Advances in Agronomy, Volume 83

VOLUME 83 Advisory Board John S. Boyer University of Delaware Paul M. Bertsch University of Georgia Ronald L. Phil...

Author: Donald L. Sparks

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VOLUME

83

Advisory Board John S. Boyer University of Delaware

Paul M. Bertsch University of Georgia

Ronald L. Phillips University of Minnesota

Kate M. Scow University of California, Davis

Larry P. Wilding Texas A&M University

Emeritus Advisory Board Members Kenneth J. Frey Iowa State University

Eugene J. Kamprath North Carolina State University

Martin Alexander Cornell University

Prepared in cooperation with the American Society of Agronomy Monographs Committee Diane E. Stott, Chair Lisa K. Al-Almoodi David D. Baltensperger Warren A. Dick Jerry L. Hatﬁeld John L. Kovar

David M. Kral Jennifer W. MacAdam Matthew J. Morra Gary A. Pederson John E. Rechcigl

Diane H. Rickerl Wayne F. Robarge Richard Shibles Jeffrey Volenec Richard E. Zartman

Edited by

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

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ISBN: 0-12-000781-9 ISSN: 0065-2113 (Series) 1 The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). W Printed in Great Britain.

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

ix xi

EFFECTS OF FUMIGANTS ON NON -TARGET ORGANISMS IN SOILS A. Mark Ibekwe I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Effects on Biological Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Enzyme Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Substrate-Induced Respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nitrogen Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Effect on Microbial Activities and Composition . . . . . . . . . . . . . . . . . . . . A. Changes in Community-Level Carbon Source Utilization by Biolog . . B. Changes in Microbial Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Changes in Soil Microbial Community Structure and Composition . . . D. Analysis of Soil Microbial Community Structure by Molecular Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Impact of Recommended Fumigants on Soil Microbial Communities and Agricultural Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Methyl Bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Methyl Isothiocyanate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. 1,3-Dichloropropene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Chloropicrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Effect on Speciﬁc Microbial Populations . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 5 5 6 7 8 8 10 12 15 20 22 25 26 27 28 29 29 29

SORGHUM IMPROVEMENT —INTEGRATING TRADITIONAL AND NEW TECHNOLOGY TO PRODUCE IMPROVED GENOTYPES W. L. Rooney I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Variation in Sorghum ssp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Sorghum Improvement—from Landraces to Cultivars . . . . . . . . . . . . . . . v

38 39 40

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IV. Mechanisms of Controlled Pollination . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hand Emasculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetic Male Sterility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Hot-Water Emasculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Control of Anther Dehiscence Control . . . . . . . . . . . . . . . . . . . . . . . . E. Cytoplasmic –Genetic Male Sterility . . . . . . . . . . . . . . . . . . . . . . . . . . V. Improvement Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Population Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cultivar and Inbred Line Development . . . . . . . . . . . . . . . . . . . . . . . C. Hybrid Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Use of Exotic Germplasm—Sorghum Conversion . . . . . . . . . . . . . . . VI. Trait-Based Breeding Efforts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Yield and Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Biotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Abiotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Grain Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Forage Sorghum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Sweet Sorghum for Syrup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Broomcorn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Biotechnology in Sorghum Improvement . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 43 44 44 45 46 49 51 52 52 56 59 60 63 75 86 89 92 93 93 95 96

CRITICAL REVIEW OF THE SCIENCE AND OPTIONS FOR REDUCING CADMIUM IN TOBACCO (NICOTIANA TABACUM L.) AND OTHER PLANTS N. Lugon-Moulin, M. Zhang, F. Gadani, L. Rossi, D. Koller, M. Krauss and G. J. Wagner I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Cadmium in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Cadmium in the Tobacco Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cadmium Tolerance in Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Root-to-Shoot Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Root and Shoot Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cadmium in Field-Grown Tobacco Leaves . . . . . . . . . . . . . . . . . . . . E. Stalk Position Versus Cadmium Accumulation . . . . . . . . . . . . . . . . . . F. Developmental Stage Versus Cadmium Accumulation . . . . . . . . . . . . G. Variation Within the Leaf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Sub-cellular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Differences Between Varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. External Factors Affecting Cadmium Concentration in Tobacco Leaves . . A. Soil Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 113 114 115 117 119 120 121 123 123 124 124 126 127 127

CONTENTS B. Agronomic Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Additional Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Options to Reduce the Cadmium Content in Tobacco Leaves . . . . . . . . . . A. Molecular and Biochemical Approaches . . . . . . . . . . . . . . . . . . . . . . B. Breeding Strategies to Reduce Cadmium . . . . . . . . . . . . . . . . . . . . . . C. Soil Cadmium Remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 130 135 137 137 137 153 154 161 162 162

THE IMPACT OF GRAZING ANIMALS ON N2 FIXATION IN LEGUME -BASED PASTURES AND MANAGEMENT OPTIONS FOR IMPROVEMENT John C. Menneer, Stewart Ledgard, Chris McLay and Warwick Silvester I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Animal Treading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Damage and Burial by Hoof Action . . . . . . . . . . . . . . . . . . . . . B. Soil Compaction: Mechanical Impedance Effects on Legumes . . . . . . C. Soil Compaction: Aeration and/or Waterlogging Effects on Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Signiﬁcance of Plant and Soil Factors, and Limits of Pasture Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Animal Grazing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Diet Selection and Defoliation Effects . . . . . . . . . . . . . . . . . . . . . . . . B. Direct Effects of Defoliation on N2 Fixation . . . . . . . . . . . . . . . . . . . IV. Animal Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Increased Soil N and Grazing Avoidance of Excreta-Affected Areas . . B. Direct Effects of Excreta N on N2 Fixation . . . . . . . . . . . . . . . . . . . . V. Strategies to Minimise the Impacts of Grazing Animals . . . . . . . . . . . . . . A. Pasture Management to Aid Legume Production . . . . . . . . . . . . . . . . B. Choice of White Clover Cultivar and Companion Grasses . . . . . . . . . C. Tactical Use of N Fertiliser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Farm-Scale Management Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Soil Management: Preventing Treading and Compaction . . . . . . . . . . B. Restricted Grazing and Supplementary Feeding in Winter/spring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Technical Based Decision Making for Improved Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

182 184 186 188 192 198 200 201 204 205 207 209 211 211 216 219 221 222 222 224 227 228 228

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SEED -FILL DURATION AND YIELD OF GRAIN CROPS Dennis B. Egli I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Seed Filling: Deﬁnition and Measurement . . . . . . . . . . . . . . . . . . . . . . . . III. Variation in Seed-Fill Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Water Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Assimilate and Nutrient Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Flower and Fruit Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Photoperiod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Plant Growth Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Genetic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Regulation of Seed-Fill Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Regulation by the Seed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Regulation by the Plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Seed-Fill Duration and Crop Productivity . . . . . . . . . . . . . . . . . . . . . . . . . A. Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Future Yield Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

244 245 248 248 249 250 251 252 252 253 254 255 256 257 262 262 265 267 268

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

D. EGLI (243), Department of Agronomy, University of Kentucky, Lexington, KY 40546-0312 F. GADANI (111), Philip Morris USA RD&E Department, PO Box 26583, Richmond, VA 23261 A. M. IBEKWE (1), USDA-ARS-George E. Brown, Jr. Salinity Lab, 450 W. Big Springs Road, Riverside, CA 92507 D. KOLLER (111), Philip Morris USA RD&E Department, PO Box 26583, Richmond, VA 23261 M. KRAUSS (111), Philip Morris USA RD&E Department, PO Box 26583, Richmond, VA 23261 S. LEDGARD (181), AgResearch Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand N. LUGON-MOULIN (111), Philip Morris International R&D, c/o Philip Morris Products SA, 200 Neuchaˆtel, Switzerland C. MC LAY (181), Environment Waikato, PO Box 4010, Hamilton, New Zealand J. C. MENNEER (181), University of Waikato, Private Bag, Hamilton, New Zealand W. L. ROONEY (37), Department of Soil and Crop Science, Texas A&M University, College Station, TX 77843-2474 L. ROSSI (111), Philip Morris International R&D, c/o Philip Morris Products SA, 200 Neuchaˆtel, Switzerland W. SILVESTER (181), University of Waikato, Private Bag, Hamilton, New Zealand G. J. WAGNER (111), University of Kentucky, Agronomy Department N212 ASCN, Lexington, KY 40546-0091 M. ZHANG (111), Philip Morris USA RD&E Department, PO Box 26583, Richmond, VA 23261

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Preface Volume 83 contains ﬁve cutting-edge crop and soil science reviews. The ﬁrst review covers aspects of fumigant effects on non-target organisms in soils including mode of action, biological processes, microbial activities and composition, recommended fumigants on soil microbial communities and agricultural practices and speciﬁc microbial populations. The second chapter is a comprehensive review on sorghum improvement using both traditional plant breeding and contemporary biotechnology. Chapter 3 is a critical review on the science and options for reducing cadmium (Cd) in tobacco (Nicotiana tabacum L.) and other plants. Discussions on Cd in the environment and in the tobacco plant, external factors affecting Cd concentration in tobacco leaves, options for reducing Cd content in tobacco leaves and soil Cd remediation are included. Chapter 4 deals with the impact of grazing animals on N2 ﬁxation in legume-based pastures and management options for improvement. Chapter 5 is a comprehensive review of seed-ﬁll duration and yield of grain crops. I am grateful to the authors for their very ﬁne reviews. DONALD L. SPARKS University of Delaware

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EFFECTS OF FUMIGANTS ON NON- T ARGET ORGANISMS IN SOILS A. Mark Ibekwe USDA-ARS George E. Brown, Jr. Salinity Laboratory, 450 W. Big Springs Road, Riverside, California 92507, USA

I. Introduction II. Mode of Action III. Effects on Biological Processes A. Enzyme Activities B. Substrate-Induced Respiration C. Nitrogen Transformation IV. Effect on Microbial Activities and Composition A. Changes in Community-Level Carbon Source Utilization by Biolog B. Changes in Microbial Biomass C. Changes in Soil Microbial Community Structure and Composition D. Analysis of Soil Microbial Community Structure by Molecular Techniques V. Impact of Recommended Fumigants on Soil Microbial Communities and Agricultural Practices A. Methyl Bromide B. Methyl Isothiocyanate C. 1,3-Dichloropropene D. Chloropicrin VI. Effect on Speciﬁc Microbial Populations VII. Summary and Conclusions Acknowledgments References

Soil fumigants are extensively used to control plant-parasitic nematodes, weeds, fungi, and insects for planting of high value cash crops. The ideal pesticide should be toxic only to the target organisms; however, fumigants are a class of pesticide with broad biocidal activity and affect many nontarget soil organisms. Soil microorganisms play one of the most critical roles in sustaining the health of natural and agricultural soil systems. The ability of soil microorganisms to recover after treatment with pesticide is critical for 1 Advances in Agronomy, Volume 83 Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved. DOI 10.1016/S0065-2113(04)83001-3

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A. M. IBEKWE the development of healthy soils. In the southwestern United States, fumigation is used to control pathogens such as Verticillium dahliae, Pythium, Rhizoctonia, or Cylindrocarpon spp. In addition to pathogen control, fumigation can also result in enhanced growth response of the plant by reducing weed pressure. The continued use of fumigants in agriculture will require more investigations of the different types of fumigants, soils, environmental conditions, and biological/microbial communities to establish both the effectiveness on target organisms and safety to the general public. q 2004 Elsevier Inc.

