to float on top of the solution in a centrifuge tube. The presence of Mn oxides, as part of the humified organic matter, contributes greatly to the stabilization of the overall humus system. In parent materials containing quantities of base-forming elements sufficient to maintain surface horizon pH values above about 5.5, reduction of Fe by organics will not occur. Manganese will be readily reoxidized after it is reduced by oxidizing organic ligands. The freshly formed Mn oxides, and especially the activated MnH supermanganese free radical, will catalyze polymerization of organic substances to form humic materials, and a strongly developed mull type of A horizon material will be developed immediately under the litter layer. The resulting soil will not be a Spodosol.

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Soils developed under conditions of very high base saturation and/or from calcareous parent materials are noted for their degree of development of mull-type surface horizons. The A horizons are dark in color, high in organic carbon, and have strongly expressed structure related to their high humus content (Lutz and Chandler, 1946). It is easy to see how Mn oxides would be maintained in a high state of oxidation in such soils and would have maximum contribution to A horizon development. Rapid rates of reoxidation of Mn( 11) following free radical-forming oxidations of organic acids and phenolic compounds by Mn oxides would lead to rapid rates of oxidative polymerization of humus components. Leaching losses of Mn( 11) would be almost nonexistent. 5 . CO, from Added Organics Versus Organic Matter Stability

In laboratory experiments, evolved C 0 2 was used as a negative index of organic matter stabilization from added organics. Organic substances, such as organic acids, phenolic compounds, and cellulose, added to soil samples, were assumed to have become stabilized as soil organic matter after rate of CO, evolution settled to the level that it was before the organic additions (Bartlett, 1990b). Compared to low-Mn oxide soil samples, those with high-Mn oxides behaved as follows: ( 1 ) High-Mn samples evolved more CO, when cellulose, glycine, and citric and gallic acids were added at field capacity moisture. Mn oxides appeared to stimulate microbial activity. (2) With flooding and organic additions, high-Mn samples remained aerobic and at first evolved less CO, . Low-Mn samples formed Fe(I1) and evolved more CO, . (3) High-Mn oxides increased CO, when microbial respiration was the predominating oxidizing vector. (4) However, high-Mn oxides decreased CO, evolution when microbial activity was suppressed in biologically relatively inert E horizon material or in the presence of Cr(V1) formed by the oxidation of Cr( 111) by Mn oxides.

XIII. WETLAND AND PADDY PROPERTIES AND PROCESSES A. PADDIES, BEAVERPONDS,BOGS,MARSHES, SWAMPLAND, AND POORLY DRAINEDSOILS Not surprisingly, a wetland is frequently wet, but it owes its characteristics not to water directly but to the effects of water-filled soil pores in

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restricting the supply of oxygen to solid surfaces in the soil body. Wetland soils are presently legally defined by agreement among the Soil Conservation Service (SCS), The Fish and Wildlife Service, the Environmental Protection Agency (EPA), and the Army Corps of Engineers. The SCS considers that a “hydric” soil identifies a wetland, and to be hydric, a soil must have a presence of free water and virtual absence of O2 in it or on it for extended periods, but not necessarily continuously. Anaerobic conditions must develop in the upper profile during these periods. This means there must be organic residues present, and temperatures need to be of the degree and duration required for microbial synthesis of reactive electron donors. Soil conditions must favor the production and regeneration of hydrophytic vegetation. Soils will tend to have high accumulations of organic matter, gley mottles near the surface, a predominance of gray or black colors below, and Fe2+ will form extensively during anaerobic periods. Unpolluted sediments at the bottom of a cold lake with low biological activity may not be a wetland. Large areas of soils in the United States, developed on level lacustrine, marine, and ground moraine parent materials and also large areas of peat and muck soils, have been artificially drained by ditching or tiling to create highly productive and valuable agricultural land. Drainage of these lands was encouraged by the Federal government for many years and has been accomplished to a considerable extent in the major humid agricultural regions of the country. Recently, tough new laws have been passed to prevent further drainage of wetlands. Consequently, emphasis is on diverting areas of precious welldrained farmland for use in development.

B. REDOX-RELATED REASONSFOR WETLAND PRESERVATION There are many valid reasons, often related to redox, for preserving any unique ecosystem in its natural state, particularly if it has plants, animals, and soils uniquely adapted to it. Preserving any soil-wetland or upland -means saving a portion of our biological heritage. Soils being used in productive agriculture have already been destroyed as parts of natural ecosystems, but their continued preservation as viable cropland soils for producing food is of extreme importance to the survival of humankind. Because of its substantial accumulation of humus, the wetland soil is a caricature or an exaggeration of a “normal” soil. Draining a wetland soil will release C02and will increase the greenhouse effect and also entropy of the earth more than disturbing a well-drained soil.

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C . REDOXINTERFACES If a wetland with standing water is undisturbed for a period of time, the saturated soil and the water above it tend to become separate entities. At the surface of the soil sediment is a thin boundary consisting of soil that is immediately below partially oxygenated free water in contact with the atmosphere and immediately above saturated soil that is almost devoid of 0,. The soil below is anaerobic and serves as a source of electron donors to the upper side of the boundary, the electron sink. The boundary itself is the interface (Bouldin, 1968). An interface is the site of coming together and it occurs anywhere that oxidative and reductive soil environments are abruptly joined together in peds, paddies, sewage lagoons, manure storage pits, landfills, septic tank leach fields, sediments, humus or clay surfaces, and in rhizospheres rootsoil boundary regions. Redox transformations are rampant at interfaces. We would not expect to find free radicals in the anaerobic zone of surplus electrons, nor will we find them in aerobic zones frequented by aerobic microorganisms equipped with dismutating enzymes. Although thin, the interface may have enough thickness to span a series or range of two or three or more levels of redox potentials, each one representing a distinctive redox transformation. Reduced Fe and Mn, N, and organic substances are abundant in the soil bulk on the anaerobic side of the interface. Soluble carbon compounds, NHt, Fe2+, and Mn2+ diffuse into the aerobic zone, where they become oxidized to nitrate and Fe and Mn oxides. Metal oxides accumulate at the interface, and C02 is lost. Some nitrate diffuses back into the anaerobic zone and is reduced. The interface provides a mechanism for denitrification (Reddy and Patrick, 1980) and also is an optimum environment for nitrogen fixation by nonsymbiotic microorganisms. Organic products of anaerobic respiration diffuse upward and provide readily available reduced carbon energy needed by the nitrogen-fixing organisms living on the aerobic side of the interface. Using cellulose as an energy source in the anaerobic zone, Magdoff and Bouldin (1970) showed that nitrogen fixation was directly proportional to the interfacial areas in soil containers. Phosphate, strongly bound in insoluble Fe( 111) compounds, can become soluble and mobile on reduction of the Fe holding it in the anaerobic zone (Russell, 1973). An adjoining wetland can be a vehicle for increasing the availability of phosphorus to a lake, as the soluble phosphates diffise into the water on the aerobic side of an interface. The Mn and Fe oxides tend to persist at the interface. In spite of the giant pool of electron donors on the anaerobic side, the metal oxides may

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meet with only very small portions of the diffusing reduced substances during any given time period, so that the Mn and Fe are reoxidized as fast as they are reduced. When Fe2+escapes into water before it is oxidized, it tends to oxidize at the air/water interface, and shiny and fragile iron oxide films are commonly seen floating on still waters. Because these films refract light, producing beautiful rainbow colors, they are often mistaken for oil floating on the water. At a touch of a finger, however, they break and scatter into tiny fragments across the water surface. The artificial drainage of wetlands, man-made or natural, often results in the production of massive blobs of red - orange and sometimes black slimy, gellike, but not unattractive, semisolids, made up of bodies and byproducts of billions of bacteria along with Fe and Mn oxides. The metals and reduced organics, preserved under anaerobic conditions, are rather quickly oxidized in the presence of air. The oxidized masses, inconveniently, may form almost overnight inside drains and drainage pipe or tile. An unforested wetland may receive the full effects of sunlight, and there will be an opportunity for the photooxidation of a variety of toxic, allelopathic, and benign organic compounds, if they diffise into the water phase of a flooded wetland soil or remain on the interfacial surface of the solid phase. Oxidized layers of soil enriched with black Mn oxides commonly are observed under the anaerobic layers in a flooded rice paddy (Russell, 1973). This layer remains oxidized during flooding because of a low energy supply of reduced carbon for microorganisms and because of an adequate amount of oxidized poising material. There are also oxidized sheaths of femc hydroxide around the paddy rice roots, even though the root systems are entirely within the reducing layer. Oxidized Cr can persist almost indefinitely in the aerated water above an undisturbed interface (Bartlett and James, 1980). However, mixing across such an interface will quickly eliminate the oxidized phase and prevent reoxidation of the Cr.

XIV. EMPIRICAL METHODS FOR CHARACTERIZING SOIL REDOX A. SOILHANDLING Sieving, mixing, and storing moist soil samples is inconvenient but absolutely essential in soil chemical laboratory experimental work related in any way to redox. Soils from the field must be kept moist. Drying soils

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alters organic and mineral redox and acidity characteristics so that behavior of a dried sample will change markedly with time during dry storage and while it is returning to its metastable moist state if water is added back to the dry soil (Bartlett and James, 1980). Rarely will stored dried soil samples [designated “lab dirt” for emphasis (Bartlett and James, 1979)] oxidize added Cr(II1). And rarely will a field-moist soil sample fail to oxidize Cr(II1). After drying, a soil will readily reduce Cr(V1) when it is remoistened, whereas, in a continuously moist soil,Cr( VI) may persist for years in the presence of stable humic and fulvic acids. Moist soil samples are most suitable for handling if near field capacity moisture. It is difficult to get moist samples to pass through a 2-mm sieve, but a 4-mm polyethylene sieve is generally suitable and has the advantage of preserving some of the soil crumb structure and aeration status of field soils during storage before analysis. Time is saved by presieving through an old tennis racquet and mixing in the field. Samples should be mixed individually before sieving. Samples will remain most stable if they are stored in double, 25-pm-thick polyethylene bags with moist paper towels between the layers in a refrigerator at 4°C. Freezing and freezedrying cause soils to change during experimentation almost as much as airdrying does. Drying in the sunlight is an unconditional never, unless you are mimicking a field condition and have considered its implications. Microbial activity appears to become stabilized after 2 or 3 months at 4”C, and most soil samples seem to reach an internal metastable equilibrium. It is safe at this time to transfer samples to tight heavy-walled plastic bags or garbage containers with lids to prevent all moisture loss. Samples should be kept in semidarkness, but temperatures as high as 10- 12°C can be tolerated in metastable moist soils. Well-mixed subsamples may be weighed for analyses after determining moisture on separate samples. For many determinations, a volume measurement, to be later corrected for dry weight, is more convenient, although somewhat less accurate. A packed and leveled teaspoon of soil is 5 cm3, approximately 5 g dry weight for many topsoils.

B. LABINCUBATIONS The empirical approach frequently involves laboratory incubations of small amounts of treated soil in polyethylene bags at about field capacity moisture. Thin polyethylene has the advantage of being quite permeable to 0, and especially CO, but not to ions and solutions (Bartlett, 1965). Water vapor will pass through slowly, however, and if you wish to prevent drying for long periods, double bagging with a moist paper towel between the layers will help.

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For monitoring CO, ,the soil can be left in a closed bag and placed inside a 4-liter pickle jar with a tight lid, along with a 100-ml polyethylene beaker containing 1 M NaOH. The base can be titrated with HCl in the beaker after adding 2 M BaCl, and phenolphthalein. Also useful are incubations in flooded beakers for somewhat restricted 0, availability and to study interface processes. These should be kept in the dark, unless you plan to monitor and control the light as part of the experiment. Samples may be removed from the supernatant solution, from interface microlayers, or from deep in the sediment.

C. DETERMINATION OF EMPIRICAL pe Redox has become associated with wet soils and platinum electrodes because electrode potentials seem to be useful only in such soils. Interpretations of redox potential measurements are thoroughly discussed in Section VII; here we simply present a practical method for measuring an empirical pe (Bartlett, 1981a). 1. Attach a bright platinum electrode (in place of the glass electrode) to the plus terminal of a pH meter with a millivolt scale and attach a saturated calomel electrode (SCE) to the negative terminal. 2. Before each reading, rinse the platinum electrode, but not the reference electrode, in a 1/ 1 6 M HCl/liquid detergent solution followed by 10% H,02 and then thoroughly rinse with distilled water. Clean in aqua regia after a few hours of use. 3. Adjust the potentiometer to read +219 mV when the electrodes are in a pH 4 suspension of quinhydrone in 0.1 M potassium acid phthalate. 4. Add 30 ml of 10 m M CaC1, or 30 m M of NaNO, to 10 g of soil (dry weight basis) in a polyethylene beaker, stir until soil and solution are well mixed, and let stand for 20 to 30 min with occasional swirling. 5. Insert electrodes so that the calomel reference electrode is in the upper half of the supernatant solution and the platinum electrode is near the bottom of the suspension. Swirl for a few seconds, let stand for at least 5 min, and without jiggling or touching the cup, read 8, in millivolts. Measure pH of the same suspension. 6. EMpe = $ (, 244 for SCE)/59.

+

Since we do not know how to interpret Pt electrode measurements made in aerobic soils, their chief value seems to be in telling us whether a soil is anaerobic. A single sniff may be as useful as an EMpe measurement. It could be worth our time to develop a method, analogous to the Munsell color chip book, for quantifymg odors. Meanwhile, common scents, aided by common sense, will have to suffice.

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D. FIELDTESTS FOR Mn

AND Fe OXIDES AND RADICALS

20 1

OXIDIZING FREE

1. Tetramethylbenzidine

A safe substitute (Liem ef a/., 1979) for carcinogenic benzidine is made (TMB) in 28.6 ml by dissolving 250 mg of 3,3’,5,5’-tetramethylbenzidine glacial acetic acid and quickly diluting it to 500 ml with distilled water. When a few drops of this reagent are added, on a spot plate, to a pinch of moist soil, containing Mn oxides, dark blue points and zones begin showing up after a few seconds, and then slowly or quickly, depending on the Mn oxide form, the surrounding solution will tend to turn intensely blue. In very acid soil solutions, ionic Fe(II1) will sometimes give a similar positive test. The TMB indicator produces a positive blue color rather slowly with NO? and still more slowly with H 2 0 2 . In each case, it appears that the TMB is acting as a weak reducing agent and forming, respectively, various free radical species, most likely Mn3+, possibly the fenyl radical, Few? (Cohen, 1985), probably NO, or NO free radicals, and the OH radical. If the supermanganese Mn3+ free radical is present to begin with, it will of course give an immediate reaction with the TMB. However, until time has been allowed for free radical formation by partial reduction of MnO, by TMB, the full blue color will not develop. Nitrite, Fe(I11), and H,02each will produce full color immediately on TMB addition if first partially reduced by hydroquinone, dipyridyl or o-phenanthroline, or peroxidase, respectively. An immediate positive TMB test results with Cr( VI). Thus, although TMB will readily form blue color in the presence of oxidizing free radicals, especially the hydroxyl free radical or superoxide, TMB is most useful as an indicator of oxidized species that have in common the proclivity for being very easily reduced to oxidizing free radicals. The first increment of TMB added sensitizes the substrate (causes it to become a free radical) by being oxidized by it so that, when more TMB is added, it will change color quickly and intensely. An intense blue color forming instantly on addition of TMB to a spot plate sample of field soil indicates the presence of available Mn( 111). If the blue color intensity builds up slowly, this means that reactive Mn( IV) and/or Fe( 111) is present. Addition of three or four drops of 0.1 A4 citric acid before addition of the TMB will prevent reaction of Fe with the TMB and will enhance the Mn color development by driving the reverse dismutation of Mn(I1) and Mn( IV) toward Mn( 111). Many soils contain Fe( 111) minerals that will not react with TMB even without the citrate, but most Mn oxide minerals found in soils will give positive TMB tests. Thus TMB is a specific test for soil Mn oxides and can also be used to indicate the

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presence of highly reactive Fe( 111). It also measures relative proportions of reactive Mn( 111) and Mn( IV) in a soil sample. 2 . Other Tests

Gum guaiac, a dye soluble in ethyl alcohol, reacts similarly to TMB as an indicator, forming a bright red color with strong oxidants. Methylene blue and tetrazolium blue are dyes that change color in alkaline solutions, to colorless or red, respectively, with strong reducing agents (e.g., ascorbic acid and phenolic compounds). Another spot plate test for reactive Fe( 111), for example, recently formed Fe(OH), or temporary Fe(II1) in a wetland soil, consists of 10 m M 2,2’-dipyridyl in pH 4.8 NH,OAc, 1.25 M acetate (Vermont buffer). A pink or red color that develops immediately is a test for Fe( 11). Color that develops after about a 1.5 hr, or in a few minutes in the sun, is indicative of Fe( 111) that has been reduced to Fe( 11) by the dipyridyl. Used at 8 X dilution with unknown or standard solutions, this reagent is useful for quantitative determination of Fe(I1) in the laboratory (at 522 nm).

E. PREPARING SYNTHETICAMORPHOUS Mn (IV) Dissolve 40 mmol of KMnO, in about 40 ml of distilled water heated to approximately 60°C and transfer with mixing into 30 ml of 2 M MnSO,. Add 80 mmol KOH dissolved in 10-20 ml of water. Mix and adjust the pH to 7.5 with KOH or sulfuric acid and let stand overnight with occasional stimng, and then adjust the pH to 6.0 with additional sulfuric acid. There should be no permanganate color remaining. Transfer into dialysis tubing and dialyze against fresh distilled water until the outside solution is close to salt free, as checked by barium precipitation or conductivity. Dilute the suspension to 500 ml, or any desired volume. The procedure may be camed out quantitatively so that there is exactly 100 mmol of Mn02,or a diluted suspension can be standardized by iodine titration as in Section XIV,I,b.

F. STANDARDCHROMIUM NETOXIDATION TEST 1. Shake 2.5 g of soil (dry weight basis or 2.5 cm3packed volume) for 15 min with 25 ml of 1 m M CrCl,. 2. Add 0.25 ml of 1 M pH 7.2 KH2P0,.K2HP0,, shake 15 sec longer, and then filter or centrifuge.

REDOX CHEMISTRY OF SOILS

20 3

3. Determine Cr( VI) by adding 1 ml diphenylcarbazide (DPC) reagent to 8 ml of extract or water, mix, and let stand 20 min, and then compare the color with that in the standards (0.5-50 p M ) at 540 nm. (Prepare the DPC reagent by adding 120 ml of 85% phosphoric acid, diluted with 280 ml distilled water, to 0.38 g of s-diphenylcarbazide dissolved in 100 ml of 95% ethanol.) 4. If cloudiness from precipitated organic or mineral matter is present, it is easily removed by filtering, following color development, using a 0.2-pm filter and syringe. The colored complex remains in solution, and filtration of standards and unknowns improves sensitivity. A portion of the Cr oxidized during the course of this test is not measured as Cr(V1) because it is reduced almost as fast as it is oxidized. Depending on the availability of easily oxidizable organic matter, some or even most of the Cr(V1) formed is reduced during the 15-min period. Leaching a sample, especially a dry one, will remove some of the low-molecular-weight reducing organics and thereby will increase the Cr( VI) quantity measured (Bartlett, 1981a). With dried and stored “lab dirt” samples, the reduction frequently equals the oxidation and no net Cr(V1) is measured. This test characterizes oxidation only to the extent that it exceeds reduction.

G. SOILREDUCINGINTENSITY 1. Shake intermittently 2.5 cm3 of moist soil I8 hr with 20 ml of pH 4.0 NH40Ac, 0.6 M with respect to ammonium, containing 0.1, 0.5, or 2.5 m M K,Cr,O,. 2. Filter or centrifuge, and determine concentration of Cr(V1) remaining (Section XI1,F). If all of the Cr(V1) is reduced, repeat with increased concentrations until the Cr(V1) remaining is measurable; 1 mol of Cr is equivalent to 6 mol of manganese plus charge. 3. To measure the tendency of a particular oxidizable organic substance to reduce Cr( VI), repeat this test after adding the organic substance to the soil sample.