I. INTRODUCTION Agricultural soils are typically treated with pesticides to provide effective control of nematodes, soil-borne pathogens, and weeds in preparation for planting high value cash crops. Fumigants are a class of pesticide with broad biocidal activity and affect many non-target soil organisms (Parr, 1974; Domsch et al., 1983; Anderson, 1993). Currently, only four registered fumigants are available in the Unites States: 1,3-dichloropropene (1,3-D), methyl isothiocyanate (MITC), chloropicrin (CP), and methyl bromide (MeBr). Methyl iodide (MeI, iodomethane) is another fumigant yet to be registered that is considered being a promising alternative to MeBr for soilborne pest control in high value cash crops. While most fumigants are known to have broad biocidal activity, their effects on non-target soil microbial communities are largely unknown, due to the lack of appropriate methods to describe microbial soil community composition. Soil microorganisms play one of the most critical roles in sustaining the health of natural and agricultural soil systems. They are a signiﬁcant component of nutrient cycling, especially of C and N, which are essential for proper plant nutrition and agricultural productivity. Changes in the microbial community composition as a result of fumigant applications may lead to changes in the functional diversity of that community and ultimately, the overall soil quality. Because of the strong relationships between microbial diversity and ecosystem function, soil microorganisms are recognized as sensitive indicators of soil health. MeBr has the ability to destroy stratospheric ozone (Yung et al., 1980; Prather et al., 1984) and a ban on its production and importation is to be completed by 2005 in the United States (USEPA, 1995). 1,3-D, MITC, and CP have been proposed as the most likely chemical alternatives to MeBr. Since little ecotoxicological information exists with respect to these (and many other

FUMIGANTS ON NON-TARGET ORGANISMS IN SOILS

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fumigants), it is essential that fumigant effects on non-target microorganisms be examined.

II. MODE OF ACTION Fumigants are extensively used to grow strawberries, tomatoes, and other high valued cash crops in California and Florida. Many studies have documented the chemistry and air pollution potential of these fumigants in the environment (Baker et al., 1996; Gan et al., 1998a,b). There are only four registered chemical fumigants available in the United States: The ﬁrst is 1,3-D, which is marketed under the trade name Telone and contains an equal ratio of cis-1,3-D and trans-1,3-D. The second, MITC, is a primary product of metam sodium (sodium methyldithiocarbamate) metabolism. CP, a third fumigant also known as trichloronitromethane, is often formulated with Telone and metam sodium. MeBr is the last fumigant available in the United States. 1,3-D, MITC, and CP have been proposed as the most likely chemical alternatives to MeBr. Figure 1 shows the structural formula of metam sodium, MITC, cis- and trans-1,3-D, CP, and MeBr. Compared to other pesticides, fumigants were found to have little or no detectable effects on soil microorganisms at ﬁeld application rates (Hicks et al., 1990; Anderson, 1993). Their effects on non-target soil microorganisms at the ﬁeld application rate are largely unknown until recently, mainly due to lack of appropriate methodology to describe the non-target population (Elliot et al., 1996; Macalady et al., 1998; Ibekwe et al., 2001a; Dungan et al., 2003a). Fumigants should only be toxic to the target organisms, be biodegradable, and should not leach into the groundwater, though this is not always the case. The widespread use of fumigants in the warm climate regions of the United States is of increasing concern. The mode of action of different classes of pesticides differs. Some pesticides are designed to affect speciﬁc or general processes in the target organisms and are more suitable for a speciﬁc target population. Fumigants are generally designed to provide effective control of nematodes and soil-borne pathogens, such as fungi and weeds. Metam sodium and 1,3-D may be very effective as fungicides, and thus may be expected to affect non-targeting soil ﬂora. The most used fungicide in Denmark is fenpropimorph, which speciﬁcally inhibits two enzymes involved in ergosterol biosynthesis (Johnsen et al., 2001). This fungicide was designed to target leaf-associated fungi and subsequently may have some effects on the soil fungi. Degradation of fenpropimorph also produces an intermediate metabolite, fenpropimorphic acid, and it has been shown that saprotrophic fungi were substantially affected by this intermediate

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Figure 1 The chemical structure of cis- and trans-1,3-dichloropropene (1,3-D), metam sodium, methyl isothiocyanate (MITC), chloropicrin (CP), and methyl bromide (MeBr).

compound, indicating that the biological activity of the fungicide may be attributed to both mother compound and the more mobile metabolites (Bjørnlund et al., 2000). Changes in the microbial composition as a result of fumigant application may lead to changes that interfere with the functional diversity and overall soil quality. The strong relationships between microbial diversity, ecosystem sustainability, and function are being increasingly recognized as sensitive indicators of soil health (Turco et al., 1994). Ultimately, the linking of information between microbial community structure/diversity and crop production will be an important step in being able to predict soil fertility. There is a signiﬁcant gap in information on the effects of fumigants on soil bacterial and their impact on major soil processes, such as organic matter transformation and pollutant degradation. This review will elaborate on the most up-to-date data available in the literature on this topic and describe some of the techniques in microbial ecology that may be helpful in understanding different processes in soil that are signiﬁcantly affected by fumigants.

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III. EFFECTS ON BIOLOGICAL PROCESSES A. ENZYME ACTIVITIES The effect of fumigants on enzyme activities was previously reported by Ladd et al. (1976) and has been shown to have an inhibitory effect on dehydrogenase activities (DHAs) (Anderson, 1978; Smith and Pugh, 1979; Tu, 1992). Fumigation of a ﬁeld soil with MeBr and CP, either individually or in combination, decreased soil enzyme activities (Ladd et al., 1976). DHA is an indicator for potential non-speciﬁc intracellular enzyme activity of the total microbial biomass. Fumigants appear to disturb the membrane-bound processes of active cells. As noted by Ladd (1978), DHA measurements may be inﬂuenced not only by enzyme concentrations, but also by the nature and concentration of added C substrates, and of alternative electron acceptors, such as NO2 3 . Zelles et al. (1997) conducted an extensive study with seven enzymes to determine the effects of chloroform fumigation on their activities and found that enzymes bound to the active microorganisms were nearly inhibited completely (dehydrogenase) or strongly reduced (arginine deaminase). These authors also reported that fumigation of soil did not change the activities of xylase, b-glucosidase and saccharase and only induced a low reduction in phosphatase activity. Recently, Dungan et al. (2003a) showed that the incorporation of compost manure to agricultural soils signiﬁcantly increased the DHA of the soil over a 12-week incubation period when compared to unamended soils. When these soils were treated with two fumigants, propargyl bromide (PBr) and 1,3-D, they observed a higher rate of fumigant degradation in the amended soils, corresponding well with the increased enzyme activity in the amended soil treatments. When treated with PBr at 10 mg kg21 or 1,3-D at 10 and 100 mg kg21, the DHA in unamended and amended soils was not signiﬁcantly reduced. At 100 mg kg21, PBr was more toxic than 1,3-D, as indicated by the reduced DHA at this concentration. At 500 mg kg21 of PBr and 1,3-D, DHA was signiﬁcantly repressed, but by week 8 in the amended soil treatments, DHA had recovered to levels similar to that of the control. DHA in unamended soils spiked with 500 mg kg21 of 1,3-D or PBr did not demonstrate signiﬁcant recovery after 12 weeks. This conﬁrmed the inhibitory effects of fumigants on DHA as previously reported (Anderson, 1978; Smith and Pugh, 1979; Tu, 1992). Decreases in DHA, in both unamended and amended soils, were probably a direct result of the adverse effects of PBr and 1,3-D on soil microbial populations. Fumigants such as metam sodium may interfere with respiratory enzymes such as pyruvate dehydrogenase due to their chelating effects on metal cations such as Cu (Corbett et al., 1984) or to toxic degradation products such as MITC (Staub et al., 1995). These authors showed that MITC is metabolized to S-methyl metam, probably by formation of S-(N-methylthiocarbamoyl)cysteine, by cysteine

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conjugated b-lyase. The alternative proposal was that metam sodium might be very sensitive to oxidation, forming reactive sulfenic and sulﬁnic acids, which might contribute to its toxic action. Assessing the toxicity of metam, its oxidative and methylated metabolites, and their contribution to the toxicity of MITC on non-target soil bacteria are complicated by their interconnected detoxiﬁcation and bioactivation pathways.

B. SUBSTRATE-INDUCED RESPIRATION Substrate-induced respiration (SIR) is mainly used to characterize microbial activity and has been used to estimate the size of the microbial biomass (Anderson and Domsch, 1978). The wide use of SIR to assess the impact of pesticides on microbial activity and biomass has been reported in the literature (Wardle and Parkinson, 1990; Harden et al., 1993; Hart and Brookes, 1996; Lin and Brookes, 1999; Smith et al., 2000; Chen et al., 2001). Results on the effects of two fumigants from a 24-h SIR experiment, evaluated as CO2 evolved (mg g21 dry soil 24 h21), were recently reported (Dungan et al., 2003a). SIR was markedly inhibited by incremental additions of PBr or 1,3-D to either unamended or manure-amended soil. Other studies show both enhancement and reduction of CO2 evolution following pesticide application, while others exhibit no effect on soil respiratory activity (Simon-Sylvestre and Fournier, 1979). In unamended soil, at the highest concentration of PBr and 1,3-D, SIR was reduced to 61 and 22% of the control, respectively. In amended soil, SIR was reduced to 50 and 25% of the control, respectively; however, SIR was 1.4 (PBr) and 2.2 (1,3-D) times higher, on average, than in fumigated unamended soil (Dungan et al., 2003a). This study demonstrated a signiﬁcant reduction of the impact of fumigant on non-target soil bacteria with soil amendments. A study by Chen et al. (2001) has shown that SIR was unaffected over a 56-day experimental period when treated with benomyl (a fungicide) at a rate of 125 mg kg21. This showed that soil microbial activity was stimulated by the addition of amendments, even when treated with PBr (at 10 and 100 mg kg21) or 1,3-D (at 10 –500 mg kg21). The effect of metam sodium has also been shown to strongly affect SIR (Macalady et al., 1998). Soil treated with 1.6 g l21 of metam sodium was reduced to 17 – 29% of the controls with no recovery after 28 days. Also, metam sodium applied at 16 g l21 eliminated respiration of added glucose for all sampling dates. The authors concluded that metam sodium had inhibitory effects on soil parameters measured even after 18 weeks. Sensitivity of soil respiration after repeated exposure to MITC (Taylor et al., 1996) suggested that fumigation with metam sodium resulted in long-term changes in the composition and activity of soil microorganisms.

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C. NITROGEN TRANSFORMATION The work of Rovira (1976) with CP and MeBr showed an increase in concentration of NHþ 4 within 28 days after fumigation, followed by a decrease after 46 days due to increase in activities of nitrifying bacteria. NO3 concentrations in fumigated soil were lower than the untreated soils after 28 days because of a lack of nitriﬁcation following fumigation. Malkomes (1995) also reported that nitriﬁcation was inhibited by fumigation for long periods of time. Metam sodium produced some inhibitory effects on nitrogen transformation as a result of depression of ammonia-oxidizing activities (Macalady et al., 1998). These authors showed a decrease in ammonia oxidation potential with increasing metam sodium dose. In a recent study, the effects of steam sterilization (SS) on soil microbial properties, including metabolic diversity of the microbial communities, were examined in a greenhouse study compared to MeBr (Tanaka et al., 2003). The numbers of nitriﬁers, both ammonium-oxidizing bacteria and nitrite-oxidizing bacteria, were severely affected by the SS and CP treatments, resulting in their virtual disappearance. The decrease in the levels of microbial biomass C and N after the treatments suggested that the SS and CP treatments eradicated the microorganisms more effectively than the MeBr treatment, and that the inﬂuence of the former persisted until the end of the experiment, 4 months after the treatments. Accumulation of NH4-N was observed after the SS and CP treatments mainly due to the partial decomposition of the dead microorganisms and the marked decrease in the number of ammonium-oxidizing bacteria. The numbers of ammonia-oxidizing bacteria were also reduced by more than four orders of magnitude in soils fumigated with metal sodium and did not show recovery 105 days later (Toyota et al., 1999). Fumigation decreased the numbers of nitrite oxidizers by three orders of magnitude, with a slight recovery after 105 days (still signiﬁcantly lower than the control). These studies have shown that several fumigants adversely affect soil N balances through their temporary inhibition of nitriﬁcation. The process of nitriﬁcation involves the conversion of ammonium to nitrite and to nitrate. The inhibition of this process through the adverse effects of fumigants on ammonia-oxidizing bacteria may result in some adverse effects on the overall soil quality. It should also be noted that once the fumigants are completely dissipated in soil, there is always a regrowth of the bacteria. It has been shown previously that autotrophic nitrifying bacterium Nitrosomonas europaea is capable of co-oxidizing numerous halogenated hydrocarbons in the presence of NHþ 4 through the action of the ammonia-oxidizing enzyme ammonia monooxygenase (AMO) (Rasche et al., 1990). As alternative substrates for this enzyme, these compounds inhibit ammonia oxidation through competitive effects. At lower concentrations these compounds may serve as an alternative substrate for AMO, or with higher concentrations bacterial population may decrease, resulting in less nitriﬁcation.