H. AVAILABLE REDUCINGCAPACITY 1. Shake intermittently 2.5 g soil, dry weight basis (or 2.5 cm3 of moist soil), for 18 hr, with 25 ml of 0.1, 0.5, 2.5, or 10 m M as K2Cr20, in 10 m M H3PO4, filter or centrifuge, and determine Cr(V1) not reduced in the extract (Section XI1,F).

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2. Begin with the lowest concentration of Cr(VI). If all of the Cr( VI) is reduced, repeat with increased concentrations until the Cr( VI) remaining is measurable but below 0.1 mM.

I. ELECTRONDEMAND BY IODIDE OXIDATION 1. As a direct titration of the reducible Mn oxides in a soil sample, the manganese electron demand (MED) determination is a direct way of estimating the oxidizing capability by the Mn oxides in a soil, without consideration of the reduction that might take place under the conditions of a less direct oxidation measurement, such as Cr( VI) (Bartlett, 1988). 2. Shake intermittently 2 g soil, dry weight basis (or 2 cm3 moist soil), for 18 hr, with 12 ml of pH 4.0 NH,OAc, 0.6 M with respect to ammonium, and 4 ml of 0.2 M KI. Add 4 drops of starch solution (0.3 g potato starch boiled with 50 ml water) and titrate to a colorless endpoint with 2 m M Na,S20, . Millimoles of thiosulfate per unit of soil are equivalent to millimoles of e- or plus charge of Mn. 3. “Total” electron demand (TED) is modified from MED as follows: 12 ml of 0.1 M HCI is added instead of pH 4.0 acetate, and the equilibration time, instead of an approximate 18 hr, is a rigid 15 min with centrifuging and titration required immediately afterwards. The time is critical in soils that contain high amounts of recently oxidized Fe, because the amount of easily reducible Fe that will react may be an ambiguous valley rather than an easily identifiable peak. A more serious problem is that iodide is slowly oxidized by 0, at low pH, and inflated TED values can result if time is not strictly limited. Thus, TED is very much an empirical measurement, although, with strict control of time, the test is amenable to calibration for measuring a particular species or fraction of reducible Fe.

J. DISMUTATION OF H,02 Manganese oxides, at pH >6, and catalase enzymes both destroy H,O, by catalyzing its dismutation [Eq. (34)]. The rate of dismutation is a better indication of quantities and activities of catalytic substances present than is amount of H,02 dismutated, because a small amount of catalyst will act on a large amount of substrate. The rate of dismutation can be evaluated by adding 5 ml of 0.5 M H,O, solution to 2.5 g of moist soil, on a dry weight basis, and clocking the time required for the soil to evolve enough bubbles to displace 24 ml of H 2 0 (1 mmol of 0,).

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K. INTERFERENCES Most colorimetric tests useful in studying redox processes in soils also involve redox reactions, and because we are trying to measure colorimetrically quantities of particular redox species in the presence of other redox species, we have many opportunities for color-interfering interactions among the various reactions. Manganese oxides produce positive interference in measuring nitrate by the hydrazine reduction method (Prochazkova, 1959) and also with brucine or diphenylamine, positive interference in nitrite by the diazonium salt method, but no interference with the diphenyl carbazide color for Cr( VI). Nitrite, citrate, and hydroxylamine negatively interfere with the Cr(VI) test, and nitrite and Cr( VI) positively interfere with determination of nitrate by brucine. Highly oxidized hypochlorite and peroxides and highly reduced substances, such as thiosulfate, sulfides, amines, and ascorbic acid, interfere with everything imaginable. Nitrate is too inert to interfere with most tests, which tells us something about the reactivity of nitrate. Wariness, ingenuity, and flexibility are required for redox titrations.

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Bartlett, R., and James, B. (1979). Behavior of chromium in soils. 111. Oxidation. J. Environ. QWl. 8, 31 -35. Bartlett, R. J., and James, B. R. (1980). Studying airdried, stored soil samples-Some pitfalls. Soil Sci. Soc. Am. J. 44, 72 1 -724. Bartlett, R. J., and Ross. D. S. (1988). Colorimetric determination of oxidizable carbon in acid soil solutions. Soil Sci. Soc. Am. J. 52, I 191 - 1 192. Berner, R. A. (1981). A new geochemical classification of sedimentary environments. J. Sediment. Petrol. 51,359-365. Blaylock, M. J., and James, B. R. (1992). Oxidation-reduction behavior of selenite in soils. Soil Sci. Soc. Am. J. (submitted). Bouldin, D. R. (1968). Models for describing the diffusion of oxygen and other mobile constituents across the mud-water interface. J. Ecol. 56,77-8 1. Bricker, 0. P. (1982). Redox measurement: Its measurement and importance in water systems. Warer Anal. 1. Brudvig, G. W., and Crabtree, R. H. (1989). Bioinorganic chemistry of manganese related to photosynthetic oxygen evolution. Prog. Inorg. Chem. 37,99- 142. Castellan, G. W. (1983). “Physical Chemistry,” 3rd Ed. Addison-Wesley, Reading, Massachusetts. Cohen, G. I. (1985). The Fenton reaction. In “Handbook of Methods for Oxygen Radical Research’’ (R. A. Greenwald, ed.), pp. 55 - 69. CRC Press, Boca Raton, Florida. Cotton, F. A., and Wilkinson, G. (1980). “Advanced Inorganic Chemistry.” Wiley, New York. Diem, D., and Stumm, W. (1984). Is dissolved Mn2+being oxidized by 0, in absence of Mn-bacteria or surface catalysts? Geochim. Cosmochim. Acta 48, 157 I - 1573. Dion, H. G., and Mann, P. J. G. (1946). Three-valent Mn in soils. J. Agric. Sci. 36, 239245.

Ehrlich, H. L. (1976). Manganese as an energy source for bacteria. Environ. Biogeochem. Proc. Int. Symp. 2nd, 2. Farrell, R. E., Swerhone, G. D. W., and van Kessel, C. (199 I). Construction and evaluation of a reference electrode assembly for use in monitoring in situ soil redox potentials. Commun. SoilSci. Plant Anal. 22, 1059- 1068. Fridovich, 1. (1975). Superoxide dismutases. Annu. Rev.Biochem. 44, 147- 159. Fridovich, 1. (1978). The biology of oxygen radicals. Science (Washinglon. D.C.) 201, 875880. Fruton, J. S., and Simmonds, S. (1961). “General Biochemistry.” Wiley, New York. Carrels, R. M., and Christ, C. L. (1965). “Solutions, Minerals, and Equilibria.” Freeman, San Francisco, California. Halliwell, B. (1974). Manganese ions, oxidation reactions and the superoxide radical. Neure tOXiCOlOgY 5, 1 13 - 1 18. Harter, R. D., and Smith, G. (1981). Langmuir equation and alternate methods for studying “adsorption” reactions in soils. In “Chemistry in Soil Environments” (D. Baker, ed.),pp. 167- 182. Soil Sci. Soc. Am., Madison, Wisconsin. Hines, M. E., Knollmeyer, S. L., and Tugel, J. B. (1989). Sulfate reduction and other biogeochemistry in a northern New England salt marsh. Limnol. Uceanogr. 34, 578590.

James, B. R. (1989). Electron activity in soils: A key master variable. Agron. Abstr., 20 1. James, B. R., and Bartlett, R. J. (1983). Behavior of chromium in soils. VI. Interactions between oxidation-reduction and organic complexation. J. Environ. Qual. 12, 173176.

Jenny, H. (1980). “The Soil Resource,” pp. 1 1 3- 195. Springer-Verlag, New York. Kittrick, J. A,, Fanning, D. S., and Homer, L. R. (1982). Acid sulfate weathering. SSSA Spec. Publ.. No. 10.

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Liem, H. H., Cardenas, F., Tavassoli, M., Poh-Fitzpatrick, M. B.,and Muller-Eberhard, U. (1979).Quantitative determination of hemoglobin and cytochemical staining for peroxdihydrochloride, a safe substitute for benzidine. ide using 3,3’,5,5’-tetramethylbenzidine Ann. Biochem. 98,388-390. Lindsay, W. L. (1979).Chemical Equilibria in Soils,” pp. 386-412. Wiley-Interscience, New York. Liu, C. W., and Narasimhan, T. N. (1989).Redox-controlled multiple-species reactive chemical transport. I . Model development. Waier Resour. Res. 25,869-882. Loach, P. A. (1976).Oxidation-reduction potentials, absorbance bands, and molar absorbance of compounds used in biochemical studies. Handb. Biochem. Mol. Biol. 3rd Ed. Loganathan, P., Burau, R. G., and Fuerstenau, D. W.(1977).Influence of pH on the sorption of Co2+,Zn2+and Ca2+by a hydrous manganese oxide. Soil Sci. Soc. Am. J. 41,57-62. Lutz, H. J., and Chandler, R. F. (1946).“Forest Soils,” pp. 140-480. Wiley, New York. McBride, M. B. (1982).Electron spin resonance investigation of Mn2+ complexation in natural and synthetic organics. Soil Sci. Soc. Am. J. 46, 1137- 1142. Magdoff, F. R., and Bouldin, D. R. (1970).Nitrogen fixation in submerged soil-sand-energy material media and the aerobic-anaerobic interface. Plant Soil 33,49- 53. Marschner, H. (1990).“Mineral Nutrition of Higher Plants,” pp. 336-337. Academic Press, San Diego, California. Masscheleyn, P. H., Delaune, R. D., and Patrick, W. H. (1991). Arsenic and selenium chemistry as affected by sediment redox potential and pH. J. Environ. Qual. 20, 522527. Matia, L., Rauret, G., and Rubio, R. (1991).Redox potential measurement in natural waters. Fresenius Z. Anal. Chem. 339,455-462. Moore, J. N., Walker, J. R.. and Hayes, T. H. (1990).Reaction scheme for the oxidation of As(lI1) to As(V) by birnessite. Clays Clay Miner. 38, 549-555. Mueller, S. C., Stolzy, L. H., and Fick, G. W. (1985).Constructing and screening platinum microelectrodes for measuring soil redox potential. Soil Sci. 139, 558-560. Pohlman, A. A,, and McColl, J. G. (1988).Organic oxidation and metal dissolution in forest soils. Soil Sci. Soc. Am. J. 52, 265-27 I. Ponnamperuma, F. N. ( I 972).The chemistry of submerged soils. Adv. Agron. 24,29-96. Prochazkova, L. (1959).Bestimmung der Nitrate in Wasser. Frescnius 2. Anal. Chem. 167, 254-260. Rabenhorst, M. C., and James, B. R. (1992).Iron sulfidization in tidal marsh soils. In “Biomineralization processes of iron and manganese” (H. C. W.Skinner and R. W. Fitzpatrick, ed.). (Catena Suppl. 21.)Catena Verlag, CremCngen-Destedt, Germany. Rabenhorst, M. C., James, B. R., and Shaw, J. N. (1992).Evaluation of potential wetland substrates for optimizing sulfate reduction. Proc. Nail. Meet. Am. Soc. Surf Min. Reclam.. Duluth, Minnesota (in press). Reddy, K. R., and Patrick, W. H. (1980).Evaluation of selected processes controlling nitrogen loss in a flooded soil. Soil Sci. Soc. Am. J. 44, 1241 - 1243. Ross, D. S..and Bartlett, R.J. (1981).Evidence for nonmicrobial oxidation of manganese in soil. SoilSci. 132, 153-160. Rowell, D.L.( I98I ). Oxidation and reduction. Chem. Soil Processes, 40I -463. Russell, E. W. (1973).“Soil Conditions and Plant Growth,” 10th Ed., pp. 670-695.Longman, London, England. Schnitzer, M., and Khan, S. V. (1972).“Humic Substances in the Environment,” p. 300 Dekker, New York. Senesi. N., and Schnitzer, M. (1978).Free radicals in humic substances. Environ. B i o g w chem. Geomicrobiol., Proc. Inr Symp., 3rd, 2,467-480. Shindo, H. (1990).Catalytic synthesis of humic acids from phenolic compounds by Mn(1V) oxide. Soil Sci. Plant Nutr. (Tokyo) 36,679-682.

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Shindo, H., and Huang, P. M. (1982). Role of Mn(1V) oxide in abiotic formation of humic substances in the environment. Nuture (London) 298, 363-365. Shindo, H., and Huang, P. M. (1984). Significance of Mn(IV) oxide in abiotic formation of organic nitrogen complexes in natural environments. Nature (London)308, 57- 58. Sillen, L. G. (1967). Master variables and activity scales. Adv. Chem. Ser. Silver, M., Erlich, H. L., and Ivarson, K. C. (1986). Soil mineral transformation mediated by soil microbes. In “Interactions of Soil Minerals with Natural Organicsand Microbes” (P. M. Huang and M. Schnitzer, eds.).pp. 497-5 19. Soil Sci. Soc. America, Madison, WI. Sparks, D. L. (1985). Kinetics of ionic reactions in clay minerals and soils. Adv. Agron. 38, 23 I -265. Sparrow, L. A., and Uren, N. C. ( I 987). Oxidation and reduction of Mn in acidic soils: Effect of temperature and soil pH. Soil Biol.Biochem. 19, 143- 148. Sposito, G. (198 I). “The Thermodynamics of Soil Solutions.” Oxford, New York. Sposito, G. (1989). “The Chemistry of Soils.” Oxford, New York. Stone, A. T., and Morgan, J. J. (1984). Reduction and dissolution of manganese(ll1) and manganese(1V) oxides by organics: 2. Survey of the reactivity of organics. Environ. Sci. Technol. 18,617-624. Stumm, W., and Morgan, J. J . (1981). “Aquatic Chemistry,” 2nd Ed.,pp. 418-504. WileyInterscience, New York. Sullivan, J. C., Gordan, S., Cohen, D., Mulac, W., and Schmidt, K. H. (1976). Pulse radiolysis studies of uranium (VI), neptunium (VI), neptunium (V), and plutonium (VI) in aqueous perchlorate media. J. Phys. Chem. 8, 1684- 1686. Sunita, J. M., Loll, M. J., Snipes, W. C., and Bollag, J. M. (1981). Electron spin resonance study of free radicals generated by a soil extract. Soil Sci.131, I45 - 150. Thompson, 1. J. (1923). “The Electron in Chemistry.” Franklin Institute, Philadelphia, Pennsylvania. Thorp, H. H., and Brudvig, G. W. (1991). The physical inorganic chemistry of manganese relevant to photosynthetic oxygen evolution. New J. Chem. 15,479-490. Vincent, A. (1985). “Oxidation and Reduction in Inorganic and Analytical Chemistry.” Wiley, Chichester, England. Wang, T. S. C., Huang, P. M., Chou, C.-H., and Chen, J.-H. (1986). The role of soil minerals in the abiotic polymerization of phenolic compounds and formation of humic s u b stances. Interact. Soil Miner. Nat. Org. Microbes Proc. Symp., 1983, 25 I - 28 1. Weaver, J. H. (1987). “The World of Physics.” Simon and Schuster, New York. Westcott, C. C. ( I 978). “pH Measurements.” Academic Press, New York. Zumdahl, S. S. (1986). “Chemistry,” pp. 931 -935. Heath, Lexington, Massachusetts.

PLANTNUTRIENT SULFURIN THE I~OPICS AND SUBTROPICS N. S. Pasricha’ and R. L. Fox’

’

Department of Soils, Punjab Agricultural University, Ludhiana, India Department of Agronomy and Soil Science, University of Hawaii at Manoa, Honolulu, Hawaii 96822

1. Introduction 11. Extent of Sulfur Deficiency 111. Forms of Sulfur in Soil A. Sulfur Transformation Products B. Sulfate Sulfur IV. Sulfur Cycling in the ‘Tropics A. Sulfur Supplies of Atmospheric Origin B. Sulfur Accession through Precipitation V. Effects of Acid Rain A. Effect on Crop Plants B. Effect on Forest Vegetation C. Effect on Soil Acidification VI. Sulfur in Irrigation Waters A. Sulfur in Streams B. Sulfur in Groundwater VII. Sulfate Retention in Soil A. Sulfate Adsorption and Desorption B. Sulfate Adsorption Curves C. Mechanism of Sulfate Adsorption VIII. Diagnosis of Sulfur Needs A. Soil Tests B. Plant Analysis IX. Critical Soil Solution Concentration X. Crop Responses XI. Sulfur Fertilization and Crop Qualiry A. Effect on Protein Oualiry B. Effect on Oil Content

Adwnrti m Abmnary. C‘ol 10 Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved

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C. Effect on Glucosinolate Content D. Effect on Nitrate Content XU. Sulfur Interactions with Other Elements A. Interaction with Phosphorus B. Interaction with Other Elements XI11. Summary and Conclusions References

I. INTRODUCTION Many generalizations of doubtful validity have been promulgated and postulated about “tropical soils.” One example is the concept that soils of the tropics are highly weathered. In fact, many soils of the tropics are so highly weathered that little remains except sesquioxides and some 1 : 1 layer silicates, but there are also numerous examples of tropical soils in which 2 : 1 clays influence, or even dominate, soil chemistry and physics. Because many soils of the tropics have reached an advanced stage of weathering, it is frequently assumed that chemical weathering in the tropics is greatly accelerated as compared with the temperate zone. However, in most of the tropics, rainfall is seasonal. The dry season may retard chemical processes just as effectively as the cool season does in the temperate zone. But even in the tropics, rejuvenating influences have operated in soils (Jackson et al., 1971; Syers ef al., 1969). Inasmuch as some concepts about tropical environments may be inappropriate, if not inaccurate, it follows that some of the concepts about the chemistry and nutritional status of soils of the tropics based on those assumptions should be reexamined. It is probably safe to say that there is nothing unique about the types of soil fertility problems encountered in soils of tropical and subtropical regions. Soil fertility problems are not necessarily more complex either, although multiple nutrient deficiencies tend to be the rule rather than the exception for leached and weathered soils of the humid tropics. A more unique feature of the tropical and subtropical zones is the magnitude of soil fertility problems in relation to the resources that are being committed, or being advocated, for alleviating those problems. Sulfur deficiencies in tropical and subtropical regions have been recognized for many years. The deficiency is particularly widespread in the semiarid and subhumid savannas of tropical America and Africa. BoleJones (1964) reviewed the early work on S deficiencies in Africa. He concluded that deficiencies are most likely on fermginous and ferrallitic soils developed on very old erosion surfaces that separate major drainage systems south of the Sahara. Sulfur deficiency was described by McClung

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21 1

and de Freitas (1958) as a major fertility problem for developing the Brazilian Campos. Sulfur was second only to P as a nutritional factor limiting growth of grasses. McClung ef al. ( 1958) believed that responses to S in central Brazil would be more common if N and P were plentifully supplied and if cropping were intensive. de Freitas et al. (1972) reported benefits from applying S fertilizer to coffee in Sao Paulo State, Brazil. Sulfur deficiencies have been reported from other tropical and subtropical areas, including islands in the Caribbean (Haque and Walmsley, 1973) and Hawaii (Fox et al., 1965). There is considerable evidence of S deficiency in subtropical regions (Kanwar and Mohan, 1962; Kanwar, 1963) and of the response to its application (Dalal et al., 1963; Chopra and Kanwar, 1966; Pasricha and Randhawa, 1971). However, there is less evidence of S deficiencies in areas of tropical rain forests in Nigeria (Kang and Osiname, 1976). The sandy surface soil therein contains a modest amount of adsorbed sulfate with a solubility of 8 p g S m1-I. The subsoil, which contains more clay, is relatively rich in adsorbed sulfate but the solubility is low (Fox and Blair, 1986). It now seems probable that better sulfur nutrition of crops in forest zones is related to the capacity of subsoils to retain sulfate. Although detailed data are lacking, savanna soils seem to be much poorer in adsorbed sulfate than are soils of rain forests. In this review we intend to give an interpretive account of developments in various aspects of sulfur as a plant nutrient in subtropical and tropical soils.