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IV.

EFFECT ON MICROBIAL ACTIVITIES AND COMPOSITION

A. CHANGES IN COMMUNITY-LEVEL CARBON SOURCE UTILIZATION BY BIOLOG The Biolog Gram-negative (GN) microtiter plate assay is often used to analyze the functional diversity through substrate utilization patterns of soil microorganisms. When the functional abilities of the heterotrophic soil microbial communities were observed over a 12-week period of time following the application of fumigants, severe alterations were seen during the ﬁrst week, especially with MeBr (Ibekwe et al., 2001a). The PCA plot (Fig. 2) from MeBr, MITC, 1,3-D, and CP-treated soil microbial communities and the control accounted for 28% of the variance on the ﬁrst component, with six PCs accounting for over 80% of the variation. The control and the 1,3-D-treated soils separated along PC1 with their coefﬁcients positively correlating to the right of PC1. Analysis of MeBr communities did not show any pattern of groupings except that communities from the ﬁrst week of treatments were positively correlated along PC2 and grouped with MITC after weeks 8 and 12. Pairwise comparison showed that the MeBr communities differed signiﬁcantly ðp , 0:05Þ from the control and 1,3-D communities. The control and 1,3-D treatments were

Figure 2 Principal component analysis performed on Biolog GN ﬁngerprints of soil extracts treated with methyl bromide (MeBr), methyl isothiocyanate (MITC), 1,3-dichloropropene (1,3-D), chloropicrin (CP), and non-fumigated soil (C). 1, 8, and 12 after the symbols indicate weeks 1, 8, and 12 and 1, 2, and 3 after the “-” sign indicates 50% below ﬁeld application rate, ﬁeld application, and 1000% above ﬁeld application rates, respectively.

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similar when compared to the other three fumigants, suggesting a lesser effect of 1,3-D on heterotrophic. In another study, effects of SS on soil microbial properties, including metabolic diversity of the microbial communities, were examined in a greenhouse study and were compared to MeBr (Tanaka et al., 2003). The authors found that the richness and average well color development (AWCD) values in the microbial communities decreased markedly immediately after treatment with MeBr but showed a rapid recovery, while those treated with CP continued to decrease until the transplanting of tomato seedlings. This was in agreement with Ibekwe et al. (2001a) who showed a sharp decrease in microbial diversity with MeBr, followed by a quick recovery of the community after 8 weeks when compared to a more sustained effect for a longer period with CP. The shifts in microbial communities observed in the Biolog assays were due to the toxic effects of fumigants on rapid growing microorganisms of high population in the soils. Analysis of microbial communities from the Biolog GN assay by DGGE conﬁrmed that carbon source utilization proﬁles obtained with Biolog GN plates do not necessarily discriminate the numerically dominant members of the microbial community used as the inoculum (Engelen et al., 1998; Smalla et al., 1998). Under ﬁeld conditions, natural ﬂuctuations in carbon substrate utilizing activity and community-level physiological proﬁles of microorganisms in low input and conventional rice paddy soils were monitored using Biolog GN plates for 2 years. The purpose was to establish criteria for assessing side effects of pesticides on soil microbial ecosystems (Itoh et al., 2002). The activity changed seasonally showing a regular pattern with more activity observed during late summer. The level of microbial activities seemed to be directly inﬂuenced by soil temperature and/or redox potential. Soil microbial communities grouped into three clusters, August – December, January – April, and May – August, based on the sampling season. Many studies have shown the effects of fumigants on microbial activities and community structure. The effect of metam sodium fumigation on community structure after a 5- and 18-week incubation showed a separation of the two communities along the ﬁrst principal component (PCA) based on treatment dose (Macalady et al., 1998). There was a signiﬁcant ðp ¼ 0:001Þ dose treatment effect at week 5, whereas at week 18 there was no dose signiﬁcant effect ðp ¼ 0:05Þ; but there was a binary variable effect ðp ¼ 0:001Þ between the treated and untreated samples. Toyota et al. (1999) compared the AWCD values and richness (number of positive wells) of different categories of the 95 substrates 105 days after fumigation with metam sodium. They found that both AWCD and richness in all the substrate groups were signiﬁcantly ð p ¼ 0:05Þ lower in the fumigated soils than in the control soils. In the radish rhizosphere and non-rhizosphere soils fumigated with CP, there was a signiﬁcant suppression in AWCD and richness in fumigated soils compared to the control (Itoh et al., 2002). It was assumed that the bacterial populations with a high substrate assimilation activity were damaged by CP fumigation and a

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different microbial community was developed in the radish rhizosphere. Cluster analysis of these communities after 24 h of incubation separated between rhizosphere and non-rhizosphere samples, and then between fumigated and nonfumigated samples, suggesting the effect of the rhizosphere by CP fumigation. After 72 h, the Biolog samples showed a clear separation between the fumigated and the non-fumigated samples in both the rhizosphere and the non-rhizosphere samples. The authors concluded that fumigants affected mostly the slowsubstrate utilizing rhizosphere microbial communities or that fast growers seemed to utilize most of the substrates during the early stages of incubation. The problem with the Biolog system is due ﬁrst to the respiratory activities of fast growing heterotrophic bacteria resulting in the stimulation or reduction of the catabolism of 95 carbon substrates (Engelen et al., 1998; Ibekwe et al., 2001a). The shifts in microbial communities observed in the Biolog assays were due to the rapid growth of organisms of their high population in the soils. For example, Pseudomonas species are found in most soil samples and they respond well in Biolog assays (Haack et al., 1994; Garland, 1996, 1997).

B. CHANGES IN MICROBIAL BIOMASS SIR (Anderson and Domsch, 1978) has been the standard method for soil microbial biomass measurements. It is based on the maximal initial response of the soil microbial biomass to a substrate amendment. Microbial biomass is assessed quantitatively by the C, N, ninhydrin-reactive compounds, ATP, quinones, and phospholipid fatty acid (PLFA) composition of the cells. Total organic carbon in the microbial biomass (biomass C) is considered as the general indicator of the amount of microorganisms in the soil, and total nitrogen is considered to be the indicator of potential available nitrogen in the soil. Ninhydrin-reactive compounds represent the labile fraction of biomass N and are metabolized to ammonia by heterotrophic microorganisms in soil. This is one of the major fractions of available N to plants and microorganisms. The concentration of ATP in soil is an indicator of the amount of microorganisms that can be readily and rapidly measured. The total amount of respiratory quinones has been shown to be an indicator of the microbial biomass since many microorganisms have only one quinone species. Bacteria contain relatively constant amounts of viable biomass as phospholipid, so this can also be used as a good biomass indicator. Many studies have shown the impact of fumigation on microbial biomass (Zelles et al., 1997; Toyota et al., 1999; Ibekwe et al., 2001a; Suyama et al., 2001; Itoh et al., 2002). Zelles et al. (1997) reported a decrease in the microbial biomass-C and -N of about 20% after chloroform fumigation. Grifﬁths et al. (2000) examined a technique based on progressive chloroform fumigation of soil to reduce soil microbial biodiversity, and measured the effects of the reductions

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upon the stability of key soil processes. The diversity of cultivable and noncultivable bacteria, protozoa, and nematodes was progressively reduced by increasing fumigation times, with the total microbial biomass less in fumigated soils than the unfumigated. Speciﬁc parameters like nitriﬁcation, denitriﬁcation and methane oxidation decreased as biodiversity decreased. Suyama et al. (2001) looked at the effects of fumigation on paddy rice soil to establish a criteria to assess the short-term effects of pesticides on soil microorganisms. They concluded that the degree of ﬂuctuation of microbial biomass and population in the paddies can be used as references to assess the degree of pesticide effects in other Japanese paddy soils. In another study, the effects of the pesticides fenitrothion, chlorothalonil, chloropicrin, linuron, and simazine on microbial biomass were monitored for 28 days for changes in respiratory quinone proﬁles (Katayama et al., 2001). Pesticides were applied to the soil at 10 times the recommended rates. Application of CP decreased the amount (an indicator of microbial biomass) and diversity (an indicator of taxonomic diversity of the microbial community) of the different quinones species during the 28-day treatment. Continuous change in the structure of the microbial community in the CP-treated soil was documented by the changes in the dominant quinone species, and there was no change in the control soil. The authors concluded that quinone proﬁle analysis is a potential method to detect the effect of pesticide on a soil microbial community and biomass. PLFA proﬁles are often used to study microbial diversity and biomass in complex communities (Zelles, 1999). PLFAs are components of phospholipids that are essential parts of membranes found in all living cells. Certain signature fatty acids in the overall PLFA proﬁle are speciﬁc for groups of bacteria, fungi, and actinomycetes (Tunlid and White, 1992). The biomass of these groups can be studied once fumigants are applied to any soil because this will represent the living component of the population. Analysis of PLFA proﬁles of soils fumigated with MeBr, MITC, 1,3-D, and CP was carried out over a 12-week period after application. Biomarker peaks were analyzed and were determined to range from a minimum of 1.3 nmol g21 dry wt for the four fumigants (week 1) to a maximum of 55 nmol g21 dry wt for the 1,3-D- and CP-treated samples in week 12 (Ibekwe et al., 2001a). The biomass contents, as indicated by the total PLFA, were signiﬁcantly different at different time points in some treatments ðp , 0:05Þ: At week 1, the biomass contents in MeBr-amended microcosms were signiﬁcantly lower than those in weeks 8 and 12 (Fig. 3), and of the three other fumigants (Ibekwe et al., 2001a). There was also a decrease in biomass of some of the Gram-negative (cy17:0, 15:0, and 18:1v7c) and the fungal (18:2v6c) biomarkers, with increases in MeBr concentration during the ﬁrst week (Fig. 3). There was a signiﬁcant increase in biomass for Gram-positive bacteria (a17:0, i17:0), fungi (18:2v6c), and actinomycetes (10me 16:0) in weeks 8 and 12. 1,3-D and CP had the strongest effects on actinomycetes, resulting in a signiﬁcant decrease in biomass for most treatments. The effects of MITC followed the same

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Figure 3 Biomass contents (nmol of PLFA g21 dry wt of soil) of samples collected after weeks 1, 8, and 12 from MeBr fumigated soils ðn ¼ 3Þ: MeBr1, -8, and -12 indicates samples were taken 1, 8, and 12 weeks after the start of the experiment. 1, 2, and 3 after the “-” sign indicates 50% below ﬁeld application rate, ﬁeld application, and 1000% above ﬁeld application rates, respectively. Error bars represent standard deviation.

trend as MeBr, except that the recovery of Gram-negative bacteria biomass did not occur during week 8. The effects of fumigants on microbial biomass may be short term with biomass recovery after a few weeks, as was seen with MeBr, MITC, and 1,3-D, or it may be long term, as was observed with CP.