11. EXTENT OF SULFUR DEFICIENCY An examination of the pattern of S deficiency on a global basis leads at once to the conclusion that areas prone to S deficiency are those which are remote from smelting industries and from heavy industrial or domestic burning of fossil fuels; areas in which weather is controlled by air masses originating from such regions; and areas which have marked wet-dry rainfall patterns giving rise to a savanna-type vegetation that is frequently burned. The situation becomes locally worse where soils are derived from basic igneous materials, especially volcanic ash, at intermediate or high elevations and some distance from the sea. Generally these are the areas in which low quantities of SO, are deposited in precipitation. Many tropical and subtropical areas fall into one or more of these categories. Thus, it is not surprising to observe that S deficiencies are frequently encountered in the tropics and subtropics. Numerous observations on a variety of crops in the tropics have demon-

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strated that incipient S deficiencies do abound. This is related to low concentrations of S in rainwaters and generally low levels of organic S in soils (Fox and Blair, 1986). Organic matter contents of tropical soils are not so low as is sometimes supposed. This fact is evidence for the stability of such organic matter that persists. It should not be assumed, however, that the availability of S in the resistant organic matter will be as readily available to plants as that which has already decomposed. The implications of this for the future are clear; organic S will be decreasingly important with increasing time of arable agriculture. To complicate the problem, some of the soils of the humid tropics adsorb S q - strongly, especially in subsurface horizons. These soils may be S deficient even though they contain much SO, (Hue et al., 1990). Sulfate solubility, and presumably its availability to plants, is related to the quantity of adsorbed SO, in relation to the capacities of soils to adsorb SO,. Most soils developed in weathered volcanic ash adsorb SO, strongly. Widespread S deficiencies in groundnut in the West Africa savanna have been recognized for 40 years and are well documented. Fox (1980a) estimated that exported S in 750,000 tons of groundnut kernels would amount to 1500 tons S per year, assuming a nominal content of 0.2% S in the kernels. Although this is not a large quantity by the standards of S reserves in most temperate zone soils, it is a serious drain for a system in which S deficiency has been chronic. The magnitude of the problem is placed in better perspective by considering that the total expected S content of rain for the groundnut-growing belt of northern Nigeria ( 1.2 million hectares) amounts to about 1400 tons, essentially equivalent to S being exported (Bromfield, 1974). Kang et al. (198 1) reported results of laboratory and greenhouse expenments comparing the S status of savanna and forest upland soils of 30 surface soil samples (0- 15 cm) from Nigeria. The S status was not related to parent material or soil type. Total and extractable S levels were in the following order: forest zone > derived savanna > Guinea savanna. Observed sulfur deficiency was most acute in soils from Guinea savanna and least acute from the forest zone. Sulfur responses are most frequently observed in the savanna zone (Enwezor, 1976). Sulfur deficiencies have been reported from several locations in the humid tropics and a few studies on the sulfur status of soils there have been reported (Bornemisza and Llanos, 1967). However, much more needs to be done before accurate predictions can be made on the magnitude of S problems, or probable requirements and effectiveness of S fertilizers. Many Central American soils, particularly those under more or less permanent crops such as coffee, cocoa, sugarcane, or pastures, have accumulated organic matter in the surface and consequently contain high organic S levels (Bornemisza et al., 1978; Burbano and Blasco, 1975; Granados,

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21 3

1972; Hardy and Bazan, 1966). However, a favorable C :S ratio is more important than a large store of organic matter for S response under tropical conditions (Bromfield et af.,1982). This is because the ratio influences amounts of S that will be mineralized (or immobilized) during microbial decomposition of organic matter. Bornemisza ( 1990) reported detailed information on S distribution and S problems in Central America. About half of the soils in the Entisol and Inceptisol orders respond to S, especially after N and P deficiencies have been corrected. Soils that are coarse textured are most responsive because of the leaching that is associated with them. Cordero et af. (1986) reported that in some of these soils, S deficiencies are the main production-limiting factor. Andisols are important in the area because of widespread volcanic ash influence, and research on S problems of these soils has been reported (Jimenez and Cordero, 1988).The more highly weathered Andisols adsorb large amounts of SO, on exchange sites below the A horizon (Gebhart and Coleman, 1974). However, SO, can be displaced by phosphate fertilizers, which can result in deficiencies, as has occurred in El Salvador (Muller, 1965). Sulfur deficiencies have been reported on Mollisols particularly after the P status was improved by P fertilization (Kass et af., 1984). Research in Panama and Costa Rica indicates considerable variation in S status of Alfisols. Research in Panama has confirmed S problems in these soils both in pot studies and in field conditions. Comparable soils in Brazil have shown strong pH-dependent SO, adsorption capacity (Couto ef al., 1979). In the Pacific lowland of Central America, S deficiency in Vertisols becomes evident only after correcting P deficiencies and consequent leaching of SO, (Bornemisza et al., 1978). In Brazil, accumulations of SO, results in increased cation retention, which can partially compensate for low exchange capacities of soils. Insufficient S usually becomes a problem for sugarcane after problems of N, P, and sometimes K have been corrected. Williams and Andrew (1970) were unable to outline any major occurrence of S deficiency in tropical Australia. It cannot be inferred, however, that S is adequate on all soils and one must look further for an explanation of this apparent sufficiency of S. The accession of S by the plant/soil system in this nonindustrialized area, if indeed it does accrue, can be considered a redistribution rather than a gain (Wetselaar and Hutton, 1963), and in any case must be low. At Townsville (near the sea) and Woodstock (20 km inland), depositions of 5.7 and 2.6 kg S ha-' annum-', respectively, have been measured (Jones et af., 1975). Other records are available from coastal stations further south in Queensland (Sedl, 197 l), where annual deposition of 3.7 to 25.2 kg S ha-' was measured. The higher figures were registered during periods of intense rainfall associated with cyclones. In tropical regions with little industrial activity, SO4-S concentrations in

214

N. S. PASRICHA AND R. L. FOX Table I Sulfate S in Soils along Tmsects in Rwanda as Determined by Repeated Phosphate Extraction" Surface Mean Transect no.

Soil

Solyinyo ( I ) Karisimbi (3) Karisimbi (2) Ruhengeri-Kigali (4) Kigali-Kibuye (5) Cyangugu-Gikongoro (6) Butare (6) Bugesera (7) Kigli-Kibungo(8) Gabiro-Ryumba (9)

Andept Andept Andept Udult Tropept/Humult Tropept/Humult Tropept Ustox/Orthox Ustox/Ustalf Ustox/Usdult

Mean

Range

Subsoil Mean

( M g-')

27 64 29 23 19 45 5.6 I1 11.5

27 26.2

2-84 6 - I36 2-66 2-46 2-54 4 - I80 0- 16 0-32 4-28 4 - 120

Range g-9

19 50 15 28 26 48 4 34 41 24

2-24 14- I38 2-44 8-50 8-72 8-94 1-12 6-62 6 - 1 372b 4- 102

29

Adapted from Vander Zaag ef a[. (1984). bvalues > 200 not included in the average.

rainwater are usually less than 1 pg m1-I. In Rwanda, SO, was generally a little more abundant in subsoil samples than in 0- to 15-cm samples (Table I). Information on availability of subsoil sulfate is limited, but deep-rooted crops are likely to benefit from subsoil sulfate. Sulfate levels determined in these soils appear to be deficient or near deficient. For soils from volcanic ash (Dystrandepts) 25 pg g-l was inadequate (Fox et al., 1965), but 810 pg g-l may be adequate for soils that do not adsorb sulfate in appreciable quantities (Kang and Osiname, 1976). Sulfur deficiency is widespread in rice fields in Bangladesh (Hoque and Hobbs, 1978; Hussain, 1990). About 44% of the cultivated land in Bangladesh is estimated to be S deficient. Crops grown on these soils respond to S application. The major cause of S deficiency seems to be extremely low redox conditions in wetland rice, although SO, is the last compound to undergo reduction, after NO3-, Mn (IV), and Fe (111) compounds, when reducing conditions set in. In soils with fine texture and appreciable decomposable organic matter, SO, reduction to H,S is likely. Decreased incidental addition of S is a probable reason why S deficiency is now appearing more frequently than formerly. Besides smaller accretions of S by rain, a drastic decrease in incidental S additions in fertilizers is

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100 h

a

0 1950

1960

1970

1980

1990

Figure I . Trends in consumption of S-bearing N and P fertilizers in India during 19501990 (Pasricha and Aulakh, I99 I ; by permission of The Sulphur Institute, Washington, D.C.)

a probable cause for the appearance of S deficiency in crops in India (Pasricha and Aulakh, 199I). In 1950, sulfur-containing fertilizers were commonly used sources of N and P. At that time 28,900 tons of N as ammonium sulfate and 4279 tons of P as single superphosphate were applied. The consumption of fertilizer N and P has increased sharply since then to 7,396,000 tons of N and 1,315,000 tons of P in 1990. However, use of N and P fertilizers that contain S has relatively decreased, thus resulting in a drastic decrease in the use of S (Fig. 1). An analysis of 1164 soil samples collected from all over India indicated S deficiency to the extent of 4 1 % (Singh, 199I). In Thailand, Hoult et al. ( 1983) estimated that 50% of the northeast plateau, 30-40% of the southeast coast, and 30-40% of the northern highland area are S deficient and would respond to S fertilization.

111. FORMS OF SULFUR IN SOIL

A. SULFURTRANSFORMATION PRODUCTS Sulfur is continuously cycled between inorganic and organic forms. Three broad fractions of organic S have been identified: (1) ester sulfate, (2) C-bonded S (mainly amino acids), and (3) residual S (Tabatabai, 1982). The nature of the compounds formed and their transformations are

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strongly influenced by biologically mediated processes, which in turn are affected by environmental conditions. Perhaps 95% or more of the total S in arable soils in temperate regions is organic S. This generalization does not apply in the tropics. Inorganic SO, vanes seasonally in soils. Castellano and Dick ( 1991) observed that during rainy winter seasons, SO, levels ranged from 7 to 13 mg kg-' in gypsum-treated plots compared with 2 to 7 mg kg-' in control plots. In the months from March to May, biomass S increased and SO, level decreased (<6 mg SO4-S kg-' soil), indicating that S immobilization occurred in the spring. Activity of aryl sulfatase (an enzyme that releases SO, from aromatic SO, esters) increased significantly in cropped plots where plant activity was greater (Tabatabai and Bremner, 1970; Castellano and Dick, 1991 ). Carbon-bonded s, which is mainly a measure of the amino acids cystine and methionine, correlates with biomass S. Generally these compounds do not accumulate in soil because they readily decompose (Fitzgerald, 1986). Residual S is resistant to hydrolysis by strong acids or bases, yet it varies with the season (Freney, 1986). However, this variation may be due to measurement errors associated with other fractions, because residual S is determined by the difference between total S and the sum of all other S fractions. Assuming the data are reliable, seasonal variation follows a pattern that appeared to be influenced by biological activity during 2 years. With increasing moisture in the autumn, residual S increases, whereas in the cool winters and springs, there is a general decrease in residual S. Residual S is least in the dry summer. Ester sulfate accumulation in the soil was associated with incorporation of inorganic SO, by Saggar et al. (198 1). Ester sulfate constituted from 20 to 65% of the total S in a group of six Brazilian soils (Neptune et al., 1975). Several short-term laboratory/greenhouse studies using 35S0, have found that ester sulfate is more transitory than C-bonded S (McLaren ef al., 1985; McLaren and Swift, 1977; Freney el al., 1975). These studies have shown that, typically, 60-90% of 35S0, added to soil is quickly incorporated into the ester sulfate fraction. A large proportion of S taken up by plants comes from the ester sulfate pool and not the C-bonded S pool. There is a tendency for the initial incorporation of applied S into ester sulfate. With time and with further S cycling, a large portion is redistributed into Cbonded and residual S pools.

B. SULFATESULFUR Many soils of the humid and subhumid temperate zones contain only small quantities of inorganic S. In such soils, plants quickly utilize S

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217

mineralized through slow and continuous oxidation of soil organic matter, together with S that accrues as dissolved SO, in rainwater and SO, absorbed directly by soils and plants from the atmosphere. In such soils, the major fraction of S is in the organic form, but in tropical and subtropical conditions, SO,-S may also be present in appreciable quantities. In fact, in many such conditions, where soils are coarse textured, the amount of organic matter is very small. Whatever little organic material is added through crop residues is rapidly oxidized and SO,-S is released. Organic S, which is sometimes referred to as reserve S, is generally an appropriate designation for soils that contain relatively little inorganic S in relation to organic S. Some soils in the tropical zone contain so much SO4-S in relation to organic S that the term reserve S seems inappropriate to describe the organic fraction. Beaton et al. (1985) mentioned especially gypsum-rich soils of arid and semiarid regions and soils developed in highly weathered volcanic ash materials. Andepts of Hawaii are examples of such soils. These may contain 0.7% SO,-S in the soil materials below 30 cm (Hue et al., 1989). Although highly weathered soils may contain great quantities of sorbed SO,, soils with properties dominated by the layer silicates sorb little SO,. Neptune et al. (1975) observed that 58% of the readily extractable S in a group of soils from Brazil was sorbed SO,, as compared with 2% in soils from Iowa.

IV. SULFUR CYCLING IN THE TROPICS Sulfur cycling has important implications because cyclic S is a source of S for crops, and yet the S being cycled out can be a major drain on the S economy of crop production systems. The most obvious illustration of S cycling is the soil - plant - rain (through-fall) pathway. Rainfall is the major contributor of S for agriculture almost everywhere. Sulfur accruing to soils and crops via that pathway (wet deposition) in the tropics is generally low in absolute terms but in relative terms it is a major factor. In much of the tropics, S in precipitation approaches background levels of approximately 0.1 to 0.2 mg liter-'. For example, the mean of I 1 sites in Northern Nigeria was 0.13 mg liter-' (Bromfield, 1974). In sugarcane-growing areas along the coast of Queensland, Australia (five sites), it was 0.88 mg liter-'. Rainwater, if it passes through a plant canopy, leaches S from the foliage. Sulfur is relatively immobile in plants. It tends to accumulate in old tissue, from which it apparently leaches with relative ease. This point has not been investigated for tropical conditions, but for a wheat canopy in England during 2 months preceding maturity, S outputs from the canopy exceeded S inputs by a factor of 2.15 (Raybould ef al., 1977).

N. S. PASRICHA AND R. Id. FOX

218

A

A

E

s

$

aid >.E \f

3

H

z

53 0 c

a

(1

' a *

" 3 :: 2 4 vr

$9

x

D

A

n 22 Transfer to ocean

3.

95

(T) Figure 2. Global sulfur cycle (units: lo6tons S/annum) (Robinson and Robbins, 1968).

Another pathway is the atmosphere-plant-soil route. This is called dry deposition and is important in industrial and residential areas where fossil fuels are burned. In the tropics, burning of vegetation is relatively more important. Areas that have a marked wet-dry rainfall pattern giving rise to savanna-type vegetation that is regularly burned no doubt lose much of the S that accrues to them in rainfall in this way. Large areas of the tropics are so affected. Burning is generally done in the dry season. Thus there is little likelihood that S volatilized by agricultural burning will be redeposited on land from which it came. A disproportionate quantity will accrue to downwind locations and to nearby areas where soils are moist and vegetation is green (Fox and Blair, 1986). Burning is likely to be important in the redistribution of S in tropical Australia. Most native vegetation is subject to fire (Tothill, 1971), but we were unable to find reports on S losses from such fires. Losses are likely to be appreciable, if data from burning of heather (36% loss) are a guide. Sulfur is one of the main components of atmospheric deposition. Atmospheric deposition impacts S cycling both directly and indirectly. The inputs of S may be relatively large (60 kg S ha-' yr-l) in some ecosystems (Johnson, 1984) and very few studies have been done on the effect of these inputs on S cycling. Increased numbers of S oxidizers have been measured,

PLANT NUTRIENT SULFUR

219

but effects on S oxidation have been mixed (Wainwright, 1979, 1980). The sulfur cycle is important in understanding the S budgets of soil. One estimate of the global S cycle (Robinson and Robbins, 1968) is presented in Fig. 2. Major components of the S cycle system are gaseous S in the atmosphere, dissolved S in rainwater, S in surface and groundwater (imgation), inorganic S in soils, organic S in soils and plant residues, and S in vegetation. In areas of coarse-textured, low-organic-matter soils that lack significant capacities for surface sorption, the limit of S uptake (S yield) by crops is approximately equal to dissolved S in precipitation. If incoming S is in the range of 1 to 4 kg S ha-', sulfur deficiency becomes a major constraint, even for low levels of production, in areas as diverse as the United States (Alabama, Nebraska, and Hawaii), Nigeria, Australia, and New Zealand (Fox and Hue, 1986). Gaseous S may contribute 20% or more of the S taken up by plants, but this S source is not a significant factor in most tropical areas.

A. SULFURSUPPLIES OF ATMOSPHERIC ORIGIN Because the atmosphere is the major S source for most upland soils, the S budget can be understood better if it is viewed in the context of the total environment. It is, therefore, important to know the S compounds in the atmosphere and their concentration and chemical behavior, the source of S in the atmosphere, and the quantity of S supplied to soils from the atmosphere. Atmospheric S is generally present in gaseous form as H,S and SO,, and as particulate forms as sulfate. Considerations of the global S budget show that HzS, SO,, and particulate SO, are present as trace constituents. These are primarily of natural origin except in polluted areas where anthropogenic emission may dominate. There are few reliable data on atmospheric H,S. Concentrations of this gas are greatest near natural swamps, anaerobic waters, industrial sources, volcanoes, geothermal wells, etc. Hydrogen sulfide has a background concentration between 5 and 50 parts in lo', parts of air by volume (Slatt et al., 1978). It has been proposed that reaction of carbaryl sulfide and CS, is an important source for both HzS ( McElroy ef af.,1980)and SO, (Logan et af.,1979). The latter author postulated that the source of atmospheric SOz formed from CS, and COS can be quite large. A major source of sulfides from the oceans is dimethyl sulfide produced by phytoplankton. Estimates of the total quantity of S released to the atmosphere in this way are greater than estimates of anthropogenic S sources. Although the quantity of this product is relatively large, the concentration is so low and the half-life is so

220

N. S. PASRICHA AND R. L. FOX

short that dimethyl sulfide is not an environmental problem. We assume that sulfide is a major contributor to background S in the tropics, without which S deficiency would be an even more serious problem than it is already. Such considerations suggest that S emission control measures such as are now being instituted, although locally effective, will be of little significance on a global basis. For SO,, an average concentration in the troposphere is about I p g m-3 STP. A generalized value for SO, is given as 0.9 ppb, but a value of 0.3 ppb is given for central Brazil. Values are higher in Panama (Lodge et al., 1973). Although the United States generates 35% of the world’s electricity and consumes a corresponding fraction of fossil fuel, its production of SO, may be less than 35% of the world’s production from energy sources (Kellog et al., 1972).We will adopt the figure 100 X lo6tons of SO, per year for total man-made contributions to the atmosphere. This corresponds to I50 X lo6 tons per year of SO,, into which most of the SO, is converted. This is a global figure. It is significant that only about 6.5% was produced in the Southern Hemisphere [Massachusetts Institute of Technology (MIT), 19701. Because seawater contains SO,, SO, concentrations in the air in coastal areas usually are higher than they are further inland. In polluted surface air the SO,: SO, ratio is about 10 times greater than in clean air (Junge, 1970). Thus, SO4 concentration does not vary as much as SO, concentration. Oxidation products of SO, are further oxidized to SO, by a variety of processes. The net residence time for SO, in the atmosphere must be similar to that of SO, because the overall ratio of SO, :SO, concentration in the atmosphere seems to be close to unity (Georgii, 1970). Quantitative estimates of global sources of S compounds in the atmosphere are presented in Table 11. The total amount of about 2 X lo8 tons/annum is of the same order as global industrial production or consumption of s. Sulfate aerosols are primarily produced naturally from sea spray over the ocean. Most of it is deposited over the ocean by rain. The large figures for H,S production were obtained indirectly by budget considerations and these may not be reliable. The range of maritime sulfate concentrations, 0.22 - 2.72 p g m-3, determined around Asia by Horvath et al. (1981) are in good agreement with results obtained over oceans in other parts of the world (Nguyue et al., 1974a,b). The value of excess SO4 (0.87 pg m-3) is similar to the value of 0.9 pg m-3 reported by Grevenhorst (1978) for the North Atlantic. Although the origin and nature of precursor gas is not well understood, one possibility is anthropogenic SO,. However, taking into account the residence time of SO, ( 1 -2 days) as well as wind direction observed during sampling rules SO, out as a major source in favor of submicron sulfate

PLANT NUTRIENT SULFUR

22 1

Table 11 Estimates of Sources of Atmospheric S per Yeara

Land (tons S)

Ocean (tons S)

-

Source

Anthrowgenic

Natural

Anthrowgenic

Total

-

-

7 0 X lo6 1 3 X lo6$'

4 4 x 106 3 0 X 106

-

3 x 106

4 4 x I06 103 X 106 73 x 106

Natural

so, H2S

so2

-

-

-

220 x 106

Grand total Adapted from Robinson and Robbins (1968). Total (natural anthropogenic).

f,

+

particles from a sulfur gas of natural origin, namely, dimethyl sulfide, the oceanic release of which is estimated at 27 Mt yr-'.