C. CHANGES IN SOIL MICROBIAL COMMUNITY STRUCTURE AND COMPOSITION Several methods to characterize microbial communities in soils that do not depend on culturing have been recently developed. These methods were based on the analysis of biomarkers, such as 16S rRNA genes, PLFA, and respiratory quinones (Morgan and Winstanley, 1997). Analysis of 16S rRNA genes in soil was used to detect the long-term effects of phenylurea herbicides on soil microbial communities (El Fantroussi et al., 1999). The analysis of PLFA was applied to detect the long-term microbial effects of heavy metals

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(Frostegard et al., 1996; Pennanen et al., 1996). However, soil microbial DNA does not reﬂect changes in soil microbial biomass, and the proﬁle of PLFA does not represent individual taxonomic groups (Katayama and Fujie, 2000). Katayama et al. (2001) showed that a signiﬁcant difference ðp ¼ 0:05Þ between the quinone proﬁles of pesticide-treated soils and the control soil at a given period of incubation was observed for CP-treated soil after 28 days of incubation. The use of quinones for microbial community structure analysis is based on the classiﬁcation of quinones into different functional groups. The group consisted of MK-8 [Gram-positive bacteria with a low guanine plus cytosine (G þ C) content and Actinobacteria ], followed by MK-7 (Cytophaga-Flavobacterium cluster and Gram-positive bacteria with low G þ C contents) and next, MK-9 (Micromonosporineae, Streptmycineae, and Streptosporangineae). The authors reported that 3 days after treatment with CP, MK-9 comprised the largest proportion of quinones, indicating a change in the dominant microorganisms. After 7 days of incubation, the largest proportion of quinones changed to MK-7, after 14 days to MK-9, and after 28 days the MK-8 represented the second largest proportion of quinones in the soils suggesting a continuous change in the structure of the microbial community in the CP-treated soils. Sigler and Turco (2002) examined the impact of the fungicide chlorothalonil on dominant bacterial and fungal populations following application to turfgrass, forest, and agricultural soils. Increased rates of chlorothalonil impacted eight bacterial populations (two inhibitions, four enhancements, and two non-speciﬁc responses) and four fungal populations (all inhibitions). Denaturing gradient gel electrophoresis (DGGE) band numbers of 16S rRNA and the Shannon –Weaver index of diversity (H0 ) indicated an altered but not signiﬁcantly different ðp , 0:05Þ bacterial and fungal community structure following chlorothalonil fumigation. Sequencing of the dominant DGGE bands indicated an impact on several groups of bacteria (Cytophaga-Flavobacterium-Bacteroides, alpha-, beta-, gamma-, and deltaproteobacteria) and two fungal taxa (zygomycota and ascomycota). The authors concluded that changes to the overall community structure of dominant species were less signiﬁcant, but a single chlorothalonil application and a short incubation period resulted in community changes including both enhancement and inhibition of a variety of dominant organisms. In another study, chloroform fumigation was used to manipulate the composition of microbial communities as a means of investigating relationships between community structure and the functioning of soil processes (Dickens and Anderson, 1999). The results showed that chloroform fumigation after 24 h caused a large reduction in total PLFAs and poor regrowth of the residual community. The effect of metam sodium on soil microbial community structure and function in two Japanese soils showed that the number and pattern of ampliﬁed 16S rRNA restriction patterns (ARDRA) fragments were changed by fumigation. Shifts were observed in the percent G þ C proﬁle toward a greater proportion of lower percentage G þ C classes in treated soils as compared to the

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untreated soils (Toyota et al., 1999). They showed that the effects of fumigation on the soil microbial community structure and function were pronounced and for some parameters were very persistent. There are several studies on the effects of fumigants on bacterial community structure and function. Some of these studies relied on cultural techniques and, more recently, on biochemical or molecular approaches. Depending upon the approach, different conclusions were reached. Macalady et al. (1998) concluded that, although community fatty acid analysis showed promise as a screening tool in soil microbial toxicity studies, more detailed information could be obtained from PLFA and other speciﬁc lipid fractions. The abundance of indicator fatty acids for bacteria after 5 weeks of incubation was correlated to MITC doses, but after 18 weeks, very few were related to the MITC dose. MITC was also observed to reduce populations of culturable organisms dramatically in the Biolog assay. Ibekwe et al. (2001a), using the same concentration of MITC and other fumigants as Macalady et al. (1998), did a detailed study of the impact of four fumigants on soil microbial communities using the Biolog assay, PLFA, and DGGE. They found that the community structures of fumigant-treated microcosms measured by PLFA analysis shifted away from the ﬁrst communities after 8 and 12 weeks. The shift was greatest with MeBr, which doubled the amount of variation in component 1 when compared to component 2. The major difference in the PLFA proﬁles between the MeBr-treated and the control microcosms was that the MeBr microbial communities contained signiﬁcantly more branched chain PLFAs [speciﬁcally, a17:0, i17:0, a15:0, and i15:0 ðp , 0:05Þ], indicative of Grampositive bacteria (White and Findlay, 1988; Heipieper et al., 1992; Tunlid and White, 1992). In the MeBr-treated microcosms, the relative proportion of PLFAs indicative of fungal biomass (Guckert et al., 1985), speciﬁcally 18:2v6c and 18:3v6c, increased over time. Comparison of PLFA proﬁles of MITC, 1,3-D, and CP-treated microcosms to the control samples showed that microbial community from week 1 was furthest away from the control, and after weeks 8 and 12, PLFA proﬁles of the three fumigants and the control were not signiﬁcantly different from each other. The major advantage of PLFA analysis over other techniques is that it has been regarded as an indicator of the total microbial biomass and certain PLFAs can be used as biomarkers for speciﬁc groups of microorganisms (Tunlid and White, 1992; Zelles et al., 1992; Vallaeys et al., 1997). The presence of large proportions of branched fatty acids (a15:0, i15:0, a17:0, and i17:0), which are markers for Gram-positive bacteria, showed that Gram-positive bacteria were less affected by the impact of these fumigants when compared to Gram-negative bacteria. Zelles et al. (1997) found that Gram-positive bacteria were less injured by chloroform fumigation and attributed it to protection by their cell wall structure, formation of spores, and ability to adapt to fumigant vapor more quickly. The total amount of PLFAs decreased by about 50% after 10 days of chloroform fumigation and monosaturated fatty acids, indicative of Gram-negative bacteria, were more heavily affected (60 – 70%).

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D. ANALYSIS OF SOIL MICROBIAL COMMUNITY STRUCTURE BY MOLECULAR TECHNIQUES Analysis of 16S rRNA sequences retrieved from environmental samples is the standard genetic technique for determining microbial community structure without bias introduced by cultivation (Head et al., 1998). The technique uses genetic ﬁngerprinting to describe microbial diversity on a community scale. This has given signiﬁcant information on environmental changes that could not have been observed by traditional techniques. However, some of the techniques still measure a fraction of the soil community or the dominant population. Marked changes in the bacterial community structure within the dominant population can be measured by a ﬁngerprinting pattern to illustrate the effects of xenobiotics. Very few studies of fumigant effects of soil bacteria have used this technique. Engelen et al. (1998) studied the effects of commercial formulations of the herbicides dinoterb (ﬁeld dose) and metamitron (10 times ﬁeld dose) on the bacterial community in a laboratory experiment on a previously unexposed soil. The impact of the herbicide treatment was monitored by 16S rRNA-TGGE of soil community DNA and other classical tests, such as SIR, dehydrogenase, carbon and nitrogen mineralization, and community ﬁngerprinting using Biolog. TGGE gels of the 16S rRNA showed that dinoterb had a marked effect on bacterial diversity. The community structure in the dinoterb soil was dominated by sequences associated with nitrite-oxidizing bacteria (Nitrospina, Nitrospira) not found in the control. In another study, El Fantroussi et al. (1999) showed the long-term effects of three phenylurea herbicides on bacterial diversity in an orchard soil. The number of bacterial colony forming units (CFUs) on R2A agar medium was affected by all herbicide treatments, emphasizing the impact of these pesticides on the microbial community. DGGE analysis of 16S rRNA revealed a clear effect of the four pesticides. DGGE was introduced into microbial ecology (Muyzer et al., 1993) as an attempt to obtain an overview of the structural diversity of microbial communities. It is based on the separation of ribosomal gene sequences directly ampliﬁed from community DNA by using conserved primers on a denaturing gel according to their melting point. DGGE analysis of 16S rRNA fragments was used to examine the effects of four fumigants on soil microbial communities (Ibekwe et al., 2001a). Figure 4a –c shows the DGGE patterns of the 16S rRNA fragments (primers P338f and P518r) ampliﬁed from the four soils 1, 8, and 12 weeks after fumigation. During the ﬁrst week, when the most drastic effects occurred, MeBr treatments did not produce any dominant bands compared to the other fumigants and the control (Fig. 4a). At week 8 there was a signiﬁcant shift in the microbial community structure of the MeBr-treated soils. As shown in Fig. 4b, more bands begin to appear in MeBr treatments. There was also a decrease in the number of bands as the concentration of MeBr increased (MeBr 8-1 to 8-3 from 8 bands to 2, with increasing concentration). At 12 weeks,

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Figure 4 DGGE analysis of 16S rRNA fragments of pooled soil samples collected from triple microcosm treated with different fumigants. Ampliﬁed products were separated on a gradient gel of 30– 70% denaturant. (a) Community structures 7 days after the initiation of the experiment. (b) Community structures after 8 weeks of treatment. (c) Community structures after 12 weeks of treatment.

the microbial communities for all concentrations of MITC, 1,3-D, CP, and the lowest concentrations of MeBr were similar to the control (Fig. 4c). A second approach was used to determine community structure of different bacterial groups based on the 16S rRNA peak height. The peak height values

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Figure 4 (continued )

generated from the sampling points (Fig. 4a – c) were integrated and analyzed using Excel (Microsoft). Data obtained were used to integrate the area under each peak for each gel lane of every treatment. Each band was presumed to represent the ability of that bacterial species to be ampliﬁed. The Shannon – Weaver index of diversity (H0 ) (Shannon and Weaver, 1963) was used to compare changes in diversity of microbial communities within the four treatments at each time period by using the following function: H 0 ¼ 2{Pi log Pi } Pi ¼ ni =N; where ni is the height of peak and N is the sum of all peak heights in the curve. This resulted in a direct comparison of the effect of each compound at one time point in one gel on the structural diversity of the four microbial communities. The Shannon –Weaver index of diversity H0 was calculated on the basis of the number and relative intensity of bands on a gel strip. The four treated samples showed different levels of diversity ranging between 0.11 and 1.26 at different sampling times. It also showed that MeBr exerted the most signiﬁcant effects on the structural diversity of the soil compared to the other three fumigants and the control. One week after MeBr application, the number of bands decreased from 16 in the control to almost undetectable numbers in MeBr-treated soil. This indicated the collapse of the microbial community due to the acute toxicity of MeBr. H0 decreased from 1.26 in the control to 0.11 in the treatment with the highest concentration of MeBr, and continued to increase slightly during week 8

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and subsequently, in week 12 to about 0.75 but remained clearly below the average control value ðH 0 ¼ 1:26Þ: To determine a shift in the microbial community structure, DGGE bands from Ibekwe et al. (2001a) were selected for reanalysis as shown in Fig. 4a – c. Figure 5 shows the phylogenetic analysis of prominent bands recovered from the DGGE gel. Band sequence was analyzed using the BLAST database (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov). Sequence alignments were performed using the PILEUP program from the University of CaliforniaRiverside Genetics Computer Group (GCG programs). Matrices of evolutionary distances were computed using the Phylip program with the Jukes – Cantor model (Jukes and Cantor, 1969). All clones extracted from fumigated soils and their accession numbers are shown in bold. Phylogenetic tree was constructed and checked by bootstrap analysis (100 data sets) using the program SEQBOOT. Bootstrap values represent the frequency of resampling that supports a speciﬁc

Figure 5 Phylogenetic tree constructed for 16S rRNA gene sequences and aligned by the GCG program from the University of California, Riverside genetic group. The tree was produced by using a neighbor-joining algorithm. Bands were cut from the DGGE gel and cloned for analysis.

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branching pattern. Two prominent bands (F1 and F2) were observed during the ﬁrst week of analysis. The derived sequences from these bands conﬁrmed F1 to be 100% similar to Pseudomonas reactans and F2 to be 99% similar to Pseudomonas putida. Mazzola et al. (2002) showed that microbial community residents in a wheat soil fumigated with MeBr-suppressed components of the microbial complex that incited replant apple disease. P. putida was the primary ﬂuorescent pseudomonad recovered from the suppressive soil, whereas Pseudomonas ﬂuorescence bv. III was dominant in the conducive soil. At week 8, four dominant bands (F3 –F7) and three other bands, F16, F17, and F31 appeared in the MeBr treatments. Communities from the other three fumigants were different from that of MeBr. Bands F3 and F6 showed relationships to the Gram-positive species Heliothrix oregonensis and Bacillus subtilis, respectively (Fig. 5). There were no signiﬁcant new bands at week 12, as the community proﬁles from the four fumigants shifted close to the control. In another closely related study, the effect of different concentrations of PBr and 1,3-D in unamended and manure-amended soil on the microbial community was evaluated (Dungan et al., 2003a). DGGE analysis of the PCR fragments (primers 63f and 518r) from weeks 1, 4, 8, and 12 showed a shift in the structural composition of the communities during the 12-week study. The effects of the fumigants on the microbial community structure were most dramatic 1 week after application, as unamended and amended soils treated with PBr or 1,3-D clustered away from the controls. Dominant 16S rRNA bands were not detected in the unamended and amended soils treated with 100 mg kg21 of PBr and 500 mg kg21 of 1,3-D. Four weeks after treatment, PBr and 1,3-D treatments began to cluster closer to the control in amended soils and in unamended soil treated with 1,3-D. The impact of both fumigants on the microbial community was less dramatic in the manure-amended soils than the unamended soil. PBr treatment was more damaging to the microbial community structure than 1,3-D, as signiﬁcantly fewer bands were found in the PBr treatments and a longer time was required for recovery. In general, the structural diversity of the dominant microbial community decreased with increasing fumigant concentration, regardless of the treatment. The Shannon – Weaver index of diversity H0 was also affected by the fumigants. H0 ranged from zero in some of the highconcentration treatments to about 1.4 in some of the controls and 10 mg kg21 treatments. In amended soil treated with 10 mg PBr kg21 the H0 differed little from the control. This was not the case in unamended soils. After week 4, the H0 values of 1,3-D concentrations were similar to the control-amended soil, but decreased to zero at 500 mg kg21. By weeks 8 and 12, the H0 values at all concentrations of 1,3-D were similar to that of the PBr treatments. They concluded that PBr is most damaging to the microbial community in unamended soil, but not in soil containing organic amendment. It should be noted that the banding patterns obtained from these two studies reﬂected the most abundant rRNA types in the community, but not the total members of the community.

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Figure 6 Cluster analysis of microbial communities generated by the analysis of DGGE 16S rRNA PCR patterns representing the genetic similarity of the microbial community proﬁles obtained by PCRDGGE. Numbers (10, 100, 500) before the letters are fumigant application rates in mg kg21. US, unamended soil; AS, amended soil; PB, propargyl bromide; 1,3-D. 1 and 12 indicate weeks 1 and 12.

Reanalysis of the results from Dungan et al. (2003a) showed that microbial communities were more affected by amendment than fumigants, as illustrated by the shift in community structure based on amendment between weeks 1 and 12 (Fig. 6). This clearly showed that the effect of fumigants on soil microbial community is very dramatic during the ﬁrst week of application and after this period, management may play a stronger role.

V. IMPACT OF RECOMMENDED FUMIGANTS ON SOIL MICROBIAL COMMUNITIES AND AGRICULTURAL PRACTICES While changes in the soil microbial population can be observed following fumigation with MeBr and other fumigants (such as higher population of heterotrophic bacteria), the soil is far from being sterilized (Ibekwe, unpublished). Ibekwe et al. (2001a) observed that the Shannon – Weaver diversity index of microbial community diversity was lowest for MB fumigated soil 1 week after application compared to CP, 1,3-D, and MITC. This index remained

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lower than the other treatments even after 12 weeks of incubation. Dungan et al. (2003a) showed the same effect with PBr and 1,3-D during a 12-week experiment where the diversity index was very low. Likewise, Schutter and Ajwa (personal communication) observed a reduction in nitriﬁcation potential in fumigated soils, with recovery in CP-fumigated soils sooner than MeBr, 1,3-D, or PBr. While these examples have focused on soil microbial populations, preliminary work with strawberry rhizosphere colonizers in fumigated compared to native soils suggests that there may be differences in deleterious and beneﬁcial rhizosphere colonizers following soil fumigation (Martin, 2003). A basic understanding of the soil and rhizosphere microbiology can simplify the identiﬁcation of speciﬁc microorganisms that can be used directly for disease management, enhancement of plant growth or altered crop management practices to enhance their populations. One excellent example is the identiﬁcation of speciﬁc bacterial rhizosphere colonizers that are capable of protecting apple roots from pathogens associated with apple replant disease and enhancing their soil populations by cropping speciﬁc cultivars of wheat (Mazzola et al., 2002). Organic amendments may also be useful for management of diseases commonly controlled by soil fumigation, although questions of cost effectiveness and ﬁeld scale practicality may need to be addressed before they are commercially feasible (Martin, 2003). The beneﬁts of adding organic amendments to soils are well documented. They can improve soil nutrition, physicochemical conditions, and crop viability (Hungalle et al., 1986; O’Hallorans et al., 1993). They have been found to be effective in reducing potentially harmful fumigant emissions (Gan et al., 1998b) and controlling soilborne pathogens by stimulating antagonistic organisms (Akhtar and Malik, 2000) or by producing toxic volatile compounds (Gamliel and Stapleton, 1997). Applications of organic amendments have also been shown to increase the soil microbial biomass and stimulate microbial activity (Perucci, 1990; Bandick and Dick, 1999; Peacock et al., 2001). In a greenhouse study to evaluate methods for management of apple replant disorder, Mazzola et al. (2002) observed that soil amendment with Brassica napus seed meal reduced the incidence of apple root infection by Rhizoctonia spp. and the lesion nematode Pratylenchus penetrans. However, in some cases it was found to increase soil populations of Pythium spp. and the incidence of disease they caused. Application of fumigants has beneﬁcial effects when the best management practice is adopted. Soil fumigation generally increases root health, growth, and fruit yields in strawberries even when major pathogens are not present in soil (Wilhelm and Paulus, 1980; Yuen et al., 1988; Duniway, 2002). Temporary inhibition of nitriﬁcation and an increase in ammonia-N in the soil may be partly responsible for the increase in plant growth (Porter et al., 1999). Soil fumigation has been shown to reduce the incidence of Pythium, Cylindrocarpon and binucleate Rhizoctonia spp. damaging to strawberry roots

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(Wilhelm and Paulus, 1980; Martin, 1998, 1999). The reduction of pathogens is the major beneﬁt of fumigation. The process does not result in soil sterilization but in some cases it results in changes in the microbial community structure. Pseudomonas sp. has been shown to survive in fumigated soil and recolonize strawberry rhizospheres rapidly and in high numbers after fumigation (Xiao and Duniway, 1998). The increase in numbers of different Pseudomonas sp. in the strawberry rhizospheres after fumigation correlated to a signiﬁcant increase in the growth of the strawberry plants in ﬁeld and greenhouse experiments (Xiao and Duniway, 1998).

A. METHYL BROMIDE MeBr is a versatile, highly effective and relatively cheap fumigant used for pre-planting fumigation. It is effective against a wide spectrum of plant pathogens and pests, including fungi, nematodes, insects, mites, rodents, weeds, and some bacteria. However, the Br2 residue is left in soil and plants after fumigation. Bromide residues produced by MeBr fumigations have importance because excessive uptake of plant materials containing Br2 is considered harmful to human beings. And in addition, some plants (mainly carnations) are sensitive to high Br2 levels in the soil (Yates et al., 2003). The concern for Br2 toxicity from edible plants grown in fumigated ﬁelds was the main reason for the suspension of MeBr in Germany (Anonymous, 1980). The generally accepted mechanism of MeBr biological activity is through a bimolecular, nucleophilic displacement (SN2) reaction with functional groups, such as NH2 and SH, in various amino acids and peptides of the target organisms (Price, 1985). In soil chemical hydrolysis and methylation through SN2, nucleophilic substitution with water nucleophilic sites on soil organic matter produces the reactive product (Gan et al., 1994). Degradation of MeBr has been demonstrated through cell suspensions of Methylococcus capsulatus as it mineralized MeBr, by its removal from the gas phase, the quantitative recovery of Br2 in the spent medium, and the production of 14CO2 from [14C] MeBr (Oremland et al., 1994). At high concentrations, biodegradation of MeBr in methanotrophic soils was inhibited due to the toxicity of MeBr itself, but became signiﬁcant at concentrations lower than 1000 ppm. Methyl ﬂuoride (MeF) inhibited the oxidation of methane as well as that of [14C] MeBr. The rate of MeBr consumption by cells varied inversely with the supply of methane, which suggested a competitive relationship between these two substrates. Soil methanotrophic bacteria, as well as other microbes, can degrade MeBr present in the environment. Miller et al. (1997) isolated a facultative methylotroph that used MeBr as a source of carbon and energy. The consumption of MeBr by the methane-oxidizing bacteria indicates that methane monooxygenases are

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responsible. Shorter et al. (1995) suggested that microbial degradation of MeBr at low concentrations (ppb) in surface soils may be important in removing MeBr from the atmosphere, thus reducing its lifetime in the atmosphere and lowering its ozone-depletion potential. This observation is limited in applicability since the effectiveness of MeBr will be reduced at concentrations below the application rate. Studies have indicated that MeBr oxidation can occur in ﬁeld-fumigated soil. High rates of 14C-MeBr oxidation to 14CO2 were observed in the ﬁrst few days following soil fumigation where the MeBr concentration was . 9.5 mg g21 soil (Miller et al., 1997). Martin (2003) reviewed the ecology of microbial rhizosphere inhabitants effected by fumigation with MeBr and CP, and how the rhizosphere may hinder or help the ability to control plant pathogens. In the absence of known pathogens, many crops have exhibited an increased growth response when planted into soil that had been fumigated with MeBr (Wilhelm and Paulus, 1980). One of the likely reasons for this observation was that fumigation altered the microbial composition of the soil, either enhancing beneﬁcial colonizers or reducing populations of deleterious rhizosphere colonizers. At a ﬁeld site where MeBr and CP were used as fumigants, the soils had 10- to 100-fold greater populations of ﬂuorescent pseudomonads and 1000-fold greater populations of total fungi than the non-fumigated soil (Xiao and Duniway, 1998). Total bacterial populations were not signiﬁcantly different in the fumigated and non-fumigated soils. Although differences in total ﬂuorescent pseudomonad populations persisted throughout the season, total fungal populations equilibrated 1 week after fumigation. Given the observed differences in microbial communities, it is likely that there were also differences in rhizosphere colonizers as well. Qualitative differences in rhizosphere colonizers of strawberries grown in MeBr- plus CP-fumigated and non-fumigated soils were observed. Differences were also observed in fungal rhizosphere colonizers, in one ﬁeld in Watsonville, CA, exhibiting much higher root colonization frequencies by Trichoderma harzianum on plants from fumigated than non-fumigated soil. In growth chamber and microcosm studies, the concentrations of human pathogenic Escherichia coli O157:H7 were signiﬁcantly higher in the nonfumigated soils during the ﬁrst 2 weeks after fumigation with MeBr and MeI (Ibekwe, unpublished). In this study, changes in microbial community structure in fumigated and non-fumigated soils were examined in microcosms without plants, in rhizospheres and in non-rhizosphere soils. The effects of the two fumigants on soil microbial community structure were greater based on the types of fumigants. Clay soil seemed to protect microorganisms better than sandy soil, since there were more DGGE bands detected in clay soil (Fig. 7a and b).