B. SULFURACCESSION THROUGH PRECIPITATION The relationship between soil sulfate and rainfall is complex. Rainfall amount seems to have influenced soil sulfate in five ways: (1 ) SO, accession, (2) SO, retention, (3) SO, utilization, (4) S immobilization, and (5) SO, leaching. Sulfate accessions are the combined effects of rainfall quantity and rainfall quality. Muller (1975) analyzed rainwater and lysimeter leachates for total S for 20 years at the Otara Research Station, Auckland, New Zealand. Losses of SO, in leachates from fertilized and unfertilized soils were also determined. An average of 13 kg ha-' S was received annually in the rainfall, with extremes of 6.6 and 17.6 kg ha-'. The proximity of tidal flats and accessions from aerial top-dressing may have been responsible for contributions not exceeding 25%. Sulfur concentration in rainwater in the continental tropics is usually low. The deposition of S in rainfall was measured at 10 locations in Central Kenya, monthly for 1 year (1977- 1978) by Bromfield el al. (1980). Amounts deposited ranged from 1.58 to 3.81 (mean 3.47) kg S ha-'. Concentrations ranged from 0.10 to 0.17 (mean 0.12) mg S liter-'. Low S concentration in rainwater and depleted organic matter reserves of soils are associated with S deficiency in the seasonally dry West African Savanna. Mean annual S in rainfall for northern Nigeria is about 1.14 kg S ha-' (Bromfield, 1974), suggesting that yields of cowpea, an important crop in the seasonally dry savanna, are limited by S deficiency (Fox et al., 1977).

N. S. PASRICHA AND R. L. FOX

222

1 .o

0.8 0.6 0.4

0.2

I

I

I

lo3

lo4

lo5

Distance (km) x altitude (m)

Figure 3. Influence of distance and elevation from the sea on the concentration of S in rainwater (Fox el al., 1983).

On the other hand, rainfall, air deposition, and particulate matter contributed approximately 10.7, 1.8, and 3.0 kg S ha-' per year, respectively at one location in the southern United States (Suarez and Jones, 1982). No relationship was obtained between applied S and crop response for several crops. Suarez and Jones (1982) emphasized the need to keep in view contributions of atmospheric deposited S when making fertilizer recommendations. Influence of the sea as a S source diminishes in proportion to a product of distance and elevation from the source (Fox et a/., 1983). Sulfur concentration in rainwater dropped exponentially with increasing distance from the coast in both New Zealand and Hawaii (Fig. 3). For example, in Hawaii, rainwater contained 4.5 mg S liter-' 0.5 km from sea, 1.0 mg S liter-' 3 km inland, and only 0.1 mg S liter-' 24 km from the coast. Estimated total S inputs from rain were 24, 10, and 1 kg S ha-' at 0.5, 3, and 24 km distance, respectively (Hue et al., 1990). Although New Zealand is only subtropical in its northern latitudes, data on the S composition of rainwater should provide a useful example of the importance of the sea in tropical regions. Maximum distance from the sea in New Zealand was 100 km. At that distance the sea was of little consequence but, even so, 0.2 mg S kg-' in rainwater has some agricultural significance. Fox et af.,

PLANT NUTRIENT SULFUR

223

1979) demonstrated that 0.2 mg SO,-S liter-’ was sufficient for approximately one-half maximum yield of banana. In the Southern Hemisphere and in the tropics generally, background atmospheric S is low because atmospheric S does not move readily across the tropical convergence zone. Thus, the influence of the oceans can be more readily discerned there than in the North Temperate Zone, where, because of pollution, the influence of oceans is relatively less important.

V. EFFECTS OF ACID RAIN The effects of acid rain (precipitation) are widely debated. Some of the chemical compounds associated with acid deposition are important nutrients for both plants and animals. Water in equilibrium with CO, in the atmosphere has a pH of approximately 5.6. As usually defined, “acid rain” is rain with pH below 5.6 resulting from the solution of other acid-forming constituents, such as SO,, directly from the atmosphere. This process is known as “wet deposition.” The term “acid deposition” represents the total deposition of acid from the atmosphere. With adequate precautions, the acidity of precipitation can be measured with reasonable ease and precision, but dry deposition is not so easily determined and little is known of the magnitude or significance of dry deposition [Council for Agricultural Science and Technology (CAST), 19851.

A. EFFECTON CROPPLANTS Experiments with simulated acid rain within the observed pH range of acid precipitation have sometimes decreased crop yields, although Irving (1983) concluded that the effects appear to be very small, and that when responses are observed, they may be positive or negative. There is no convincing evidence that acid precipitation as such is detrimental to crops in the field. Increased incidence of blossom-end rot of tomatoes has been associated with volcanic activity in Hawaii, suggesting that acid rain may have brought on a Ca deficiency. Faller ( 1 97 l), observed that crop yields increased with increasing concentration of SO, in the atmosphere (Table 111). For tobacco, total dry weight increased up to 48%. The yield of leaves and stems increased by SO%, reflecting reduced root yield frequently associated with improved S status. Additions of SO, increased plant inorganic sulfate. Simulated acidic rain on radish plants decreased hypocotyl growth but not shoot growth

N. S. PASRICHA AND R. L. FOX

224

Table 111

Effect of Atornospheric SO,on Relative Dry Matter Yields of Sunflower, Cow and Tobacco" Relative yield, dry weight (per x mg SO, M-' air) Crop Sunflower TOP Root Corn TOP Root Tobacco TOP Root

N=O

0.2

0.5

1.o

1.5

100 100

I49 93

I52 89

I78 45

I59 60

100

112

100

100

I24 98

I I7 87

I I3 87

100 100

147 99

165 79

I77 75

I85 75

Adapted from Faller ( 197I); by permission of The Sulphur Institute, Washington, D.C.

0

3

6

9

1 2 1 5

Internal flux of SO, (nrnol ern-' hc')

Figure 4. Relationship between the internal deposition of SO, and the inhibition of net photosynthesis in Viciu fubu (Black and Unsworth, 1979;Reprinted with permission from Nuiure (London), Macmillan Magazines Limited.)

PLANT NUTRIENT SULFUR

225

(Jacobson et al., 1986). Heggestad and Lesser (1990) studied the effects of SO, in concentrations from 0.005 to 0 . 2 2 4 ~ 1liter-’ (4 hr day-’, 5 days week-’) from seedlings to mature soybean. They observed a negative impact on bean yields and seed size. Black and Unsworth ( 1979)observed that CO, assimilation in Viciafaba associated with low levels of SO, exposure was highly flux dependent, exhibiting first-order reaction kinetics at fluxes less than 1 nmol cm-* hr-I (Fig. 4). At higher fluxes, CO, assimilation was typical of second-order reaction kinetics achieving a trend indicative of saturation at fluxes greater than 8 nmol cm-, hr-’.

B. EFFECTON FORESTVEGETATION Much of the literature on SO, deposition has been reviewed by Voldner ef al. (1986). While most of the measurements were done on field crops, studies on watersheds in forests suggest that pollutant effects on ecosystems can be more important than direct effects on vegetation. Near large anthropogenic sources of SO,, accretions of SO, by forests may be the largest source of S in the watershed S balance. Even at some distance from these sources, inputs from dry deposition of SO, may be nearly as great as the input from acid rain (Garland, 1977). For this reason, it is necessary to make accurate estimates of the input of SO, to forested watersheds and to know how the S from this source is redistributed in the forest system. Gay and Murphy (1989) attempted to measure the deposition and fate of 35S02 in a pine plantation. Sulfur dioxide is a much discussed air pollutant in relation to a “new type” of forest damage, but owing to the spatial distribution of SO, deposition, it cannot be singled out as the causative agent. Atmospheric concentrations of SO, are highest in industrial and densely populated areas, but the most serious forest damage occurs in remote areas. In fact, SO, concentrations in urban areas have decreased significantly (Kandler, 1985). A decreasing SO, emission trend over western Germany has been shown by Huettl ( 1989). In areas where forest damage is observed, SO, concentration may be below well-established International Union of Forest Research Organization (IUFRO) standards (1983). Thus direct SO, damage is not the overall cause for “new type” forest damage. This conclusion is supported by histological observations by Fink (1986). So far there are no research results indicating that acid precipitation, within the range of pH values usually encountered, directly damages forest vegetation [Council for Agricultural Science and Technology (CAST), 19851.

226

N. S. PASRICHA AND K. L. FOX

C. EFFECTON SOILACIDIFICATION The evidence for widespread accelerated acidification of soils by acid precipitation is not very strong. Acid soils are predominant in humid regions from the tundra through the tropics, irrespective of their proximity to industrial emissions. They have been acid for a long time. Even strongly acid precipitation in a humid region with well-buffered soils probably would be detectable only after several decades. However, in Hawaii, and in many tropical areas, some of the soils are so delicately poised with respect to pH that only small amounts of acid might push them over the edge (Fox and Hue, 1986).

VI. SULFUR IN IRRIGATION WATERS

A. SULNR IN STREAMS Surface waters and groundwaters may be important sources of S for irrigated or flooded crops. Sulfur in imgation waters varies greatly depending on whether it is surface water or groundwater. Much runoff water from high mountains is S deficient, and so is water from some aquifers. Sulfur deficiencies have been discovered in newly reclaimed Swamp soils in the lower Amazon Basin (Wang, 1978). A general deficit in S in the environment of tropical South America has been known for many years (McClung and de Freitas, 1959; McClung ef al., 1959). The amount of S in a stream depends on contributions from geologc formations and other inputs contributing to the water. For example, the upper Yakima river in Washington State contains only 0.7 mg liter-' of S and crops irrigated with it respond to S fertilization, but the lower reaches of the river, contain 1.4 mg liter-' and irrigated crops do not respond to S applications. At its mouth, the river water contains 5.1 mg liter-' (Dow, 1976). Yoshida and Chaudhry (1972) observed that imgation waters carrying more than 2.7 mg S liter-' met the S requirement of rice. Wang el al. (1976) suggested this limit is 6 mg liter-'. Severe S deficiency in rice in South Sulawesi, Indonesia, was not prevented by imgation with waters having 2.8 mg liter-l (Blair ef al., 1978). Generally, irrigation waters carrying more than 4 to 6 mg liter-' S will supply enough S for most crops. Applicability of this guideline will depend on (1) the amount of water applied per growing season, (2) crop requirement, (3) redox potential of the system, and (4) yield expected.

PLANT NUTRIENT SULFUR

227

Sulfate contents of nine selected Indonesian irrigation waters ranged from 1.28 to 20.2 mg liter-' of SO,-S with all but two samples being 6. I7 mg liter-' or less (Ismunadji and Zulkamaini, 1978). These two were associated with extractable SO4-S of < 4 mg kg-l. A survey of 254 rice fields indicated that 31% were deficient and 42% were marginal in S (<0.1% plant S, indicative of S deficiency).

B. SULFURIN GROUNDWATER In Punjab State, India, sandy alluvial soils, although they may be low in extractable S, generally do not respond to applied S because irrigation waters from groundwater sources contain considerable SO,. Mean S concentration in groundwater used for irrigation in selected villages was a p proximately 8 mg liter-' (Cheema and Arora, 1984), which would provide approximately 24 kg S ha-' to a wheat crop during a typical season. Although mean values for the various villages ranged from 4.3 to 13 mg liter-' S, in one village the range for 10 different tubewells was 0.7 to 18 mg liter-l S. In years of high rainfall, when irrigation requirements are low, crops are S deficient and respond to S application (Pasricha et al., 1987).

VII. SULFATE RETENTION IN SOIL A. SULFATEADSORPTION AND DESORPTION In general, surface soil materials adsorb less sulfate than subsoil materials do. No doubt illuviated clay-sized material is responsible for this in some cases, as is illustrated by the Piiia soil of Puerto Rico (Table IV) and the Alagba soil of Nigeria (Table V). But even in soils in which eluviation of clay is of little consequence, sulfate accumulation is clearly seen, as in the case of Oxisols (Table IV). One of the striking features of most highly weathered soils is that large quantities of SO, have accumulated somewhere in the profile, assuming of course, that there are sufficient mineral surfaces to hold the sulfate. Typically, adsorbed SO, increases as a function of depth until a maximum is reached at about 100 cm. Usually, water solubility of this sulfate is low, but much of the SO, can be desorbed by repeated extractions with a 500pg ml- P solution (Table IV). Thus, many soils of the humid tropics contain

'

228

N. S. PASRICHA AND R. L. FOX Table IV

Extractable Sulfate Sulfur, Solubility of Sulfate, and Calculated Sulfate Adsorption Maxima of Some Highly Weathered Soils of Puerto Rico'

soil lnceptisol (Picacho)

Ultisol (Los Guineous)

Torres

Catalina

Pirla

Nipe

Depth increment (cm)

0- 18 18-35 35-60 60-85 85-130 0-8 8-25 25-45 45-65 65-90 90-150 0- 10 10-30 30-50 50-90 90- I25 0- 16 16-35 35-60 60-80 80- 120 120- I30 0-20 20-34 34-60 60-90 90- I25 0-25 25 -40 40-70 70-95 95-120

Sulfate adsorption

SO,-Sb

SO,-S,' 0- 120 cm

Sulfate S solubility

maxd

Saturation

(Pgg-')

(WW

(ppm)

(pg S g-' soil)

('W

4Ooo

0.5 1.5 I .o 0.8 0.8 0.4 0.4 1.2 4.2 3.2 4.0 19.0 10.0 9.4 1.6 2.8 6.0 16.0 16.0 2.0 5.0 3.0 I .7 1.7 7.0 7.5 7.0

94 280 640 710 970 98 209 670 830 1330 870 270 990 800 9 10 830 930 I180 1430 I720 1730 860

Extractable

16 16

60 340 597 20 80

4540

190

326 452 441

280 819 740 589 537

9210

444

10780

1080 1310 1267 I225 195 2 8 210 260 I30 40 255 373 46 3 792

2370

7330

0.5

1.6 1.1

4.0 7.7

17

6 10

48 62 20 40 28 25 34 41 104

10 10

262 280 235 200 360 430 770 1250

83 82 65 65 45 32 92 74 71 23 20 80 80 93 81 20 71 87 60 63

Adapted from Fox (1982) with permission. Sulfate extracted with 500 pg ml-1 P. Sulfate in saturation extract. Maximum sulfate adsorbed as determined from an isotherm based on the Langmuir equation.

an abundance of sorbed sulfate. This is not always evident from published data because most investigations of sulfate in tropical soils have been restricted to relatively shallow depths. Also, the extraction procedures most frequently used have not effectivelyextracted sulfate, nor has the extracted SO, been effectively determined (Fox et al., 1987). Nevertheless, in assess-

PLANT NUTRIENT SULFUR

229

Table V Phosphate-Extractable SO,-S in Profiles of Selected Soils from the Humid Tropicsa Depth increment (cm) Location Australia Cape York Columbia Carimagua Nigeria Alagba Puerto Rico Carracas Catalina Hawaii Hanipoe Made Akaka Wahiawa

0- I5

15-30

30-60

60-90

90-120

5

5

9

35

31

3

18

22

5

15

25

18

56

112

55

200 I040

360 I270

1250

300 1220

17 950 5480 I60

7 960 4520 200

410 25

18

16

150 220 135

320 1210 66

830 3720 146

340

a Adapted from Fox and Blair (1986). Values in table body are in kilograms per hectare.

ing the available SO4supply, the quantity of sorbed SO, in the subsoil, in addition to that contained in the surface horizons, should be considered, because adsorbed SO, is a feature of subsoils in most highly weathered soils of the tropics. However, deep root penetration is a precondition for adsorbed SO, utilization in such soils (Probert and Jones, 1977). Sulfate accumulates to very high levels in some soils, especially in weathered Andepts, which contain gellike materials, and acid soils, which contain oxides of iron and aluminum. This is illustrated in Table IV. Phosphate-extractable S from 19 New Zealand soil profiles to a depth of 2 m was as follows:

SO,-S range (kg ha-')

20-200 200- 1000 1000-2000 2000-5000 10.000- 15,000

15,000-20,000

Number of soils

230

N. S. PASRICHA AND R. L. FOX

All soils that contained more than 10,000 kg SO4-S ha-' developed in volcanic ash and cinders. All were from the North Island and were highly weathered. Incipient S deficiency is encountered in the area. Soil profiles that contained less than lo00 kg S0,-S ha-' were from the South Island, from areas where S deficiency is a major nutritional problem for white clover -ryegrass pasture production. Sulfate does not accumulate in large amounts in profiles developed in volcanic ash until weathering is well advanced. Coarse textured sandy soils of the subtropics, especially those developed on alluvium with neutral to alkaline reaction, adsorb little sulfate (Bahl and Pasricha, 1984). In general, surface soil materials adsorb less sulfate than do subsoil materials. No doubt, eluviation of clay-sized materials is partially responsible, but organic matter and phosphate accumulations in surface horizons, which block sulfate sorption sites, are major factors. Even when eluviation of clay is of little consequence, the relationship is clearly seen, as in the case of Oxisols. Sulfate from surface horizons is easily leached to lower depths, where it accumulates in large quantities, especially in highly weathered Oxisols and Dystrandepts (Fox, 1974). Sulfate distribution in the Nipe profile is typical of highly weathered Oxisols that have been examined (Table IV). The fact that SO, solubility continued to increase with increasing depth, even though saturation percentage decreased, suggests that adsorbed SO, and saturation percentage are not the only factors controlling SO, availability. For Andepts in Hawaii and a diverse group of Puerto Rico soils, sulfur solubility was about 5 pg ml-' when saturation was 60-80% (Hasan et al., 1970; Fox, 1982). Sulfur nutrition is probably borderline deficient for some crops when soil solutions contain 3 - 5 mg liter-' SO,-S. Upland soils of the Llanos of Columbia contain little adsorbed SO,. The mean is about 150pg g-I and sulfate solubility is low (Fox, 1974).These soils require about 60 pg S g- of soil to attain 5 pg ml-' S in solution (Fig. 5). Sulfate is readily desorbed from soils by phosphate and hydroxide. Organic anions also compete for adsorption sites with sulfate and in this way may account for the low quantity of sulfate found in surface soils. Desorption of SO, results in decreased concentration of SO, in solution. Repeated desorption produces a relatively smooth desorption curve over the concentration range usually encountered in leached tropical soils (low solution concentration). This, we believe, is evidence for sulfate (ligand) adsorption as the principal mechanism for SO, retention by such soils. At higher concentration, other mechanisms of SO, retention probably operate, including electrostatic adsorption associated with positive charge. In any case, adsorption -desorption of sulfate proceeds relatively rapidly as compared with phosphate. For example, a 24-hr equilibration time was

PLANT NUTRIENT SULFUR

23 1

I

m

I

I

I I

I I

I I

1.o

10

sod-s in solution (pg m ~ ' ) Figure 5. Sulfate sorption by some diverse soils from tropical America: 0, Typic Dystrandept, Costa Rica;X, Nipe, Puerto Rico; A, Llanos, Columbia; 0, Terra Roxa, Brazil; A, Eutropept, Costa Rica; 0, 1963-1965 ash, Costa Rica (Fox, 1974; by permission of the publishers, Buttenvorth-Heinnemann Ltd. 0 )

more than adequate for obtaining a constant concentration of sulfate in solution (Hasan ef al., 1970), whereas, for phosphate, up to a 6-day equilibration time was required (Fox and Kamprath, 1970). Data in Fig. 6 suggest that at levels of extractable SO,- S above 1000 mg/kg, the solubility of SO, decreases abruptly. Each of the two points represent five soil samples (all subsoils), two from a highly weathered ash layer (60- 100 cm) and the remainder developed in weathered basalt scoria. These data suggest one or more of the following criteria: 1. Up to approximately 100 mg S kg-' soil, availability is controlled by SO4- S in solution by weakly adsorbed SO4. 2. In the range of 100- lo00 mg S kg-' soil, SO, adsorption assumes an important role and availability increases with quantity adsorbed. 3. In the range of 1000-3500 mg S kg-' soil, availability is considerably depressed.

Perhaps decreased availability at high levels of extractable SO, is because adsorption capacity increased more than SO, quantity, or perhaps because of precipitated iron or aluminum sulfate compounds of low solubility (Wolt and Adams, 1979; Wolt ef al., 1992).

N. S. PASRICHA AND R. L. FOX

232 0.200

I

I

0.180

\

r

O

0: I I

0)

0.140 C

-m a

0.120 -

I

, I

I I I

I

I,/I

0.100

0

I /

2

I

8

I

1

32

1

I

128

I

I

1

512

1

I

2048

/

0.080 I

Mean extractable SO., -S (mg S/kg)

Figure 6. Sulfur contents of ryegrass indicator plants in relation to SO, extracted with phosphate from New Zealand soils (R. L. Fox, P. M. Cooper, and W. M. H. Saunders, unpublished).

Indicated yield potential of ryegrass at 0.175% plant S is approximately 80% of the maximum attained (approximately 0.22% for maximum yield). What do these data indicate about S solubility? Information is not sufficient for us to make a firm statement about ryegrass, but if data for maize apply here, approximately 5 mg liter-' of S in solution is adequate for maximum yield and only 1 to 2 mg liter-' should be adequate for 80% yield. Thus, it appears that these soils are undersaturated with SO, (assuming that solution concentration is being controlled by adsorbed SO,). Given proper environmental conditions and time for precipitating aluminum hydroxy sulfate, lower concentrations may be expected, thus, greater quantities of extractable SO,.

B. SULFATE ADSORPTION CURVES Adsorption curves are useful for describing, studying, and managing the SO, status of soils. They integrate and reflect many aspects of mineralogy, chemistry, and management history of soils. The concentration of SO, in solution, as predicted by sulfate sorption - desorption curves (the equilibrium concentration when SO, is neither sorbed or desorbed), provides valuable information on plant nutrition. It indicates the immediate concentration at which sulfate should be available to plants and the concen-

PLANT NUTRIENT SULFUR

233

tration of sulfate in water that drains from that horizon (Fox, 1982). It also provides an explanation for the fact that crops may be adequately supplied with S, which usually is 3-5 mg SO4-S liter1, even though the soil is being leached with rainwater that contains much less S than that. An example of this has been reported for Hawaii by Hasan et al. ( 1970). Akaka surface soil materials equilibrated at 5 mg SO,-S liter-' and subsoil material at 3 mg SO4-S liter-' even though predicted SO,-S in rainfall at that location is approximately 0.35 mg liter1.Annual rainfall is approximately 4500 mm and contains 16 kg S ha-'. Sugarcane and pasture vegetation growing at this and similar locations give no obvious evidence of S deficiency. Evapotranspiration probably does not concentrate the leachate more than 50%. There is, therefore, a 10-fold difference in the concentration of rainwater and predicted soil solution concentration. A probable explanation for this discrepancy is that SO, in rainwater is augmented by S leached from vegetation, from leaves, stems, and roots, and from dry deposition in the plant canopy and on the soil. Further reference to this effect was made in Section IV dealing with S cycling. Adsorption curves can also be used to follow the course of soil development. A good example is sulfate adsorption by highly weathered soils on volcanic ash in the humid tropics, where soil materials and climatic conditions promote rapid removal of silicon and accumulation of hydrated iron and aluminum oxide. Sulfate concentration in solution is related to the degree of saturation of the exchange complex with sulfate or with other specifically adsorbed anions. Sorption maxima for highly weathered soils are usually associated with 10-2Opg SO,-S ml-I (Fox, 1982). Studies of SO, adsorption by some West Indian soils by Haque and Walmsley (1974) indicate that higher solution concentrations do not conform to the Langmuir equation. Therefore, SO4-S concentrations of approximately 10 pg ml-* or less should be used for calculating adsorption maxima in such soils. Some specifically adsorbed anions and structural anionic impurities can give oxide surfaces a net negative charge, thus shifting the point of zero net charge (PZNC) to lower pH values. The PZNC of synthetic hematite, corundum, boehmite, and geothite occurs at pH values greater than 7 and hydration can increase the pH at zero net charge (Parks, 1965). The PZNC of surface horizons of ferruginous soils is, however, usually below pH 5.0. Mekaru and Uehara ( 1972) demonstrated that phosphate adsorption increased the CEC of highly weathered soils, resulting in negative adsorption of NO; and CI-. Such variable-charge soils, in which the sign and magnitude of charge on the solid phase are determined by the chemical environment, adsorb significant amounts of Sot- (Barrow, 1967; Fox, I980b).

234

N. S. PASRICHA AND R. L. FOX

Variable-charge colloids dominate the mineralogy of most soils of the humid tropics because weathering and leaching processes dedicate soil, forming minerals enriching them in hydrated oxides of Fe and Al. Materials such as these, together with kaolin clays and organic matter, give rise to variable charge. In soils in which permanent negative charge predominates, practically all of the soil SO, is in the soil solution. But in variable-charged soils, especially if they are sufficiently acid to be near the point of zero net charge, most of the SO, is sorbed. Thus, SO, sorption by soils requires positive charge. Net positive charge is not required. To attain net positive charge in whole soils requires variable-charge colloids and lower pH than is common to highly weathered soils. The final result, however, depends on at least three possibilities: (1) the point of zero net charge for certain components of the soil system may be sufficiently high for net positive charge to develop on that component at soil pH values near 7.0, (2) adsorbed SO, may be held as ligands or by mechanisms other than electrostatic attractions, or (3) sulfate may be retained in compounds or complexes that are relatively insoluble at low pH but are soluble in phosphate extractants.

C. MECHANISM OF SULFATE ADSORPTION Soils derived from volcanic ash may adsorb large amounts of SO, owing to hydrous oxides of Fe and Al and allophanic clays (X-ray amorphous A1 silicates) present in them (Hingston el al., 1972). Rajan (1979) observed that SO, is adsorbed on a net positive charged surface as a bidentate forming a six-membered ring, displacing either two aquo or hydroxyl ligands. On neutral or negative surfaces, SO, is adsorbed as a monodentate, displacing one aquo or one hydroxyl ligand. This makes the surface charge more negative. Organic ligands play an important role in determining the SO, adsorption capacity of soil and subsequent amount of OH released (Fuller ef al., 1985). Evans (1986) noted that increases in dissolved organic carbon resulted in increased SO, transport in soil columns at pH 4.6. Similarly, Gobran and Nilsson ( 1988) found that forest floor leachates containing dissolved organic ligands inhibited SO, retention by a Spodosol soil at SO4 levels less than 7 mM. The ability of soils to adsorb SO, is an important factor in determining the effect of acidic deposition on the transport of H+ and cations in terrestrial ecosystems. Inskeep ( 1989) showed that inhibition of SO, adsorption was related to the quantity of oxygen-containing functional groups rather than soluble C. These results indicate that organic

PLANT NUTRIENT SULFUR

235

acids compete for SO, adsorption sites and that the presence of organic acids in soil solution will influence SO, adsorption capacities. The input of acidic deposition containing mobile anions like SO, may be responsible for increased cation leaching from soils to ground and surface waters in some forest ecosystems (Seip, 1980; Van Breeman ef al., 1983). However, mobility of SO, has received considerable attention because net SO, retention by soils can result in decreased cation leaching. Where an equivalent displacement of OH or other anions does not occur during the SO, retention process, cations are coadsorbed with SO, (Khanna and Beese, 1978; Singh ef al., 1980). Highly weathered tropical soils, which adsorb substantial amounts of SO,, are considered to be resistant to accelerated cation leaching (Huete and McColl, 1984). The mechanism of SO, adsorption involves an exchange of OH coordinated with an Fe atom of the oxide structure, resulting in increases in pH at higher levels of sorption. When SO, is specifically sorbed by goethite or hematite, negative charge on the surface increases, increasing the ability of the material to retain cations. In tropical soils, this aspect of SO, sorption could be valuable, given the extremely low cation exchange capacities at the low pH of many of these soils. Couto et al. (1979), however, did not observe a constant increase in pH per unit SO., sorbed for different soil samples. The Ap horizons sorbed small amounts of SO, and the effect on pH was not marked, whereas B2 horizons sorbed high amounts of SO,, and sorption clearly decreased as the pH of the equilibrium solution increased. The differences between horizons were probably due to the higher organic matter content of the Ap horizons, because no clear changes in mineralogical composition were observed. Blocking of positively charged sites may occur as a result of occupation of these sites by organic anions or coating of oxide surfaces by organic matter. Phosphate quantity and intensity also are almost invariably greater in surface horizons. We may well suppose that organic matter and its decomposition products are involved. Adams and Rawajfih ( 1977) have suggested that SO, is precipitated as insoluble basic A1 and Fe sulfates in these kinds of soils. Acid soils that have been limed or those that have naturally high pH, such as the Tropudalf studied by Couto ef al. (1979) and alluvial soils studied by Bahl and Pasricha (1984), may require SO, application as a consequence of their lowered SO, sorption capacities. In addition, liming acid soils enhances movement of SO, out of the limed zone of soils. Sulfate retention involves more than one mechanism. Our results (Fox, 1984) suggest that mechanisms of SO, sorption and phosphate sorption are similar, and that both ions compete for the same sorption sites, although sorbed SO, does not compete strongly with phosphate (Gebhart and Coleman, 1974). As a consequence of phosphate fertilization, sulfate may be

236

N. S. PASRICHA AND R. L. FOX

0.5

1

2

5

10

20

50

so4 in supernatant (vg mi-’) Figure 7. The residual influence of phosphate fertilizers on SO, sorption by an Oxisol in Hawaii (Fox ef a!.. 1971).

displaced from some soils. This effect may persist for several years as is demonstrated by the shift in a SO, sorption curve that resulted from a massive phosphate application 12 years earlier (Fig. 7). Phosphate can extract relatively large amounts of SO, from variablecharge soils, although this may not be obvious if only surface horizons are investigated (Neptune ef al., 1975). Even CaC1, extraction in a wide soil :solution ratio may desorb substantial quantities of SO, from highly buffered soils without depressing SO, concentration below an adequate level, as indicated by vigorously stirred solution culture experiments. Thus the external SO, requirement of plants growing in acid, variable-charge soils and permanent-charge soils may be distinctly different. Phosphate-extractable SO,- S in profiles of selected soils from the humid tropics are presented in Table V. It is obvious from such diverse data that it is unwise to generalize about the quantity of SO, in soils of the humid tropics, except to say that somewhere in the profile of most of these soils there is a significant quantity of SO,. Whether such sulfate is available to plants cannot be inferred from the quantity of SO, alone. Growth of stylosanthesis on leached sandy soils of tropical Queensland has indicated that some species can utilize sulfate from considerable depth in such soils (Gillman, 1973).

PLANT NUTRIENT SULFUR

237

VIII. DIAGNOSIS OF SULFUR NEEDS A. SOILTESTS 1 . Extractable Sulfur

Estimates of plant-available S in soils have been attempted by several scientists using a variety of methods. Phosphate-extractable S using monocalcium phosphate (Fox et al., 1964) or monopotassium orthophosphate ( KH2P04)(Ensminger and Freney, 1966) extracts soluble plus adsorbed SO,. Repeated extraction is required for quantitative removal of sulfate from many highly weathered soils (Hasan et al., 1970). Hot-water-soluble sodium acetate (NaOAc, pH 4.8) and sodium bicarbonate extracts include some organic S also. Phosphate extracts correlate best with crop yields or S uptake by plants (Barrow, 1967; Scott, 1981) and soil tests appear to be most valuable on sandy soils. Beaton et al. (1985) reported that soils with the same characteristics but with soil sulfur concentrations between 7 and 15 kg ha-' would be expected to respond to applied S in dry years but not in wet years. Brogan and Murphy (1980) did not find soil testing for S to be promising. They observed, however, that soils with more than 50% sand and less than 3% organic carbon would likely respond to applied S. Determining SO4in soil extracts is fraught with difficulties. The methylene blue method of Johnson and Nishita (1952), although sensitive, has many disadvantages (Lee ef al., 1981; Fox et al., 1987). Ion chromatographic methods are sensitive and more reliable (Kalbasi and Tabatabai, 1985) and are advantageous for simultaneous measurement of other anions. However, equipment is expensive. Turbidimetric methods have been widely used for SO, assay of soil extracts since Chesnin and Yien (1950) introduced one such procedure. Hesse ( 1957) used a sodium acetate extractant. He attributed interference to colloidal organic matter. Fox el al. (1964) attempted to overcome this difficulty by extracting with Ca(H,PO,), instead of NaOAc and digesting the extracts with oxidizing agents such as nitric-perchloric acid; but even this may not eliminate the problem of BaSO, precipitate suppression in extracts of some soils (Vander Zaag et al., 1984). Introduction of BaSO, seed crystals as proposed by Tabatabai (1974) and adding additional Ba as solid BaCI, in phosphate extracts that have been evaporated and digested with nitric-perchloric acid produced higher values. Searle ( 1979) also discovered soil extracts that failed to yield satisfactory precipitates. Absorbance values less than the zero standard were obtained. Additions of a SO, spike to these extracts produced spuriously low results and several activated carbon treatments

238

N. S. PASRICHA AND R. L. FOX

did not overcome the interference. Fox et al. (1987) proposed a more reliable, although more complicated, turbidimetric method for determining phosphate-extractable sulfate in tropical soils. This method consistently yielded more SO, than other turbidimetric procedures. Inconsistencies between soil tests for S and crop performances have been reported widely. These inconsistencies may result from seasonal effects on extractable S. Castellano and Dick (1991) observed great seasonal variability in SO, levels even in control plots that have not received any S applications for at least 20 years. Critical levels as high as 10 mg SO,-S kg-' soil have been reported for the production of winter rape in the Pacific Northwest (Murray and Auld, 1986). One might reach different conclusions, depending on the time of sampling. Current S soil test recommendations are usually based on samples derived from surface horizons. In environments where there are periods of significant evaporation, movement of SO, from subsurface to surface may affect soil test results, depending on time of sampling. Additionally, plants usually develop roots below 60 cm and subsoils vary in their SO, contents. Leaching is an even greater problem. So also is knowing how to evaluate adsorbed sulfate and subsoil sulfate, i.e., whether it is adsorbed or not. 2. Optimum N :S Ratio in Soil

Because S is an important component of protein, balanced N :S fertilization is important in obtaining optimal yields and protein contents. If the N : S ratio is too great, protein synthesis may be restricted and N may accumulate in plants in nonprotein forms (Pasricha and Randhawa, 1975). Applications of N to soils deficient in S may lead to decreased yields (Janzen and Bettany, 1984; Nyborg et al., 1974). The optimal fertilizer N :S ratio vanes among soils because of differences in available soil N and S levels. However, one estimate of a suitable available N: S ratio [(soil NO, N fertilizer N)/(soil SO4-S fertilizer S)] is approximately 7 for upland conditions (Janzen and Bettany, 1984). This probably exceeds S requirements in the tropics. A suitable N:S ratio in solution for sugarcane growth is approximately 10: 1 (Fox, 1976). As a first approximation, this ratio could be a guide to fertilizer applications for nonlegumes in those areas of the tropics where S removal by crops is great in comparison with soil and rainfall S (Fox and Blair, 1986). However, because SO, is not so easily leached or so easily reduced as NO,, and because the internal N :S ratio is greater than 10, it seems reasonable to suppose that a 10: 1 N:S ratio in fertilizer, consistently applied, will more than adequately meet the S requirement of nonlegumes.

+

+

PLANT NUTRIENT SULFUR

239

95 I I

Sulfur content in younger leaves (mg S g-’ )

Figure 8. Utilization of fertilizer N by oliseed rape based on the S status of plants (net utilization by seeds) (Schnug, 1991; by permission of The Sulphur Institute, Washington, D.C.)

Leaching of underutilized NO3 can create serious environmental problems. Nitrogen and sulfur are both involved in protein synthesis, thus a shortage of S in relation to N leads to poor N fertilizer efficiency (Fig. 8) (Schnug, I99 1).

B. PLANTANALYSIS 1. Plant Sulfur

Sulfur deficiency in plants results in pale yellow-green leaves. Unlike N deficiency, S deficiency symptoms first appear in young leaves. Older leaves may accumulate S and, if mobile nutrients are adequate, may develop normal color. These symptoms persist even after adequate N application. Sulfur-deficient plants are often spindly with short and slender stalks. Four criteria of assessing the S status of plants have been used: ( 1) total S, (2) SO,-& (3) N: S ratio, and (4) SO,-S: total S ratio. The concentration of S in plants is a direct consequence of S supply; thus total plant S may be the first choice for evaluating the adequacy of the S supply. Ulrich and Hylton (1968) concluded that SO,-S content of blades or stems is useful for diagnosing the S status of rye grass (Loliurn mulfij7orurnLam.). Critical

240

N. S. PASRICHA AND R. L. FOX

values of S in plants are affected by stage of plant growth, plant part analyzed, and amount of defoliation previous to sampling, as in the case of rapeseed mustard and grazed or mowed pasture crops (Jones et al., 1975). Sulfate, N:S ratio, and SO,-S:total S ratios may be less influenced by these side issues than total S. However, plant sampling for diagnostic purposes is usually restricted to a relatively short period of time and to specific tissues so that the problems enumerated above are not as serious as is sometimes assumed. The usefulness of foliar analysis is greatly enhanced if nutrients other than S are adequate. Because this is often not the case, the use of ratios of nutrients is helpful in diagnosing problems and making recommendations. Sumner ( 1981) has vigorously advocated the “Diagnosis and Recommendations Integrated System” (DRIS) for that purpose. The system appears to have merit when a large data base of yields and chemical compositions is available. But even for major crops, S data are scarce and for many crops in the tropics and subtropics data are almost totally lacking. However, pasture crop yield data from thousands of sites are not available and would be difficult and expensive to obtain (Beaton et al., 1985). Data for foliar diagnosis cannot be transferred with confidence among various sampling systems. Therefore, standard procedures should be agreed on that would serve as reference against which other procedures could be calibrated. One such procedure has been suggested for banana and calibration work on S has been published (Fox et al., 1979). 2. Optimum N :S Ratio in Plants

The N :S ratio in plant material has been used as an index to determine the probability of crop response to N, and more particularly, S fertilization. Although total N :total S ratios have been used successfully to diagnose S needs over a wide range of S nutrition, considerable fluctuations in the ratio have been reported as S levels in plants approach adequacy (Pumphrey and Moore, 1965). One of the problems of using N :S ratios is that S is a relatively immobile nutrient in plants. Older leaves tend to be higher in S than young leaves. Research with sugarcane, banana, and macadamia demonstrate that very well (Fox et al., 1976). Nitrogen is mobile; young leaves tend to have higher N concentrations than old leaves. So it is not correct to assume that the N :S ratio is more stable with age, or stage of maturity, or plant tissue sampled, than total S or SO,-S is. If the critical level of S in the ear leaf or maize is about 0.24%,a critical N : S ratio of about 12 is indicated. Daigger and Fox ( 1971) observed that most of the N : S ratios of nonS-fertilized sweet corn were little greater than 12 (Table VI). It is evident from their data that yield versus N :S ratios do not show

24 1

PLANT NUTRIENT SULFUR Table VI

Yields of Irrigated Fresh Sweet Corn, and N:S Ratios, in Relation to N and S FertilizationD

SO,-S applied (kg ha-') 22

0

66 ~~

N applied (kgha-I)

Yield (103kgha-')

45 90 I35 180 225

9.8 11.4 12.9 12.8 13.5

N:S

Yield (103kgha-')

N:S

Yield (IO'kgha-I)

10.2 13.4 13.6 11.9 14.9

11.2 13.0 13.4 13.2 13.7

10.0 11.4 12.2 12.6 12.0

11.4 12.2 13.4 13.2 13.2

N:S ~~

8. I 10.0 8.2 10.4 9.3

'Adapted from Daigger and Fox (197 1). any notable trend. The correlation coefficient for the relationship was only 0.277. For apparently adequate nutrition (yields 13.0 to 13.7 tons ha-'), ratios ranged from 8.2 to 14.9. There are other problems. When no S was applied, 225 kg N ha-I was required for maximum yield (13.5 tons), but when 66 kg S ha-' was applied also, yield was maximum ( 1 3.4 tons ha-') at 135 kg N ha-'. Thus, a case for the N : S ratio could not be made from the data examined. Perhaps a better system, at least it seems so for banana, is to compare the S in young leaves to the S in old leaves. If the ratio approaches unity, S nutrition is adequate. This system is consistent with the general observation that S deficiency produces a chlorosis of young leaves whereas a N deficiency produces a chlorosis of old leaves.