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B. METHYL ISOTHIOCYANATE MITC is the primary active ingredient of metam sodium in soil and is a broadspectrum fumigant with activity against plant pathogenic nematodes, weeds, and a range of pathogenic fungi (Kreutzer, 1963). Metam gained notoriety in 1991 when a spill into the Sacramento River in California resulted in human exposure and an environmental disaster (CA EPA, 1992). The toxic action of this fumigant involved ﬁrst the decomposition or metabolism of metam and dazomet to MITC as the activation product with derivatized critical biological thiols and amines (Kaufman, 1977; Sinha et al., 1988; Tomlin, 1994; Ware, 1994). In living cells MITC is metabolized by the mercapturate, N-acetyl-S(N-methylthiocarbamoyl)cysteine via the GSH conjugate. The GSH serves as a potential carrier for the later release of MITC (Mennicke et al., 1983; Baillie and Slatter, 1991; Lam et al., 1993). S-methylation is another alternative in which metam might be methylated and MITC and dazomet metabolized to S-methyl metam. S-methylation is a bioactivation mechanism for metam and metabolites of MITC and dazomet in cells (Staub et al., 1995). The conversion of MITC to Smethyl metam and its oxon is believed to involve conjugation with glutathione, hydrolysis to 50 -(N-methylthiocarbamoyl)cysteine, cleavage by cysteine conjugate, lysis to release metam, and methylation and oxidative desulfuration (Staub et al., 1995). Riffaldi et al. (2000) evaluated the extent to which metam sodium (MS) was applied at two different recommended rates and how its degradation product, MITC, could affect soil respiration. The results suggested that MS degradation to MITC was complete within 4 h. MITC decomposed quickly in a few days, except in the soil containing high organic matter where it was still present after 15 days. Metam sodium does not move through soil like MeBr, so a thorough mixing of the soil is needed to ensure even distribution and avoidance of “hot spots” where high concentrations of MITC do not fully dissipate and can result in phytotoxicity problems (Martin, 2003). In addition, uniform watering is needed to activate all products in the soil prior to planting or phytotoxicity problems may be encountered later when subsequent irrigations activate residual products. Nonuniform distribution may also result in poor degradation of MITC. The degradation of MITC is a result of both chemical and biological mechanisms, since degradation of MITC in sterile soil is signiﬁcantly slower than in non-sterile soil (Gan et al., 1999; Dungan et al., 2003b,c). Smelt et al. (1989) demonstrated

Figure 7 DGGE analysis of 16S rRNA fragments of duplicate soil samples collected from microcosm treated with different fumigants in clay and sandy soils. Samples were collected 7 days after fumigation. Ampliﬁed products were separated on a gradient gel of 30– 70% denaturant. (a) Community structure 7 days after the initiation of the fumigation in clay soil. (b) Community structures 7 days after the initiation of the fumigation in sandy soil.

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enhanced degradation of MITC in soils that had been previously treated. This implies that microbial degradation of MITC is occurring. Microorganisms responsible for the enhanced degradation of MITC may speciﬁcally target the isothiocyanate functional group, which enables the degradation of the isothiocyanate compounds at an accelerated rate. There is little information available in the literature about the nature of the degradation products of MITC. The concern with the products is that they could be more toxic and mobile than MITC. More research is needed to provide detailed information on the environmental conditions that may enhance toxicity of the products.

C. 1,3-DICHLOROPROPENE The fumigant 1,3-D (Telonew, Dow AgroSciences) is an effective nematicide used either as a stand-alone fumigant (Telone II, 94% 1,3-D) or in mixture with 17 or 35% CP (Telone C-17 and Telone C-35, respectively). The purpose of the mixture is to improve efﬁcacy against soil-borne fungal pathogens. Registration of 1,3-D was suspended in California in 1990 because of air quality concerns in Merced County, but was reinstated in 1994 (Martin, 2003). Concern for safety and air quality led California’s Department of Pesticide Regulation (2002) to institute buffer zone requirements and place limits on the amount of this fumigant that can be applied in a township. In ﬁelds that are cropped to strawberry at least once every 3 years, this can inﬂuence the growers’ ability to fumigate some portions of their ﬁelds, especially in agricultural areas faced with urban encroachment. The township cap varies with area and season, and will restrict the use of 1,3-D as an alternative fumigant to MeBr. These restrictions take into consideration potential groundwater contamination, worker exposure, and air emissions for potential chronic exposure. The toxicity of 1,3-D to soil microorganisms at the recommended rate may not be a serious concern (Ibekwe et al., 2001a). What is of concern to farmers is the high degradation rate with repeated application (Gan et al., 1999). A decrease in ﬁeld performance for 1,3-D following repeated application has been reported (Smelt et al., 1989; Ou et al., 1995, 1997). This loss in efﬁcacy is associated with an increase in microbial degradation in the adapted soils. Adaptation of such soils to fumigants may be due to the selection of microbial populations with high degradative potentials (Van Dijk, 1974; van Hylackama and Janssen, 1992; Verhagen et al., 1995). Field experiments with continuous potato cropping found that sustained annual applications of 1,3-D led to insufﬁcient control of potato-cyst nematodes (Solanum tuberosum L.) (Lebbink et al., 1989). Pseudomonas sp. may be one of the most abundant bacterial species in soil. There are many reports of this species’ ability to degrade 1,3-D (Lebbink et al., 1989; Verhagen et al., 1995). Fifteen bacterial strains with the capacity to degrade 1,3-D (of which four were Pseudomonas sp.) were isolated from

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enrichment cultures grown from adapted soils (Verhagen et al., 1995). One strain, Pseudomonas cichorii 170, was shown to completely degrade 1,3-D to 3-chloroallyl alcohol (Poelarends et al., 1998). The characterization of the genes involved in the complete metabolism of 1,3-D was identical to the dhaA of the Gram-positive bacterium Rhodococcus rhodochrous NCIMH13064 and the dhiA genes from Rhodococcus sp. strain m15-3 (Bosma et al., 1999).

D. CHLOROPICRIN CP and possibly its dechlorination products are lacrimators, respiratory irritants, and toxicants and must be used under containment conditions. CP is a preplant soil fumigant, a warning agent for other fumigants, and a former war gas. Its mode of action is unknown but presumably related to its facile metabolic dechlorination on reaction with biological thiols (Sparks et al., 1997). The reaction of 14CCl3NO2 with GSH yields the di- and monochloro derivatives, GSSG, and a small amount of nitrite. The toxicity of CP (CCl3NO2) is probably due to disruption of multiple targets by its cascade of dechlorination products. The reactivity of CCl3NO2 with biological thiols has been known since the 1940s, but no products or mechanisms have been identiﬁed (Bacq, 1942; Desreux et al., 1946). CP is known to be metabolized by Pseudomonas (Castro et al., 1983; Castro, 1993). In aerobic soil CP will be degraded to produce CHCl2NO2, CH2ClNO2, and CH3NO2 (Wilhelm et al., 1996): Cl3 CNO2 ! Cl2 CHNO2 ! ClCH2 NO2 ! CH3 NO2 The reactions of CP in cells involve rapid dechlorination to CHCl2NO2 and conversion of GSH to GSSG, plus possible adduct formation with thiol proteins, e.g., Hb-SH (Sparks et al., 1997). CP then oxidizes protein thiols with formation of disulﬁde bonds that may disrupt multiple targets by its dechlorination products. CP stability in soil is short term, with microbial degradation primarily responsible for inactivation of the fumigant (Gan et al., 2000). In these studies degradation was shown to follow ﬁrst-order kinetics ðr 2 . 0:87Þ: Total degradation of CP was higher in compost-amended soil and enriched with CP, than the unamended soil and it was signiﬁcantly lower in the sterile soils (Ibekwe et al., unpublished). The degradation capacity of these samples was predominantly of biological origin. In unamended, compost-amended, and compostamended soils treated with CP, the k values varied from 0.21, 1.12, and 3.50 day21, respectively. This corresponds to half-lives of 3.40, 0.62, and 0.20 days. Degradation of CP is very rapid compared to other fumigants. For instance, halflives was 2 days for 1,3-D from compost-amended Arlington sandy loam soil (Ibekwe et al., 2001b). Degradation of CP in sterile soil was signiﬁcantly inhibited suggesting an important role of soil microorganisms in CP degradation

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(Gan et al., 2000). Microbial degradation accounted for 70, 57, and 83% overall CP degradation in compost-amended, unamended, and compost-amended treated soils, respectively.

VI. EFFECT ON SPECIFIC MICROBIAL POPULATIONS Fumigants are used for the control of plant pathogens. Some fumigants may be toxic to some microbes, and this may enhance the selection of others, which may be beneﬁcial to plants. Since the majority of research has been concentrated on microorganisms that are easy to manipulate in culture, our understanding of microbial interactions and their impact on plant health is not yet well understood. The ecological signiﬁcance of Mycobacteria, a group of organisms that are not amenable to standard laboratory enumeration techniques, is currently being evaluated (Bull, unpublished data). Mycobacteria from fumigated and nonfumigated soil and from strawberry roots were evaluated to determine interactions with biocontrol agents and pathogens (Shetty et al., 2000; Bull et al., 2002). Some microorganisms are more amenable to study than others due to the complexity of the microbial community evaluated. Studies in our laboratory have shown the effect of MeBr and MeI on heterotrophic bacterial growth to be insigniﬁcant in the rhizosphere and non-rhizosphere of lettuce grown in a growth chamber and irrigated with E. coli O157:H7 contaminated water. After an initial decline, there was a fast regrowth of the heterotrophic bacteria (after 3 weeks). This was also observed with the E. coli O157:H7 colonies. Toyota et al. (1999) showed insigniﬁcant differences in culturable bacteria between the control and fumigated soils 7 days after fumigation in two Japanese soils. They were able to quantify a 50% reduction in total number of bacteria 15 days after fumigation, but at 23 days, the numbers were at the normal level. Both fungal and bacterial biocontrol agents, including Gliocladium virens, Pseudomonas chlororaphis, Pseudomonas ﬂuorescens, Pseudomonas aureofaciens, Bacillus cereus, and Streptomyces isolates, were included in the trials to determine the effects of MeBr to speciﬁc microbes (Martin, 2003). Fumigation promoted the survival of this species. Ibekwe et al. (2001a) and Dungan et al. (2003a) were able to show that fumigation with MeBr and PBr resulted in changes in microbial community leading to the emergence of new communities dominated by Pseudomonas sp. and Bacillus sp. The approaches that ﬁrst describe the microbial ecology of fumigated soils and of the roots in these soils should provide sound information for targeting screening efforts to identify speciﬁc microbes capable of controlling root diseases. Investigations evaluating individual components of MeBr plus CP and non-fumigated rhizosphere communities indicated that some strains can have beneﬁcial or deleterious effects on strawberry plants (Martin, 1997). Some rhizosphere colonizers were found to enhance strawberry yields in the ﬁeld.

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Trials were conducted in test plots managed by a commercial grower in a ﬁeld where the predominant pressure was from generalized root pathogens associated with black root rot, such as Pythium, binucleate Rhizoctonia, and Cylindrocarpon spp. After 2 years, yields were different from both years due to environmental conditions. Itoh et al. (2000) showed that Fusarium oxysporum was not detected in soil 3 weeks after fumigation, but this varies with fumigant, with CP having a stronger effect than MITC. In another study, Tanaka et al. (2003) showed more vigorous growth of tomato plants after CP treatments than those treated with MeBr. The result was attributed to an increase in NH4-N supply at that stage.