IX. CRITICAL SOIL SOLUTION CONCENTRATION Data on the external SO4 concentration required by plants have been accumulating for several years and have now become sufficiently numerous to provide a basis for predicting adequacy of the soil S supply and S fertilizer requirements. Spencer ( 1975) cites data showing 3- 5 pg ml-' S adequate for growth of many species, although rape and lucern were somewhat higher in their requirements at 8 p g ml-I S. From the work done with several crops under a variety of conditions (Hasan et al., 1970; Daigger and Fox, 1971; Fox et al., 1976, 1977, 1979), it appears that the external S@- S requirement for several crops is approximately 5 pg m1-I.

242

N. S. PASRICHA AND R. L. FOX

Several attempts have been made to identify sulfur-deficient soils on the basis of total and/or extractable sulfate, and some of these attempts have been successful in the temperate zone. This approach may be less than satisfactory in the tropics. One reason for this is the large quantity of “sorbed” sulfate in many soils of the tropics (Hue et al., 1990). For example 58% of the readily available S in a group of soils from Brazil was sorbed sulfate but only 2% of the readily available S in Iowa was due to sorbed sulfate (Neptune ez al., 1975). A problem arises because of uncertainty about the nature and availability of extractable sulfate (Adams and Rawajfih, 1977); although it is clear that plants can utilize adsorbed sulfate to some degree (Barrow, 1969) and availability of basaluminite has been demonstrated (Wolt and Adams, 1979), it has been observed that S deficiency may develop in crops growing on tropical soils that contain more than 1000 kg ha-’ of SO,-S within the root zone. Sulfate sorption by soils is concentration dependent, and the reciprocal is also true, so that sulfate in soil solution, the availability of which plants must depend on, can be less than 1 p g ml-’, even if adsorbed S is high, but sorption capacity is even greater (Table VII). Sulfate in some tropical soils is held so strongly that it is sometimes considered virtually insoluble. The fact that it persists in these soils regardless of leaching is evidence of low solubility. But it is by no means insoluble. Concentrations of SO, usually exceed those of PO, by one or two orders of magnitude. Subsoil S frequently is less soluble than S in the surface soil (Lund and Murdock, 1978). If the hypothesis that plants derive sulfate from soil solution is valid, and if an adequate concentration of S in solution is in the range of 2 - 5 pg ml-’, then it is clear from Table VII that some subsoils, even though they may be rich in adsorbed SO,, will not support sulfate concentration in the soil solution sufficient for adequate plant nutrition. However, the importance of subsoils as a S source has been recognized for a long time. Probert and Jones ( 1977)accurately distinguished fertilizer S-responsive sites from nonresponsive sites by using weighted profile means of extractable S to a depth of 1 m or more (Fig. 9). That this sulfate is also positionally available is evident from data on the uptake of subsoil SO,-S using a radioisotope dilution technique (Goh et al., 1977; Gregg et al., 1977). This research indicated uptake of S by grass and clover roots to depths of at least 1 m in one soil and 50 cm in another. A 1982 study, as yet unpublished by R. L. Fox, P. M. Cooper, and W. M. H. Saunders, evaluated the availability of soil sulfate in a set of samples taken at 20-cm increments to a depth of 200 cm from 19 New Zealand soils. Some of the samples contained > 1000 mg SO,-S kg-I (Fig. 6). These soils were well leached and acidic. Sulfur was extracted with

24 3

PLANT NUTRIENT SULFUR Table VII Status of Sulfate S in Some Tropical Soil Profilesa Sulfate as sampled

Saturation at 5 pg l i t e r ' in solution

s g-9

(%I

(%I

32 27 145 220

78 67 39 51

herto Rico, Dagney (Orthoxic Tropohumult) 0- 10 10 0.6 10-30 I70 3.0 250 I .5 30 - 60 60-95 373 I .5

51 435 548 67 I

20 39 46 56

herto Rico, Catalina (Tropeptic Haplothox) 0- 16 414 6.0 16-35 1080 16.0 35-60 1310 16.0 60 - 80 I267 2.0 80- 120 1225 5.0 120-130 195 3.0

930 I I80 1430 I720 1730 860

45 92 92 74 71 23

Hawaii, Hanipoe (Typic Dystmndept) 0- I 5 25 0.4 15-30 18 16 30 - 60 17 60-90

220 -

11 -

-

-

-

-

Depth increment (cm)

Adsorbed sulfate (pg g-')

Sulfate in solution (pg s m1-9

Nigeria, alagba (Oxic Paleustalf) 0- 15 25 15-30 18 30 - 60 56 60 - 90 I12

8.0 4.5 I .2 0.7

Hawaii, Akaka (Typic Hydmndept) 0-15 220 15-30 1210 30 - 60 3730 60 - 90 5480 -

Sulfate adsorption maxima h 3

Adapted from Fox ( I980a); by permission of The Sulphur Institute, Washington, D. C.

Ca( H2P04)2solution and by short-term growth of ryegrass seedlings. Plant S percentage increased linearly with increasing log SO,-S until soil S reached 250 mg kg-'. Maximum uptake was attained at 1000 mg kg-I, after which uptake decreased. Apparently, the low-concentration mechanism of SO4 retention (presumably adsorption) began to phase out after soil SO4 reached 250 mg kg-' and a second mechanism (presumably a compound of low solubility) controlled availability (solubility) as soil

N. S. PASRICHA AND R. L. FOX

244 1 .o

0.8

0.6 9 al

.-x al > .-c

04

m -

2 0.2

0 0

2 4 6 8 Phosphate-extractable S (pprn)

10

Figure 9. Relationship between relative yield of legumes and weighted profile mean of extractable sulfer. Fitted curves are as follows: A = I - Y d Y , = exp(-0.7228 S ) , R = 0.43. 19 D F B = 1 - Y d Y , = exp(-0.7262 S ) , R = 0.58, 18 DF. 0, Siratro; 0, various Stylosanthes spp. (Probert and Jones, 1977).

SO,-S content approached 1000 mg kg-'. Further studies of this type are needed, but it is probable that both adsorbed and precipitated SO, are major factors in the S economy of highly weathered soils. That probability offers a plausible explanation for why S deficiencies have been slow to develop in such soils. In negatively charged soils that may have no sulfate buffering capacity, the magnitude of external S is such that movement of S to roots by mass flow in the transpiration stream is suggested (SO, concentration in soil solution X transpiration ratio = plant S). In variable-charge soils, the concentration of SO,-S may be lower than necessary for adequate nutrition, even though deficiency of S may not be obvious (Fox, 1984). Yield response curves that relate plant growth to the external SO,-S concentration demonstrate that substantial yields can be made at solution concentrations that require SO4 movement to roots along concentration gradients. These observations suggest that in some well-buffered soils, adsorbed SO, is moving to plant roots by diffusion. A constant but low SO, concentration in solution in relation to large amounts of P-extractable SO, from chemically similar soils suggests that S-bearing minerals, such as basaluminite, are controlling soil solution concentrations (Fig. 10). However, a smooth plot of concentration versus

PLANT NUTRIENT SULFUR

245

22

B+ Q

20

s -J

Qa

18

-

Jurbanite

I

1

1

I

I

I

I

quantity suggests adsorption. There are reasons to believe that both mechanisms are involved in controlling sulfate solubility in highly weathered soils. Sulfate adsorption isotherms of volcanic ash soils generally show biphasic properties and suggest that 40-8Ofig SO4-S g-' is required to maintain 3 - 6 mg SO,-S liter-' in soil solution, a concentration range considered adequate for growth of most crops (Hue et al., 1990). A rational approach to S nutrition is to detem'ine the required sulfate concentration in the soil solution (the external S requirement) and the amount of S fertilizer needed to produce that concentration. Investigations to define specific external S requirements of plants have been conducted using four lines of approach: 1. The minimum S content of imgation water associated with near maximum production (Blair et a/., 1979; Wang, 1978; Yoshida and Chaudhry, 1972). 2. Yield curves as a function of SO4-S in solution based on sulfate sorption curves. This approach is appropriate for soils that have a high capacity for sulfate sorption (Hasan et al., 1970). 3. Solution or sand culture experiments with SO, concentration as a variable (Fox, 1976). 4. Frequent leaching of soils with dilute sulfate solutions to establish and

246

N. S. PASRICHA AND R. L. FOX

maintain a range of SO, concentrations in “soil solutions,” in which plants are grown (Fox ef al., 1976, 1977, 1979). From results of these investigations, using several crops, it seems reasonable to generalize that the external S requirements of crops in the subtropics and tropics are approximately 2 - 5 mg liter-’ S in solution.

X. CROP RESPONSES Numerous papers have been published on responses to S by crops growing on highly weathered or intensely leached soils. An extensive listing has been prepared by Blair (1979). That S deficiency is a problem in the tropics and subtropics, and that the deficiency has a potential for becoming worse, is abundantly clear from papers on sugar, fiber, and oil crops (Aulakh and Pasricha, 1988; Pasricha el al.. 1987, 1988, 1991; Pasricha and Aulakh, 199 1 ; Braud, 1969; Stanford and Jordan, 1966; Fox, I976), legume forages (Metson, 1973; Pasricha and Randhawa, 197 1, 1975), grain legumes (Fox ef al., 1987; Aulakh and Pasricha, 1986; Aulakh et al., 1990; Pasricha ef af.,1987; 1991), rice (Wang, 1978; Mazid, 1986), corn (Kang and Osiname, 1976; Pasricha et al., 1977a), coffee (de Freitas et al., 1972), and banana (Fox ef al., 1979). Sulfur deficiencies in tropical, subtropical, and warm temperate areas are being reported with increasing frequency (Blair, 1974; Jones ef af., 1975; Tandon, 1991). Sulfur probably is the fourth most limiting nutrient in highly weathered soils, and if only the tropics are considered, it probably ranks third. Furthermore, if effectively nodulated legumes are being grown, S moves up one place in the ranking to third or second. The relative adequacy of 12 nutrients for clover on seven mountain soils from Equador is summarized in Table VIII, which clearly shows that S is second only to P for legume growth in these soils. Fox et al. ( I 977) observed that the S content of the seeds of cowpea increased with increasing S fertilization of soil. The levels of S adequate for seed yield were also sufficient for near maximum S content in the seed. Sulfur percentage associated with 95% of maximum yield was 0.26%. Seed yield increased 15-fold as soil solution concentration increased from near zero to 1.8 mg m1-I. The S: N values were in the range 0.03-0.04 at S concentrations less than 2 mg liter-’, to 0.07-0.08 at > 5 mg liter-’. These results imply that to obtain maximum yield of cowpeas in the tropics, S fertilization will be required in many areas. The S concentration in rainwater in northern Nigeria during high-rainfall months is about 0.2 mg

247

PLANT N U T R I E N T SULFUR Table VIII Mean Relative Yields of Trifolium mpens as Intluenced by Withholding Nutrients from Plants Grown in Seven Mountain Soils of EquadoP

Relative yield (%) Nutrient(s) withheld None (all added) All (none added) P S K Ca B

General responseb

Mean

-

Range

100

-

Deficient, 7 soils

16

Deficient, 6 soils NS'

41 95 92 96

0-45 0-46 17-84 80- I15 67- I17 66-134

NS

Deficient, I soil Toxic, 1 soil

17

-

-

Calculated from data of Poultney (1975). ' No statistical evidence that Mg, Zn, Fe, Mn, Mo, or Cu increased yield.

'NS, Not statistically significant.

liter-' (Bromfield, 1974), a level far below adequacy for either good yield or high seed S content. However, S contents of rainwater that infiltrates the soil are augmented by S leached from the standing crop and crop residues and are further concentrated by water evaporation and transpiration. The final concentration may be more favorable than is indicated by rainwater composition. Laurence et a/. (1976) observed that in Malawi, S applied either as a foliar dust or soil treatment increased yields of groundnut, although the effects of complementary treatments were not fully additive. Besides improving yield, S application also produced kernels of better size and quality than untreated samples. There is great pressure to achieve breakthrough in human nutrition by introducing new foods into diets or by developing new cultivars of staple crops that have high protein contents and at the same time produce high yields. The improbability of accomplishing either goal (much less a happy combination) on a sustained basis given the S nutrition constraints of vast areas in the tropics is evident from research in West Africa (Bromfield, 1974; Fox ef al., 1977). On the other hand, it is possible to increase the S amino acid content of cowpea, groundnut, and mustard and at the same time achieve greater yields of grain by increasing the S supply (Pasricha et al., 1970; Evans ef al., 1977; Fox ef al., 1977). Likewise, S fertilization

N. S. PASRICHA AND R. L. FOX

248

Table IX Elemental Composition of 11 Cultivars of Cowpea Grain Produced at IITA, Ibadnn, Nigeria and Calculated Quantities in Harvested Grain for Two Levels of Production'

Elemental yield (kg ha-') Concentration (%) Element

Mean

Range

N P K Ca Mg S Zn

3.97 0.47 I .63 0.10 0.23 0.25 0.0044

3.64-4.36 0.44-0.54 1.50- 1.80 0.10-0.1 I 0.21 -0.23 0.2 I -0.28 0.0038-0.0051

Typical yield'

Agronomicall possible yield

8.9

59.7 1.4 24.4 I .5 3.4 3.8 0.07

1.1

3.7 0.2 0.5 0.6 0.01

i

Adapted from Fox et al. ( 1977). A typical yield is 224 kg ha-I; 1500 kg ha-l is believed to be agronomically feasible.

improves the quality of rice (Jones el al., 1975; Yoshida and Chaudhry, 1972). Summerfield et a/. (1974) placed cowpea (Vigna unguiculata) among the grain legumes, with immediate potential for alleviating human malnutrition in the tropics. Cowpea is relatively rich in protein. The leading area of production is in the West African Savanna. Per season yield in Nigeria was 224 kg ha-', but it is feasible to produce much higher yields. It is obvious from Table IX that such agronomically feasible yields will require much higher outlays of nutrients. Fox ef al. (1977) examined what these numbers mean with respect to S supply. Assuming typical yields of 224 kg ha-', a mean sulfur percentage of 0.2596, and 50%partition of plant sulfur into the grain, a projected value of 1.12 kg S ha-' in the cowpea is obtained. Mean annual sulfur in the rainfall for northern Nigeria is about 1.14 kg ha-' (Bromfield, 1974). These estimates suggest that cowpea production in the dry savanna is already limited by a nutritional (S) constraint. To attain near-maximum yield, the indicated requirement for sulfur will exceed natural inputs by an order of magnitude. Obviously, production cannot be sustained with such a deficit. Few studies have been reported on responses of millets to S. Relatively large difference between yields of millet fertilized with single superphosphate and triple superphosphate after 7 years of cultivation (Fig. 1 1 ) are evidence of the need for S in the soils of West Africa. The implications for

PLANT N U T R I E N T SULFUR

1400 1200

1 -

249

Single superphosphate

1000 800 .-C

s

600 400 L

200

I

I

I

I

I

4.4

80

130

175

P-applied (kg P/ha)

Figure 11. Effect of P sources and rates of application on pearl millet grain yield at Sadore, Niger. Rainy season, I988 (Bationo and Mokwunye, I99 I ; Reprinted by permission of Kluwer Academic Pub1ishers.l

S fertilization are discussed by Friesen ( I99 I ). Single superphosphate, rather than more concentrated fertilizers, may be a preferred source for crops that require both P and S to increase crop yields (Aulakh and Pasricha, 1988; Aulakh et al., 1980b; Pasricha et al., 1987, 1991). Table X presents a summary of yield responses to sulfur applications in Bangladesh. In most cases, 500 ppm P extracted approximately 10 p g g-' soil. Yield increases in various crops attributable to S were 5-95% where S was applied in conjunction with N, P, and K. Yield responses to applied S were observed in corn grown in the Dominican Republic (Pierre et al., 1990). Sulfur deficiency is intensified by burning, which volatilizes up to 75% of S contained in residues (Sanchez, 1976). Because much of the total S in soils of subhumid regions is in organic forms (Tabatabai, I982), total and available S are expected to be low in conventionally tilled soils where residues are burned. In Thailand, S applied along with NPK fertilizers significantly increased yields of cassava, corn, and sesame growing on coarse-textured soil with less than 8 - 12 mg kg-I phosphate-extractable SO,-S (Parkpian el al., 1991). Intensive cropping and use of S-free fertilizers has caused 60 - 70% of rice in South Sulawesi, Indonesia, to become S deficient; pastures respond to S

Table X Average Yield Increase of Crops Due to S Application in Bangladesh'

Yield (tons ha-l)b Crop

Increase in yield (%)

Response ratio (kg extra yield per kg S)

Trials

Locations

NPK

NPKS

Rice

50

8

4.26

4.53

6.34

9

Wheat Maize Mungbean Chickpea

39 10

10

3.2I 4.62 0.59

1 .oo

9.93 40.85 9.26 9.64

45 2 6

10

1

1

3

1

2.92 3.28 0.54 0.84

Mustard

4

3

0.81

1.27

56.79

15

Groundnut Potato

3 3

3

I

1.69 22.I3

1.85 22.80

9.41 3.03

5 22

Sugarcane

6

3

16.39

81.31

6.44

164

Conon

5

1

1.67

1.83

9.58

5

18

7

2.31

2.42

2.1 1

18

4

1

1.91

2.00

4.7I

4

Jute Tobacco

a

2

Reference(s) Bangladesh Agricultural Research Council (BARC)(1981,1982, 1983) -do-doAhmed er al. ( 1 984) Talukder er 01. (1984)and Bangladesh Agricultural Research Council (BARC) (1981,1982) Bangladesh Agricultural Research Institute (BARI) (1981,1982) Noor and Islam (1983) Bangladesh Agricultural Research Council (BARC) (1981,1982) Bangladesh Agricultural Research Council (BARC) (1981,1982, 1983) Bangladesh Agricultural Research Council (BARC)(1981, 1982) Bangladesh Agricultural Research Council (BARC)(1981, 1982, 1983) Bangladesh Agricultural Research Council (BARC) (1981,1982)

Adapted from Hussain (1990). NPK, Addition of nitrogen, phosphorus, and potassium; NPKS, addition of nitrogen, phosphorus, potassium, and sulfur.