VII. SUMMARY AND CONCLUSIONS The phase-out of MeBr has generated a lot of public awareness of fumigants and the large use of these compounds in agriculture. Since there is no single, registered fumigant that is as effective as MeBr, other compounds need to be developed and tested. The actual registration procedure includes evaluations of the impact of herbicides on the environment by testing for non-target organism effects on a single species or on microbial communities. The impact on soil microbial communities is evaluated in view of their role in sustaining the global cycling of matter and their varied functions in supporting plant growth. Internationally, there are various protocols that are required before a new pesticide is granted registration.

ACKNOWLEDGMENTS Thanks to Ms Pamela Watt for reviewing and assisting in literature searches, and Drs Scott Yates, Sharon K. Papiernik, and Frank Martin for providing helpful materials. This review was supported in part by the 206 Manure and Byproduct Utilization Project of the USDA-ARS. The mention of trademark or propriety products in this review does not constitute a guarantee or warranty of the property by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.

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SORGHUM IMPROVEMENT— INTEGRATING TRADITIONAL AND NEW TECHNOLOGY TO PRODUCE IMPROVED GENOTYPES W. L. Rooney Department of Soil and Crop Science, Texas A&M University, College Station, Texas 77843-2474, USA I. II. III. IV.

V.

VI.

VII. VIII.

Introduction Variation in Sorghum ssp. Sorghum Improvement—from Landraces to Cultivars Mechanisms of Controlled Pollination A. Hand Emasculation B. Genetic Male Sterility C. Hot-Water Emasculation D. Control of Anther Dehiscence E. Cytoplasmic – Genetic Male Sterility Improvement Methodology A. Population Improvement B. Cultivar and Inbred Line Development C. Hybrid Development D. Use of Exotic Germplasm—Sorghum Conversion Trait-Based Breeding Efforts A. Yield and Adaptation B. Biotic Stress C. Abiotic Stress D. Grain Quality E. Forage Sorghum F. Sweet Sorghum for Syrup G. Broomcorn Biotechnology in Sorghum Improvement Conclusion References

Sorghum (Sorghum bicolor L. Moench) is a major cereal grain crop grown throughout the semi arid regions of the world. Depending on the region of production, the type of sorghum and the purpose for its production varies widely. Whether they are breeding varieties or hybrids, the primary focus of sorghum breeders throughout the world are yield, adaptation and quality. In addition to breeding for these factors, reducing losses due to stress is equally important. Most breeding 37 Advances in Agronomy, Volume 83 Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved. DOI 10.1016/S0065-2113(04)83002-5

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W. L. ROONEY programs consistently select for tolerance to abiotic stresses (such as drought and low temperatures) and biotic stresses (such as sorghum midge, grain mold, anthracnose, and charcoal rot). Finally, the integration of molecular genetic technology is enhancing sorghum improvement by providing a genetic basis for many important traits and through marker-assisted selection. Sorghum improvement in the future will require effective utilization of all the available tools in order to develop sorghum genotypes suitable for the needs of their producers and end-users. q 2004 Elsevier Inc.

I. INTRODUCTION Sorghum (Sorghum bicolor L. Moench) is one of the most important cereal grain crops in the world. In 2001, sorghum was produced on approximately 50 million hectares with an average yield of 1280 kg ha21 worldwide (FAO, 2001). Average yields for sorghum production are generally low because the crop is widely grown in environments where abiotic and biotic stresses are common and limit production. While the worldwide average sorghum yield is low, average yields vary widely among countries (FAO, 2001) and the maximum recorded grain sorghum yield was 21.5 t ha21 (Wittwer, 1980). Most sorghum production is located in semi-arid tropical and subtropical regions, but production occurs in some temperate regions where rainfall is limiting. Depending on the location, sorghum is grown for many different purposes. The grain is used for food, feed, and industrial purposes. The vegetation is important in many production systems where it is used as forage. The location of production often deﬁnes the ultimate end use and the speciﬁc types of sorghum that will be grown. For example, in many regions of Africa, sorghum is a vital food grain and the stalk and leaves are valued for building and forage. In these production systems, small farmers demand pure-line cultivars that are tall with speciﬁc food quality parameters and stable production under stress. In developed countries, sorghum is grown as a feed grain with high input and management. The production system is mechanized and demands sorghum hybrids with high yield potential, relatively short, lodging resistant, and responsive to favorable environmental conditions. Because of diversity within the species and the inﬂuence of selection, many different types of sorghum have been developed for speciﬁc uses and purposes throughout the world. Modern sorghum improvement programs have been faced with the challenge of using these genetic resources in combination with modern technologies to produce productive and useful sorghum genotypes for future use. The speciﬁc goals of each program are dependent on the purpose and location of

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the program and the resources available to the program. The purpose of this review is to summarize the goals and methods of current day sorghum-breeding programs. While this discussion will focus on sorghum improvement programs that emphasize hybrid development, it is necessary to review the origins of sorghum diversity and initial improvements in sorghum genotypes.

II. VARIATION IN SORGHUM ssp. The genus Sorghum is incredibly diverse with members of the genus present throughout the semi-arid tropics. The center of origin is in Eastern Africa; a signiﬁcant amount of diversity still exists in this region today (Dahlberg, 2001). Based on phenology and genetics, Garber (1950) and Celarier (1959) subdivided the genus into ﬁve subgenera: Sorghum, Chaetosorghum, Heterosorghum, Parasorghum, and Stiposorghum. The cultivated sorghums are included in the Sorghum subgenera and Snowden (1936) completed the classiﬁcation of this subgenera. Celarier (1959) reported that the base chromosome number in the Sorghum subgenera is n ¼ 10 and most members of the subgenera were diploid ð2n ¼ 2x ¼ 20Þ; but several members were polyploid ð2n ¼ 4x ¼ 40Þ: de Wet (1978) further classiﬁed the Sorghum subgenera by recognizing three distinct species: S. propinquum, S. halepense, and S. bicolor. Furthermore, S. bicolor was divided into three subspecies: drumondii, bicolor, and verticilliﬂorum. All of the cultivated sorghums are classiﬁed as S. bicolor subsp. bicolor. Finally, Harlan and de Wet (1972) partitioned the primary gene pool of S. bicolor L. Moench into ﬁve basic races (designated Bicolor, Guinea, Caudatum, Kaﬁr and Durra) and 10 intermediate races from the combinations of the ﬁve basic races. These races are used today for the classiﬁcation of sorghum germplasm collections. In addition, these races are reﬂective of different patterns of production and utilization in speciﬁc geographic regions. Dahlberg (2001) provides a recent and complete review on the classiﬁcation and characterization of Sorghum. Cultivated sorghum (S. bicolor subsp. bicolor) is a very diverse species with signiﬁcant variation for many important traits. As a crop, sorghum is managed as annual, but sorghum growth is indeterminate if weather permits. The plant is a grass with culms that grow up to 5 m in height, often branching and tillering. Culm thickness and density vary widely. The plants have a ﬁbrous root system that can consistently penetrate up to 2 m into the soil proﬁle. Leaves of sorghum vary extensively in length and width but can be up to 90 cm long and 12 cm wide. Inﬂorescences develop from a terminal growing point and panicle shape ranges from open to compact and can be up to 40 cm long and 20 cm wide. Flowers and seed (following pollination) develop on spikelets within the panicle. Wide variation exists for seed size and shape. The races of S. bicolor subsp. bicolor described by Harlan and de Wet (1972) are delineated using

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panicle shape, glume shape and coverage and seed size and shape. Sorghum is a predominantly self-pollinated crop, with outcrossing ranging from none to 30%. The amount of outcrossing is contingent on the speciﬁc genotype being grown and the environmental conditions encountered prior to and during anthesis (Schertz and Dalton, 1980). The greatest diversity of cultivated and wild sorghum is in Africa (Doggett, 1988). This variation is probably due to the self-pollinated nature of the crop and the fact that variation, when it occurred could be ﬁxed through selfpollination and then maintained in a true breeding landrace. While the exact mechanism is unknown, it is likely that the diversity of S. bicolor was created through disruptive selection followed by isolation and recombination in the extremely variable habitats of northeast Africa and the movement of peoples carrying the crop throughout the continent (Kimber, 2001). This process resulted in the diversity that is currently classiﬁed in the ﬁve basic races that are described by Harlan and de Wet (1972). The intermediate races deﬁned by Harlan and de Wet (1972) arose later out of hybridization, recombination, and ﬁxation of the intermediate genotypes. It is from this germplasm that initially indigent peoples selected the sorghum genotypes of value to them and eventually these genotypes were used to form the basis of modern sorghumbreeding programs.

III. SORGHUM IMPROVEMENT—FROM LANDRACES TO CULTIVARS Human involvement in the improvement of sorghum began when they identiﬁed and selected plants with desirable characteristics. These new genotypes arose from random outcrosses or mutations that were ﬁxed due to the selfpollination of the new type. The people who found the particular trait(s) to be of value then maintained the landrace varieties that possessed them. This approach to improvement was utilized throughout every sorghum-growing region of the world until the rediscovery of Mendel’s laws when basic genetic principles were applied to agricultural research and improvement. The best documented example of the development of the crop is the introduction of sorghum into the US. From its introduction as a novelty, sorghum developed into a major crop grown as a pure-line cultivar where producers saved seed to replant. The identiﬁcation and development of the cytoplasmic –genetic male sterility (CMS) transitioned sorghum from cultivars to commercial hybrids where seed production is completed by commercial seed companies. Producers are willing to pay for the seed because of its added productivity and stability. This development is also important as it documents the genetics behind the transition to hybrid production.

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The ﬁrst documented introduction of sorghum to the US was Chinese Amber, which was a sweet sorghum brought to the US from France in 1853 (Martin, 1936). Soon after, additional sweet sorghums were introduced and eventually approximately 20 introductions formed the basis of early sweet sorghum cultivars and derivatives of these lines are still used in the Southeast US for sorghum syrup production. There is little doubt that the ﬁrst grain sorghums introduced to the US arrived with the West African slave trade. These sorghums were likely guinea types and hence the name “guinea corn” was applied to these types, which were commonly grown in the Southeast US. However, these types eventually disappeared and did not contribute directly to the development of the crop in the US Great Plains. The ﬁrst grain sorghum types to contribute to the development of grain sorghums in the US were White and Brown Durra from Egypt in 1874 (Vinall et al., 1936). Additional Durra and Kaﬁr types were introduced in the late 19th century and were adopted and utilized in US production. Smith and Frederiksen (2001) provide a more detailed history of sorghum introductions in the US and the reader is referred there for more information. Early sorghums were typically tall, late, easily lodged, and had marginal yield potential. Early improvements were farmer selections resulting either from random outcrossing, or genetic mutations in their production ﬁelds. These unique types were selected and increased by the producer and eventually adopted by other farmers as well. Some of the signiﬁcant shifts included a shift from tall cultivars to shorter cultivars through selection of natural mutation of dwarﬁng genes, a shift to earlier maturing cultivars and a shift from recurved to straight peduncle cultivars (Smith and Frederiksen, 2001). The discovery of genetic inheritance and the development of plant-breeding principles in the early 20th century were rapidly adopted by sorghum breeders and utilized to address critical issues in sorghum breeding. The ﬁrst sorghum cultivars produced from intentional crosses were “Chiltex” and “Premo,” which were developed from crosses of Feterita/Kaﬁr and released in 1923 by Vinall and Cron (Quinby and Martin, 1954). From that point onward, breeding programs were responsible for improvements in plant height, maturity, disease resistance and yield by making intentional crosses and selecting pure-line cultivars from progeny. Pure-line cultivars of sorghum were used until an economically feasible method of producing hybrid seed was developed. Interest in hybrid sorghums was increased by the development of hybrid corn and its acceptance by corn producers. Stephens and Quinby (1952) documented the enhanced yield potential, reduced maturity, and stability of yield of sorghum hybrids produced by emasculation. Even though researchers knew of this advantage, there was no economical way to produce hybrid seed in quantities suitable for production. This problem was solved with the discovery of cytoplasmic male sterility systems in sorghum (Stephens and Holland, 1954). The system was rapidly incorporated and adopted, initially using standard