PLANT NUTRIENT SULFUR

2s 1

4

z Figure 12. Response in Centrosema pubescence to sulfur applications in South Sulawesi, Indonesia (Blair ef a/.. 1978).

application (Blair et al., 1978), and responses to these applications were greater than to P applications (Fig. 12). In the Sahelian and Sudanian zones of West Africa, soil organic matter is being exploited to supply S to crops (Bationo and Mokwunye, 1991). Friesen ( 1991) reported on the fate and efficiency of S fertilizer applied to food crops in West Africa. Sulfur fertilizer increased grain yields from 10 to 65% in 14 out of 20 site-years in semiarid and subhumid West Africa. Substantial leaching losses resulted in low crop recovery of fertilizer S. Low S fertilizer rates were required, which suggests that S deficiencies in the region can be corrected at relatively low cost. Leaching losses probably explain the poor residual value of sulfate fertilizers on highly permeable soils of West Africa. The low organic matter content of soils provides a very small sink for S immobilization. Most of the residual S (about 71%) remained as SO, in the profile (Fig. 13) and is again subject to leaching at the onset of the next rain. The high mobility of SO, in this region is a result of sandy soil texture. Considerable interest has developed in acid subsoil amelioration by

252

N. S. PASRICHA AND R. L. FOX Fertilizer-S(% applied)

0

10

20

30

15

30

5

v

45

%.a n

60

75

Figure 13. Soil profile distribution at harvest of S derived from phosphogypsum applied to millet at Sadore, Niger (Friesen, 1991; Reprinted by permission of Kluwer Academic Publishers.)

applying large quantities of gypsum on the surface of highly weathered soils and letting natural leaching move Ca and SO4 into the subsoil. The primary purpose, A1 inactivation, deals only indirectly with S as a nutrient, but S in solution and S nutrition of crops will be influenced, irrespective of the primary purpose. The physical chemistry and mineralogy of what happens in the subsoil is complicated (Fig. 10) and crop performance may be influenced in ways that are unexpected (Farina and Channon, 1988). In this regard, it will be appropriate to remember that SO4-S concentrations in solution greater than 15 mg liter-' have depressed the growth of banana (Fox d al., 1979) and cowpea (Fox et al., 1977).

XI. SULFUR FERTILIZATION AND CROP QUALITY The contribution of soil fertility to crop quality should not be overlooked. In areas of highly weathered soils, but especially in tropical areas

PLANT NUTRIENT SULFUR

253

where leached and weathered soils constitute the majority of soils used for local food production, crop quality becomes especially important. One concern is amino acid deficits in diets. It is in the tropics, with low anthropogenic additions of S and intensively leached soils, that S deficiency is most likely; and it is in the tropics and subtropics, generally, that protein intake is low and primary dependence is placed on plant protein sources, usually grain legumes and cereal grains.

A. EFFECTON PROTEIN QUALITY Sulfur applied to crops grown on S-deficient soils not only increases crop yields but also favorably affects crop quality. Concerns about protein quality have led to interest in increasing the sulfur amino acid content of edible legumes (Luse er al., 1975; Pasricha er al., 1991). Pasricha ef al. (1970) observed increased S-containing amino acids in response to S fertilization of groundnut and mustard. For some lupine varieties, S fertilization increases the S amino acid content of seeds. This increase is associated with a change in the proportion of various proteins with differing amino acid ratios (Blagrove ef al., 1976). Sulfur-containing amino acid content is a better predictor of protein efficiency than is total S (Sandhu er al., 1974), but relative ease of determination makes the N: S ratio desirable for screening purposes. For human nutrition, legume seed proteins are deficient in sulfo-amino acids. A possible remedy for this deficiency is to increase the sulfo-amino acid levels in seeds by S fertilization. Concerns about protein quality have created interest in using N : S ratios of cowpea cultivars as a tool for screening for protein quality (Porter et al., 1974). The protein S : protein N ratio of IVu 76 cowpea meal increased 27% over the control when cowpea was supplied with 5 mg liter-' of SO4-S, and that of variety Sitao Pole increased 100%when cowpea was supplied with 1.8 mg liter-' of SO,-S (Evans er al., 1977). Further details are presented in Table XI. Sulfur concentrations, S : N ratios, and S amino acid contents in cowpea seeds increased with increasing levels of S fertilization. For cowpea variety Sitao Pole, concentrations of methionine and cyst(e)ine increased approximately twofold as adequacy of S supply increased from severe deficiency to sufficiency for maximum yields (Evans er al., 1977). For variety IVu 76, methionine content was increased by 14%, cysteine increased 32Y0, and S-methyl-L-cysteine increased 470%. Of the 53% increase in S percentage associated with 5 mg liter-' SO,-S, 16% was derived from increased methionine plus cysteine and 3 I % from increased S-methyl-L-cysteine.

254

N. S. PASRICHA AND R. L. FOX Table XI

Ratio of S in Methionine

+ Cysteine to Amino Acid N in Cowpea Varieties under Various Sulfate S Fertilition Levels'

Recovered Cowpea Treatment amino acid N Met + Cys cultivar (SO,-S mg liter') (g/IOO g meal) (a100g meal)b %Met + Cys):N(amino acid) IVu 76

0 0.2 0.6 I .8 5.0 15.0 45.0

3.38 3.77 3.62 3.68 3.19 3.13 3.33

0.1 10 0.104 0.1 14 0. I29 0.134 0.122 0. I52

0.033 0.028 0.03 I 0.035 0.042 0.039 0.046

Sitao Pole

0 0.2 0.6 I .8 5.0 15.0 45.0

3.90 4.36 4.22 3.6 I 3.85 3.48 4.04

0.070 0.093 0.1 I I 0.128 0.143 0.133 0.150

0.018 0.02I 0.026 0.036 0.037 0.038 0.037

'Adapted from Evans et a/.(1977). Dry weight basis of cowpea.

B. EFFECTON OILCONTENT Improving the S nutrition of S-deficient oil seed crops increases oil contents in peanut (Aulakh et al., 1980b; Singh, 1968), Brussica species (Aulakh er af.,1980a; Pasricha el af., 1988), linseed (Aulakh et af., 1989), and soybean (Aulakh et af., 1990) (Table XII).The relative concentration of different fatty acids in some oilseeds determines their use. Sulfur fertilization with an adequate supply of N and P resulted in a large decrease in percentage of stearic, oleic, and linoleic acids with a concurrent increase in the content of linolenic acid (Aulakh et af., 1989).

C. EFFECTON GLUCOSINOLATE CONTENT Plant S is the major factor in the glucosinolate content of oilseed rape (Zhao et ul., 1991). Excessive S can result in unacceptability due to high glucosinolate levels and inadequate S may substantially decrease yields. Both situations markedly reduce the profitability of oilseed rape crops. Therefore, the effects of S application should be quantified for both yield and quality in order to obtain optimum benefits.

255

PLANT NUTRIENT SULFUR Table XI1 Influence of Applied S on the Oil Content, Protein Content, and Oil Yield of Dierent Oil Crops'

Oil content

Oil yield (kg ha-')

(%)

Protein

Crop

No S

S

No S

S

No S

Peanut Brassica juncea Brassica compestris Linseed Soybean

39.0 36.4 41.5 41.6 21.7

48.0 42.6 47. I 43.2 23.6

659 540 300 1285 317

859 670 450 1480 412

-

-

22.5 23.3

30.8 28.1

28. I

31.9

-

S

-

Adapted from Pasricha and Aulakh (1991); by permission of The Sulphur Institute, Washington, D. C.

D. EFFECTON NITRATE CONTENT Sulfur plays an important role in secondary plant metabolism, which is related to parameters determining the nutritive quality of vegetables (Schnug, 1990). Nitrate concentration in vegetables has become an important criterion for food quality (Corre and Breimer, 1979; Schuphan, 1976; Vetter, 1988). A shortage of S adversely affects utilization of N during plant metabolism. Thus S deficiency causes an accumulation of nonprotein N compounds, including NO3 (Fig. 14). Such a condition indicates severe S deficiency and is invariably associated with S deficiency symptoms

Y = 69.35 Exp(-l.l28X)+ 0.643

0

1

2

3

4

5

6

S-Content (mg g-')

Figure 14. Nitrate concentration in the dry matter of lettuce as influenced by plant S status of the plant (Schnug, 1990; by permission of The Sulphur Institute, Washington, D.C.)

2 56

N. S. PASRICHA AND R. L. FOX

(Schnug, 1990). Murphy (1 990) observed that S fertilization affected N : S ratios and significantly reduced NO3 contents. An inadequate S supply hinders protein formation and results in accumulation in forage crops of soluble N compounds such as nitrate N and amide N (Pasricha and Randhawa, 1975).

XII. SULFUR INTERACTIONS WITH OTHER ELEMENTS A. INTERACTION

WITH

PHOSPHORUS

The fertilizer P and S interaction may be positive or negative depending on (1) the level of each when applied in combination and (2) soil conditions that control availability of each nutrient. If applied P induces SO, leaching in soils in which the S level is marginal, onset of S deficiency may be hastened. In such cases the interaction is antagonistic. On the other hand, in highly weathered soils that may retain adsorbed SO,, added P may mobilize the SO,, increasing its availability in the soil. In such a case, application of S along with P may be without benefit. For example, crop responses to applications of P and S were synergistic at fertilizer rates of 20-40 kg P and 43 kg S ha-' (Pierre et al., 1990), but others have shown antagonistic effects (Barrow, 1969; Aulakh et al., 1990).

B. INTERACTION

WITH OTHER ELEMENTS

Sulfur fertilization may lower the concentration of B and Mo in plants. This antagonistic effect has been used to suppress Mo in forages growing on Mo-toxic soils (Pasricha and Randhawa, 1971, 1972; Pasricha et al., 1977b). On coarse-textured soils with marginal to low amounts of B and Mo, S fertilization of Brassica species can create deficiencies for these crops (Schnug and Haneklaus, 1990). Sulfur fertilization is a feasible technique by which to decrease plant uptake of some toxic or otherwise undesirable elements on polluted soils. In areas where Se toxicity exists, Se uptake can be suppressed by S fertilization (Dhillon and Dhillon, 1991). Antagonistic relationships between S and anionic trace elements such as arsenic, bromine, and antimony have been reported. Grill et al. ( 1990) reported that excess S fertilization may also increase concentrations of cations such as Cu, Zn, and Cd in roots, while reducing levels in shoots. This results from stimulated production of phytochelatines (metallothioneins) in roots, induced by metals in the growth medium, and

PLANT NUTRIENT SULFUR

257

perhaps by enhanced S supply. By this mechanism, plants may avoid excess uptake. Thus, S fertilization may be a feasible technique to enhance the quality of crops grown on polluted soils.

XIII. SUMMARY AND CONCLUSIONS Sulfur deficienciesin the tropics and subtropics have been recognized for more than 50 years, but even today the extent and magnitude of the problem is ill-defined. In recent years S-deficient areas of considerable extent have been discovered and delineated, including, for example, much of Bangladesh and South Sulawesi. Sulfur deficiency has been slow to develop, or at least slow to be recognized, for several reasons: the atmosphere is a ubiquitous source of S; other nutrients, especially N and P, are usually even more deficient than S; S has been applied in irrigation water and as adjunct to other nutrients (a factor that is rapidly decreasing in importance); SO, is more efficiently used by plants than NO3, with which it is frequently compared; as soil organic matter is exploited, S cycling between organic and inorganic forms is net positive for inorganic S; adsorbed SO,, which is usually abundant at some depth in profiles of highly weathered soils, is continually being released. The pattern of S deficiency on a global scale leads at once to the conclusion that areas prone to S deficiency are those that are remote from industrial and domestic burning of fossil fuels, areas where weather patterns are controlled by air masses originating in remote regions, and areas that have marked wet-dry seasons giving rise to savanna-type vegetation that is burned frequently. Much of the tropics and subtropics is included in one or more of these categories. Sulfur sources in much of the continental tropics are meager. Long-term yields there will not exceed those that can be supported by the incoming S supply. In some areas S yields in crops are approximately equal to incoming S in the rainfall. In the case of soils that do not adsorb sulfate, S supply is controlled by S currently accruing as rainfall (wet deposition) and directly from the atmosphere (dry deposition), plus S mineralization from organic matter. Other sources may be locally important: irrigation water, fertilizers, animal manure, and plant residues. Adsorbed SO, and/or sparingly soluble SO,-containing minerals are major factors in the S supply of highly weathered subtropical and tropical soils. In most highly weathered soils, large quantities of SO, have accumulated somewhere in the profile. Usually the accumulation approaches maximum at about a 1-m depth. Total SO,-S in some leached profiles

258

N. S. PASRICHA AND R. L. FOX

exceeds 16,000 kg ha-'. These forms of SO, are usually associated with acid soils that contain hydrated oxides of iron and aluminum. Adsorbed SO, can be extracted with phosphate solutions, presumably by ligand exchange. This has led to the use of phosphate solutions as extractants for soil-testing purposes. Success has been mixed; the availability of SO, so extracted should not be inferred from quantity alone. Sulfate concentration in highly weathered soils is low, but, although many of these soils contain copious amounts of adsorbed SO, within the root zone, and although availability to plants of adsorbed SO, has been demonstrated, crops may be mildly S deficient. Sulfur concentrations in rainwater and irrigation water can be used as a rough guide to the level of S nutrition of crops and long-term requirements for S fertilizers. It is obvious that sustained production cannot remove more S than has been put into the system. In remote areas of the subhumid tropics in the Northern Hemisphere, S inputs are in the range 1 -2 kg ha-'. Even lower values can be expected in similar situations in the Southern Hemisphere. Estimates of S being removed in some crops (cowpea and peanut) in subhumid tropical Africa are approximately equal to S inputs. It is reasonable to believe that, without additional S inputs, there can be no significant yield increases unless additional S is introduced. The oceans are important sources of S. However, the influence of oceans decreases rapidly with distance and elevation from the coast. Global estimates of S inputs suggest that biological sources, among which those of the oceans are dominant, contribute more S to the atmosphere than manmade pollutants. Sea spray across the sea-land boundary contributes relatively little to the total global system. The list of crops for which S fertilizer has been beneficial is almost as long as the list of cultivated crops. Some crops that formerly were not considered to be susceptible to deficiency, rice, for example, are now considered as being so. Seasonal burning of vegetation during the dry season is widely practiced in the tropics. Without doubt, burning represents a severe drain on already meager S resources. Probably much of the S volatized is recovered in adjacent areas of green vegetation and accounts for the relatively better S status of these areas. Because S accrues to plants from numerous sources, instances of acute S deficiency are not common in the field. Even in S-deficient areas, typical yield increases resulting from S fertilization are in the range of 5 - 20%. Thus much evidence for S deficiency can be overlooked by an ultraconservative approach to data interpretation. As a first approximation the fertilizer requirement should be that which will establish and maintain 3-5 mg SO,-S liter-' in solution. For me-

PLANT NUTRIENT SULFUR

2 59

dium-textured soils that contain little adsorbed S, this amounts to approximately 10- 15 mg SO,-S kg-' on a dry soil basis. For soils in which adsorbed SO, controls SO, concentrations in soil solutions, a rational approach to predicting whether S fertilizer is required, and how much, can be based on sorption curves. The approximate fertilizer requirement is that which will establish a level of SO, in solution appropriate for the crop being grown. Most soil testing for advisory purposes uses turbidimetric methods for SO, analysis. Most of these procedures are not satisfactory for extracts of highly weathered soils. Some substances inhibit BaSO, precipitate formation in extracts of such soils. Many of the data on SO, in tropical and subtropical soils are probably underestimates. A more reliable, although more complicated, method has been developed. Although SO, concentration in rainfall can be used as a rough guide to the adequacy of sulfur supply, it should not be taken at face value. Wet deposition of S is augmented by dry deposition as rainwater passes through plant canopies, plant residues, and into soils, and it may be concentrated further in the soil by surface evaporation. Sulfur contents of plants increase with increasing concentrations of S in soil solutions. For many crop species maximum yield requires approximately 0.2% S in leaves. Although crop yield and plant composition are sensitive to the level of S supply, foliar diagnosis of the S status has been little used in the tropics and subtropics. For survey work foliar analysis is probably superior to soil analysis, and seed analysis has advantages over both. Best results require that all appropriate tools be used. This is especially true for evaluating the S status of crops in the tropics, where, in many areas, background information is lacking. Care must be exercised in selecting tissues for foliar analysis. Because S is one of the less mobile nutrients, it may accumulate in old tissues even though young tissues are deficient. Deficiency symptoms, if they are expressed at all, can be confused with symptoms of other nutrient deficiencies. Because S is relatively immobile in plants, upper leaves are first to show symptoms of deficiency-just the reverse of N. However, S-deficient plants are often more distinctly yellow than are N-deficient plants. An interveinal chlorosis develops in maize leaves that is similar to Zn or Fe deficiency. The external requirements for SO,-S in soil solutions is in the range of 3-5 mg S liter-' for some important crops of the tropics and subtropics; however, yields of approximately 80% of the maximum attainable yield may be obtained with as little as 1 mg liter-'. A need for special attention to S in the tropics and subtropics arises from the importance of S for human nutrition. The essential S-containing

260

N. S. PASRICHA AND R. L. FOX

amino acids of foods are of particular concern. Their concentration in plant products can be enhanced by appropriate use of S fertilizers. Finally, a word about the environmental impact of anthropogenic S in the atmosphere on S as a plant nutrient. On a global scale, excess S appears as local problems. In the subtropics, and especially in the tropics, levels of S from all sources are below those that are optimum for plant nutrition. From this perspective, burning low-sulfur fuel to avoid contaminating vast areas is nonsense.

ACKNOWLEDGMENTS The senior author is grateful to Dr.M. S. Bajwa, Professor and Head, Department of Soils, Punjab Agricultural University, Ludhiana for providing facilities. Special thanks are due to Mr.Subhash Chander Gossain for typing the manuscript.

REFERENCES Adams, F., and Rawajfih, Z. (1977). Basaluminite and alunite: A possible cause of sulfate retention by acid soils. Soil Sci. SOC.Am. J. 41,686-692. Ahmed, 1. V., Rahman, S., Islam, M. S., and Sultana, Z. (1984). Effect of phosphorus and sulphur application on the growth and yield, and phosphorus, sulphur, and protein contents of moongbean. Bangladesh J. Soil Sci. 20.25 - 30. Aulakh, M. S., and Pasricha, N. S. (1986). Role of sulphur in production of grain legumes. Fert. News 31 (9), 3 1-35. Aulakh, M. S., and Pasricha, N. S. (1988). Sulphur fertilization of oilseeds for yield and quality. Sulphur in Indian Agriculture. Fert. Assoc. India, The Sulphur Institute, Washington, D.C., S( I I)/3( I - 14). Aulakh, M. S., Pasricha, N. S., and Sahota, N. S. (1980a). Yield, nutrient concentration, and quality of mustard crops as influenced by nitrogen and sulphur fertilizers. J. Agric. Sci. 94,545-549. Aulakh, M. S., Pasricha, N. S., and Sahota, N. S. (1980b). Comparative response of groundnut (Arachis hypogaea L.) to the phosphatic fertilizers. J. Indian Soc. Soil Sci. 28, 342-346. Aulakh, M. S., Pasricha, N. S., Azad, A. S., and Ahuja, K. L. ( I 989). Response of linseed (Linum usitatissimum L.) to fertilizer nitrogen, phosphorus, and sulphur and their effectson the removal of soil sulphur. Soil Use Manage. 5, 194- 198. Aulakh, M. S., Pasricha, N. S., and And, A. S. (1990). Phosphorus-sulphur interrelationships for soybeans on phosphorus and sulfur deficient soils. Soil Sci. 150,705-709. Bahl, G . S., and Pasricha, N. S. (1984). Adsorption and desorption of sulphate by soils pretreated with different cations. Int. J. Trop. Agric. II, 143- 150. Bangladesh Agricultural Research Council (BARC) (1981, 1982, 1983). Coordinated soil test crop response correlation studies. Annual Report 1980-81, 1981 -82, 1982-83. BARC Soils Irrig. Publ. No. 9. Bangladesh Agricultural Research Institute (BARI) (1981). Annual Report 1981 -82. Joydebpur, Gazipur, Bangladesh.

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Index A Acidification, soil, 226 Acid rain, effects on crop plants, 223-225 forest vegetation, 225 soil acidification, 226 Adsorption SO,, mechanism, 234-236 sulfate by soils, 227-232 Adsorption curves, sulfate, 232-234 A horizons development, 191 - 192 oxidative polymerization by Mn oxides, 193 stabilization of organic matter in acid soils, I93 - 194 CO, from added organics, 195 in near-neutral soils, 194- 195 Albugu candida white rust, in Brassica spp., 28-32 Alfalfa, chloride deficiency, I10 Amphidiploids, among oilseed brassicas, 5 a-Amylase, chloride activation, I I I Anther culture, oilseed brassicas, 41 -42 Antibiotic resistance, intrinsic, Bradyrhizugbum japonicum and DNA homology groupings, 74-75 methods, 72-73 phenotype diversity, 72 - 74 and symbiotic performance, 74 Asparagine synthetase, chloride activation, Ill Atmosphere, sulfur sources, 219-221

B Eacillits thicringiensis toxin, production by oilseed brassicas, 46 Barley, chloride deficiency, I10 Beans, chloride deficiency, I10 Blackleg, resistance in oilseed brassicas, 2428

27 1

Black spot, resistance in oilseed brassicas, 34 - 36 Boron, sulfur fertilization effects, 256 Bradyrhizogbum japonicurn dissimilatory nitrate reduction and DNA homology groupings, 80 methods, 78 phenotype diversity, 78 and symbiotic performance, 79 fatty acids, 93 genotypic groupings, 68-69 hemoproteins, 92 - 93 hydrogenase system and DNA homology groupings, 78 phenotype diversity, 75-71 and symbiotic performance, 77- 78 intrinsic antibiotic resistance and DNA homology groupings, 74-75 methods, 72-73 phenotype diversity, 72-74 and symbiotic performance, 74 nitrogenase activity ex planfa, 92 phenotype/genotype relationships, summary, 94 plant growth-regulating substances and DNA homology groupings, 91 - 92 phenotype diversity, 90-91 and symbiotic performance, 9 I protein profiles and DNA homology groupings, 88 methods, 87 SDS-PAGE phenotype diversity, 8788 and symbiotic performance, 88 rhizobiophage typing and DNA homology groupings, 89-90 phenotype diversity, 89 and symbiotic performance, 89 rhizobitoxine and DNA homology groupings, 83-84 phenotype diversity, 80-82 and symbiotic performance, 82-83 serology and DNA homology groupings, 72

272

INDEX

methods, 69 phenotype diversity, 69-70 and symbiotic performance, 70-72 surface polysaccharides and DNA homology groupings, 87 methods, 84-85 phenotype diversity, 81 -85 and symbiotic performance, 85-86 taxonomic status, 93-95 Brussicu spp., oilseed, see Oilseed brassicas Breeding programs, for Erussicu seed yield, 10-13 Bromoxynil tolerance, oilseed brassicas, 39

C Cabbage, chloride deficiency, 109- I10 Cadmium sequestration by transformed oilseed brassicas, 47 sulfur fertilization effects, 256 Canker, stem, in oilseed brassicas, 32-34 Carbon pe-pH diagrams, 170- 172 reduction half-reactions, 163 Cation transport, chloride role, 1 I2 Chitinase, production by Erussicu spp.. 47 Chloride fertilizers, 134- I35 Chloride, in plants additions with no effects, 124 biochemical functions, 109- I I I enzyme activation, I I I photosynthesis, 110- I 1 I crop development responses, 124- I25 deficiencies, 109- 1 10 disease interactions, I I7 - I24 enhanced host tolerance and, 112- 114 IOSS~S, I33 - I34 manganese interactions, 1 16- 1 17 nitrogen interactions, 114- I I5 osmoregulatory functions, I 12- 1 13 phosphorus interactions, I I5 - I I6 quality responses corn, 129-131 oats, 129 potatoes, 132 soybeans, I3 I - I32 research needs, I4 I - 143 sources, 133- I34 suppression common root rot, I 19

foliar diseases, 1 19- I22 take-all root rot, I 18- I I9 uptake, 113-114 yield responses, I25 - I26 barley, I26 - 128 wheat, 126- 128 Chlorsulfuron tolerance, oilseed brassicas, 38 Chromium cycle, soil, 187- 188 Chromium, net oxidation test interferences, 205 protocol, 202-203 Coconut palm, chloride deficiency, I10 Copper, sulfur fertilization effects, 256 Corn chloride deficiency, 1 10 chloride effects, 129- I 3 1 Crop responses acid rain effects on plants, 223-225 to chloride developmental effects, I24 - I25 plant analyses, I35 - I38 soil testing, 138- 140 to sulfur, 246-252 Cultivars, oilseed brassicas, canolaquality, 7 Cytoplasmic male sterile-nuclear restorer system, in Erussicu spp., I7 -20

D Deposition dry, 218 wet, 2 I7 Desorption, sulfate by soils, 227 - 232 2,2’-Dipyridyl tests, for Fe oxides, 202 Disease resistance, in Brussicu spp. blackleg, 24 - 28 black spot, 34-36 light leaf spot, 36 stem rot, 32-34 turnip mosaic virus, 36 Verticillium wilt, 36- 37 white rust, 28-32 Dismutation, of H,O,, 204 Dissimilatory nitrate reduction, Brudyrhizogbumjaponicum and DNA homology groupings, 80 methods, 78 phenotype diversity, 78 and symbiotic performance, 79 DNA homology groupings, Erudyrhizogburn japonicum. phenotypes

27 3

INDEX dissimilatory nitrate reduction, 83 - 84 fatty acids, 93 hemoproteins, 92-93 hydrogenase system, 78 intrinsic antibiotic resistance, 74-75 nitrogenase activity ex plunfu,92 plant growth-regulating substances, 9 1 - 92 protein profiles, 88 rhizobiophages, 89-90 serogroups, 72 surface polysaccharides. 87 DNA markers, in oilseed brassica breeding random amplified polymorphic, 48 restriction fragment length polymorphisms, 48 Doubled-haploid technique, for oilseed brassicas, 4 I - 42

E Electrons activity in soils, thermodynamic relationships, 155-158 characteristics, I53 - I55 Equilibrium constant, for redox equilibria, I59 Erucic acid, in extracted seed oils, 7- 10

F Fatty acids Brudyrhizogbum japonicum, 93 oilseed brassicas, 8 -9 Fertilizers chloride-containing, 134- I35 sulfur-containing, 252- 256 Foliar diseases, chloride effects, I 19- I22 Free radicals behavior, I77 - I78 formation in soils, 176- I77 oxidizing, field tests for. 20 I - 205

G Gametocides, for Erussiccl spp., I5 - I6 Gel electrophoresis. SDS-polyacrylamide. soybean bradyrhizobia phenotypes, 87 88 Genomic relationships, among oilseed brassicas. 5

Ghcosinolate, sulfur fertilization effects, 254 Glyphosate tolerance, oilseed brassicas, 38 Groundwater, sulfur in, 227 Gum guaiac, tests for Mn and Fe oxides and oxidizing free radicals, 202

H Heavy metals, sequestration by Brussica SPP., 47 Hemoproteins, Bradyrhizogbum juponicum, 92-93 Herbicide resistance, in oilseed brassicas, 37 - 39 Heterosis, Brussicu F, hydrids, I3 - I5 Horizons A. see A horizons E, development, 191 Host tolerance, enhancement by chloride, 112-114 Humic substances A horizon development, 19 I - 195 E horizon development, I9 I manganese role, 190- I9 I Hybridization in identification of genetic groupings of Bradyrhizogbum juponicum, 68 interspecific, oilseed brassicas, 5 , 40-4 I Hydrogenase system, Bradyrhizogbum j u p onicirm and DNA homology groupings, 78 phenotype diversity, 75-77 and symbiotic performance, 77 - 78

I lmidazolinone tolerance, oilseed brassicas, 38 Insect control, gene-based, in Brussica spp., 46 Iron catalytic oxidation of organics, 180- I8 I disproportionation, I8 I - I82 oxides, field tests for, 20 I -205 pe-pH diagrams, 170 proportionation, I8 1 - I82 redox system, I79 reduction half-reactions, 162- 163 Irrigation water, sulfur contents, 226-227

2 74

INDEX L

Leaf diseases, chloride effects, I I9 - 122 Leaf movement, chloride role, I 13 Leaf rust, chloride effects, I2 1- I22 Lepfosphaeria maculans blackleg, resistance in oilseed brassicas, 24 - 28 Lettuce, chloride deficiency, 109 Light leaf spot, resistance in oilseed brassicas, 36 Linkage mapping, oilseed brassicas, 48-49

M Manganese chloride interactions in plants, I 16- 1 17 as electron acceptor, I8 I field tests, 20 I - 205 humus formation, 190- 191 Mn-nitrogen transformations, 186- I87 Mn(II1)-organic acid reductants, 182184 oxidation in soils mechanism, 185- 186 oxygen restriction effects, 184- I85 oxides dismutation of H,O,, 204 interferences, 205 pe- pH diagrams, I67 - I69 redox system, I79 reduction half-reactions, 162- 163 synthetic amorphous Mn(1V) preparation, 202 Manganese electron demand (MED) determination, 204 Metals, heavy, see Heavy metals Methionine, levels in oilseed brassicas, 46 Microspore culture, oilseed brassicas, 41 -42 Molybdenum, sulfur fertilization effects, 256 Mutagenesis,with haploid Brassica spp., 42

N Nitrate reduction, dissimilatory, see Dissimilatory nitrate reduction Nitrification, inhibition by chloride, 114 Nitrogen -chloride interactions in plants, 114- I15 -manganese transformations, 186- I87 pe-pH diagrams, 166- 167

reduction half-reactions, 162 sulfur fertilization effects on plant nitrate content, 255 - 256 Nitrogenase, activity in Bradyrhizogbumjaponicum, ex planta, 92

0 Oats, chloride effects, I29 Oil quality Brassica spp., Agrobacierium-mediated transformation techniques, 44 - 45 sulfur fertilization effects, 254 Oil quantity Brassica spp., Agrobacleriurn-mediated transformation techniques, 45 -46 oilseed brassicas, 22 - 23 sulfur fertilization effects, 254 Oilseed brassicas disease-resistant cultivars, 24- 37 DNA markers, 48 -49 fatty acid compositions, 8-9 genomic relationships among species, 5 haploid production in viiro, 4 I -42 herbicide-resistant cultivars, 37 - 39 heterosis and F, hybrids, 13- I5 interspecific hybridizations, 40-41 oil yield, 22-23 plant descriptions, 6-7 pollination control systems, I5 - 22 modes, 7 production, 2-4 protein yield, 22 - 23 protoplast fusions, 43 -44 quality, improvements in, 7- 10 seeds, descriptions, 6- 7 seed yield breeding methods, 10- 13 components, 10- I3 somaclonal variations, 42-43 transformation, 44-48 world socioeconomic importance, 2- 3 Organic compounds, catalytic oxidation by iron, 180- 181 Osmoregulation, chloride role, I 12 Osmosis, adjustments, chloride role, I I2 Oxidation - reduction reactions, see Redox reactions

INDEX Oxygen pe-pH diagrams, I65 - I66 reduction half-reactions, 162

P PP

definition, I56 empirical, determination, 200 PP-PH diagrams carbon species, 170- I72 iron species, 170 manganese oxide species, 167- I69 nitrogen species, I66 - I67 oxygen species, I65 - I66 sulfur species, 170- I72 thermodynamic information from, 160165 PH definition, 156 pe-pH thermodynamic data, 160- 165 Phage typing, Bradyrhizogbum japonicum and DNA homology groupings, 89-90 phenotype diversity, 89 and symbiotic performance, 89 Phenotype, oilseed brassicas dissimilatory nitrate reduction, 78-80 fatty acids, 93 hemoproteins, 92-93 intrinsic antibiotic resistance, 72-75 nitrogenase activity ex planta, 92 plant growth-regulating substances, 90-92 protein profiles, 87-88 rhizobiophage typing, 88 -90 rhizobitoxine, 80- 84 serology, 69 - 72 summary of relationships, 94 surface polysaccharides, 84-87 uptake hydrogenase, 75-78 Phosphinotricin tolerance, oilseed brassicas, 39 Phosphorus, interactions with chloride in plants, I I5 - I I6 sulfur in plants, 256 Photosynthesis, chloride role, 110- I I I Plant analyses crop response to chloride, 135- 138 sulfur deficiency, 239- 24 I

275

Plant growth-regulating substances, Bradyrhizogbumjaponicum and DNA homology groupings, 9 I - 92 phenotype diversity, 90-91 and symbiotic performance, 9 1 Pollen control, in Brnssica spp. cytoplasmic male sterile- nuclear restorer system, I7 - 20 gametocides, I 5 - I6 genic male sterility, 20-21 self-incompatibilitysystems, I6 - I7 transgenics, 2 1 -22 Pollination, oilseed brassicas control systems, 15 -22 modes, 7 Pollutants, reduction half-reactions, 163 Polysaccharides, surface, Bradyrhizogbum japonicum and DNA homology groupings, 87 methods, 84-85 phenotype diversity, 8 I - 85 and symbiotic performance, 85-86 Population diversity groupings, Bradyrhizogbum japonicum dissimilatory nitrate reduction, 78-80 fatty acids, 93 hemoproteins, 92-93 hydrogenase system, 75-78 intrinsic antibiotic resistance, 72- 75 nitrogenase activity explanta, 92 plant growth-regulatingsubstances, 90-92 protein profiles, 87-88 rhizobiophage typing, 88-90 rhizobitoxine, 80-84 serology, 69 - 72 summary of genotype/phenotype relationships, 94 surface polysaccharides, 81 -85 Potatoes chloride deficiency, I I0 chloride effects, I3 1 - 132 Precipitation, and soil sulfate, 22 I -223 Protein profiles, Bradyrhizogbumjaponicum and DNA homology groupings, 88 methods, 87 SDS-PAGE phenotype diversity, 87-88 and symbiotic performance, 88 Protein quality crop responses to sulfur, 253-254 oilseed brassicas, 46

INDEX

276

Protein yield, oilseed brassicas, 22-23 Protons, characteristics, 153- 155 Protoplast fusion, in oilseed brassicas, 43 -44

Q Quality response to chloride corn, 129-131 oat, 129 soybeans, 131 - I32 to sulfur, 252 - 256

R Redox reactions characterization, empirical methods empirical pe determination, 200 lab incubations, 199-200 soil handling, 198- 199 free radicals behavior, 177- 178 formation, 176- 177 iron, 178- I79 log K determination, 157 manganese, 178- 179 measurement in soils electrochemical relations in reverse, 174-175 empirical pe values, 175- I76 platinum electrodes, I72 - I74 pe, definition, 156 pH, definition, I56 photochemical transformations in soil and water, I88 - I90 reduction half-reactions, 155- 156 thermodynamic parameters for electron activity, I55 - I58 kinetic derivation, 158- 160 pe-pH information, 160- 165 wetlands interfaces, 197- 198 preservation, I96 Reducing capacity, soil, 203 - 204 Reducing intensity, soil, 203 Reduction half-reactions characterization, I55 - 156 for N, 0, Mn, Fe, S, C and pollutants, 162- I63

nitrate, dissimilatory, see Dissimilatory nitrate reduction -oxidation reactions, see Redox reactions Rhizobitoxine, Bradyrhizugbum japonicum - related and DNA homology groupings, 83 - 84 phenotype diversity, 80-82 and symbiotic performance, 82-83 Root rot common, chloride effects, 119 take-all, chloride effects, I I 8 - I 19 Rot, see Root rot; Stem rot Rusts, see specific types ofrust

S Sclerotinia sclerotiurum, stem rot, resistance in oilseed brassicas, 32 - 34 Seeds, oilseed brassicas, 6 - 7, 10- I 3 Selenium, sulfur fertilization effects, 256 Self-incompatibility systems, for Brassica spp. pollination control, 16- 17 Serology, Bradyrhizugbum japonicum and DNA homology groupings, 72 methods, 69 phenotype diversity, 69 - 70 and symbiotic performance, 70-72 Somaclonal variations, in oilseed brassicas, 42-43 Soybean brad yrhizobia, see Bradyrhizugbum japonicum Soybeans, chloride effects, I3 I - I32 Spinach, chloride deficiency, 109 Stem canker, in oilseed brassicas, 24-28 Stem rot, Sclerutinia sclerutiorum, resistance in oilseed brassicas, 32- 34 Sterility, genic male, in Brassica spp., 2021 Stomata operation, chloride role, 1 I2 - 1 13 Streams, sulfur in, 226-227 Stripe rust, chloride effects, 120- 12 I Subtropics, sulfur deficiency, 2 1 I -2 15, 257-258 Sugar beets, chloride deficiency, 109 Sulfur critical soil solution concentration, 24 1 246 crop responses, 246-252 fertilization effects, 252-256

277

INDEX interactions with phosphorus, 256 with various elements, 256-257 in irrigation water, 226- 227 pe-pH diagrams, 170- I72 reduction half-reactions, 162 requirements of plants, 241 -246 SO, adsorption mechanisms, 234-236 sulfate form accession through precipitation, 22 I 223 adsorption by soils, 227-232 adsorption curves, 232-234 characterization, 2 16- 2 I7 desorption by soils, 227-232 transformation products, 2 15 -2 16 Sulfur cycling accession through precipitation, 22 I -223 supplies of atmospheric origin, 219-22 I in tropics, 2 I7 - 2 I9 Sulfur deficiency plant analyses for, 239- 24 I soil tests for, 237-239 tropical/subtropical, 21 1-215, 257-258 Sulfur tetroxide, adsorption mechanism, 234-236

T Tanspot, chloride effects, I22 Tests, soil crop response to chloride, I38 - 140 sulfur deficiency, 239-24 I Tetramethylbenzidine, tests for Mn, Fe oxides, and oxidizing free radicals, 20 1 202 Thermodynamics, redox reactions electron activity, I55 - I58 kinetic derivation, I58 - I60 pe-pH information, 160- 165 Tomato, chloride deficiency, I09 Transformation ARrobactprium-mediated, in Brassicu spp., 44-48 disease resistance, 47 heavy metal sequestration, 47 insect control, 46-47 molecular farming, 47-48 oil quality modifications, 44-45

oil quantity modifications, 45 -46 protein quality, 46 redox, photochemical, 188- 190 Triazine tolerance, oilseed brassicas, 37- 38 Tropics sulfur cycling, 2 17- 2 I9 sulfur deficiency, 21 1-215,257-258 Turnip mosaic virus, resistance in oilseed brassicas, 36

V Vegetation, forest, acid rain effects, 225 Verticillium dahliae wilt, in Brassica spp., 36-37

W Water irrigation, sulfur contents, 226 -227 ground-, sulfur in, 227 Wetlands characterization, 195- 196 preservation, redox-related reasons for, I96 redox interfaces, 19?- 198 White rust, resistance in oilseed brassicas in Brassica napus, 3 1 - 32 race 2 resistance, 29 - 3 I race 7 resistance, 3 1 selection for, 32 Wilt, see Verticillium dahliae wilt

Y Yellow rust, chloride effects, I 19- I20 Yield oilseed brassicas oil, 22-23 protein, 22-23 seeds, 10- 13 wheat and barley responses to chloride, 126- 128

Z Zinc, sulfur fertilization effects. 256