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cultivars as parental lines. The ﬁrst hybrid sorghums were sold in 1956 and within 4 years, hybrid sorghums were planted on over 90% of the area planted to grain sorghum. Soon after the development of grain sorghum hybrids, forage sorghum hybrids for both hay and silage were developed and adopted by producers. The rapid adoption and development of hybrid sorghum brought instant interest and development of a hybrid seed industry. With this development, sorghum-breeding programs switched from a few publicly supported breeders to numerous privately supported breeders. This increase in sorghum-breeding programs increased not only the size and commitment to research, but also the scope of research. Traits such as yield under high inputs (water, fertilizer) became important as was the protection of yield potential from both biotic and abiotic stresses. These factors formed the basis of the modern sorghum-breeding programs that are now working to improve the crop. These efforts have led to substantial improvements in yield potential, grain and forage quality and the protection of this potential through abiotic and biotic stress tolerances. Speciﬁc information on the breeding successes in these areas are described in the trait improvement section. While hybrid sorghums are commonly grown in many regions of the world, for economic or cultural reasons other regions have not adopted hybrid sorghums. Consequently, the goals and products from sorghum-breeding programs depend largely on the funding, goals, and the target area. More recent developments in biotechnology have also affected the implementation and breeding schemes used by sorghum breeders. While there are many approaches and methodologies that may be used for improvement of the crop, all phases of a sorghum breeding program begin with hybridization to create segregating populations that can be used for selection. In the case of hybrid sorghums the method also ends with hybridization to create the sorghum hybrids that are produced.

IV. MECHANISMS OF CONTROLLED POLLINATION Methods of hybridization, whether for creating populations or producing hybrid seed, provide the basis for sorghum improvement programs. Because grain sorghum is a self-pollinated species that occasionally outcrosses, special manipulations must be completed to make controlled crosses between parents. Likewise, because sorghum will outcross naturally at low frequencies, breeders must maintain purity of lines through self-pollination. Morphologically, the sorghum panicle contains spikelets (i.e., the multiﬂowered subdivisions of the inﬂorescence), usually in pairs, with one of the pair being sessile, bisexual, and fertile. The other spikelet is staminate, or sterile, and borne on a short pedicel. The fertile ﬂoret will contain a lemma and palea, two lodicules, three stamens, and an ovary with two long styles with plumose stigmas

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(Freeman, 1970; Doggett, 1988). Flowering begins within 3 days of emergence of the panicle from the boot. It starts at or near the apex and proceeds toward the bottom of the panicle, being complete in 4 – 7 days. Schertz and Dalton (1980) reported that stigmas are receptive up to 2 days before blooming and can remain receptive for up to 16 days in the absence of pollination. Anthesis usually occurs after sunrise but has been noted during the night hours, even as early as 10 p.m. (Stephens and Quinby, 1934). Viable pollen is typically shed until about noon. Based on these ﬂowering habits, sorghum breeders have developed several methods of pollination control. In fertile sorghum lines where self-pollinated seed increases are desired, bagging the plant prior to ﬂowering ensures self-pollination and eliminates cross-pollination. To facilitate crossing, several methods of crosspollination have been developed, and each may be used to meet various objectives within the breeding program. To create segregation for breeding and selection, Schertz and Dalton (1980) suggested four methods for use in preparing the female ﬂower for fertilization by the male parent: (1) hand emasculation, (2) genetic male sterility, (3) hot-water emasculation, and (4) anther dehiscence control by use of humidity. For commercial production of hybrid cultivars, CMS is used. A brief description of each method and its basis for use follows.

A. HAND EMASCULATION Flowers are emasculated the day before anthesis. Such ﬂorets occur below and within about 3 cm of opened ﬂorets in a sorghum panicle. All open spikelets are removed with scissors. Panicles and equipment should be washed to remove any pollen prior to emasculation, especially if the emasculation occurs outdoors. There is less likely to be such pollen movement in greenhouses, but such rinsing of panicles and equipment should be conducted to avoid unwanted outcrossing. All ﬂorets except those that are to be emasculated are removed, leaving only the ﬂorets that are expected to open the next day. The three anthers are coaxed out of the enclosing lemma and palea by inserting a sharpened pencil or similar pointed instrument. Care must be taken not to break the anthers, and if the anther is breached, that ﬂower should be removed and instruments rinsed to avoid contaminating the next ﬂoret. Every anther must be removed before the set of ﬂorets is “completely emasculated.” The presence of one anther will cause pollination of one or more ovaries prior to the transfer of pollen by the breeder. After the ﬂorets are emasculated, a paper bag is placed over the emasculated panicle until the ﬂorets are pollinated 1 – 2 days later. Field emasculation usually is carried out during the afternoon in an attempt to avoid stray, viable pollen from other plants.

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An experienced person can emasculate 10 –25 panicles per day (with each panicle having 10 –20 emasculated ﬂorets). If more than one cross is desired with the female parent, the panicle can be trimmed in such a manner that speciﬁc sections of ﬂorets can be emasculated and pollinated 1– 2 days later. Regardless of the number of emasculations per panicle, emasculated ﬂorets are pollinated by collecting pollen from the male parent and then dusting it on the exposed stigmas of the emasculated ﬂorets 1 –2 days after emasculation.

B. GENETIC MALE STERILITY A series of nuclear recessive male sterility genes, designated as ms1 through ms7, have been characterized in sorghum. These mutations, in the recessive condition, result in a male-sterile plant that can be used for hybridization (Rooney, 2001). Because these plants are completely male sterile, there is no need to emasculate and larger numbers of seed can be made more easily. However, the inability to produce true-breeding, uniform genetic steriles eliminates the use of genetic male sterility for hybrid seed production. Consequently, genetic male sterility has been used in sorghum-breeding programs to facilitate population improvement programs in sorghum. The use of genetic male sterility facilitates hybridization, but it also requires close management of the population during anthesis. Once improvement is completed, lines must be derived and the recessive ms alleles must be eliminated or they will produce sterile progeny in the lines in future generations. Lines segregating for genetic male sterility can be maintained by self-pollination of random panicles or bulk pollination of sterile panicles with pollen from heterozygous and male-fertile plants in the same row. To use this system, malesterile plants must be identiﬁed at tip ﬂowering. Anthers in male-sterile plants are smaller, thinner, and do not shed viable pollen. Upon identiﬁcation, the tip of the male-sterile panicle should be removed and the panicle bagged to avoid open pollination. The panicle can then be pollinated 3 –5 days later with pollen collected from the desired male parent. Hybrids from these crosses can be used for population improvement or to begin another plant-breeding scheme, such as pedigree selection for producing improved pure lines. Opportunities for the utilization of genetic male sterility are well developed as breeders have developed genetic male sterility stocks in many elite sorghum germplasms and parental lines (Pedersen and Toy, 1997).

C. HOT- WATER EMASCULATION Stephens and Quinby (1934) developed this method of emasculation to produce a larger number of F1 seed prior to the availability of cytoplasmic male

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sterility. Open ﬂorets of the selected panicle are removed and the entire panicle is enclosed in a waterproof sleeve of rubber or plastic tied securely around the peduncle. The panicle is immersed in water heated to 42 –488C for 10 min. This treatment kills the majority of the pollen grains but does not damage the ovary. The head is allowed to dry and then covered with a paper bag. Pollen from the selected male parent is dusted onto the sterilized panicle 3 –4 days after the hotwater treatment. Users of this approach should be aware that there will be a few escapes (i.e., self-pollinations). This system is simple to use in the greenhouse, but it is not commonly used today because other simpler methods such as genetic male sterility and cytoplasmic male sterility have been identiﬁed and developed.

D. CONTROL OF ANTHER DEHISCENCE CONTROL Schertz and Clark (1967) developed a method to control anther dehiscence using the humidity created from covering the panicle with a plastic bag prior to ﬂowering. This method, also known as plastic bag emasculation and/or poured crossing, is commonly used to create segregating populations for breeding and selection because it allows a breeder to make large numbers of crosses in a short amount of time. Usually done in the ﬁeld, plants selected for use as females in poured crosses have ﬂowered approximately 2.5 – 5 cm from the panicle apex. The portions of the panicle that have ﬂowered are removed (Fig. 1). The bottom ﬂorets in the panicle are also removed, so that 3 –5 cm of the panicle remains. This panicle is covered with a plastic bag and then covered with a pollinating bag to shade the panicle and reduce the temperature under the plastic bag. The bags remain on the plant for 2 – 3 days during which the panicle completes anthesis. These bags create a highly humid atmosphere in which the moisture content inhibits anther dehiscence. To complete pollination, pollen from the male parent is collected, the plastic bag is removed, the head is “rapped or jarred” to remove excess condensate and pendant anthers, and the pollination is made immediately thereafter. Because all of the anthers are not removed, a certain level of self-pollination will occur in seed from a poured cross. In most cases, the proportion of progeny that are F1 hybrids will vary based on the speciﬁc genotype used as a female parent, the fecundity of the pollen parent, and the environmental conditions during the process. To identify F1 hybrids, seed from the poured cross is planted in a progeny row in the next generation. F1 hybrid plants must be identiﬁed by the breeder on some speciﬁc phenotypic or genetic basis. This is typically accomplished by using a simply inherited phenotypic trait or heterosis that occurs between parents of disparate origin. This method should be avoided when no markers are available and plants derived from selﬁng cannot be distinguished from F1 plants.

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Figure 1 Photographic series which depicts the plastic bag emasculation process for making breeding crosses in sorghum. (A) Panicle at mid-anthesis which is ideal for setting up the cross. (B) All sections of the panicle which have already ﬂowered are removed and any excess sections which will not ﬂower in the next 2 days are removed from the lower part of the panicle. (C) The cut panicle is bagged with a plastic bag, and covered with a paper bag. (D) Two to three days later, pollen from the male parent is collected, the paper bag is removed and the female panicle is rapped to remove excess anthers. The plastic bag is removed and the panicle is pollinated immediately. (E) Seed from the cross is grown the following generation and in this row hybrids are easily detected by heterosis and seed color (F1s are red while female parent is white).

E. CYTOPLASMIC – GENETIC MALE STERILITY Unlike the methods previously described, the CMS system is not used for population development, but CMS is the mechanism that makes the production of the hybrid sorghum seed economically feasible. The CMS system relies on a set of male sterility-inducing cytoplasms that are complemented by alleles at genetic

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Table I Summary of the Different Cytoplasmic–Genetic Male Sterility Systems Identiﬁed in Sorghum bicolor L. Moench Cytoplasm Group A1

A2

A20 A3 A4

Source

Restorer source

Genetics of restoration

Simple, dominant, Numerous, including Tx430, sporophytic Tx7078 IS12662C Numerous, Simple, dominant, including IS2801C, sporophytic Tx430 IS3063C, IS2801C Simple, dominant, IS1056C sporophytic IS1112C, IS1112C Simple, two genes, IS12565C gametophytic IS7920C IS2801C Unknown

Milo

Reference Worstell et al. (1984) and Klein et al. (2001b) Worstell et al. (1984)

Worstell et al. (1984) Worstell et al. (1984) and Tang and Pring (2003) Worstell et al. (1984)

loci in the nuclear genome that either restore fertility or maintain sterility. There are many different CMS systems documented in sorghum, each caused by a different mutation in the cytoplasm and each is complemented by different nuclear restoration loci (Table I). For most CMS systems the interaction of cytoplasmic and nuclear genes deﬁnes whether all speciﬁc lines are fertile or sterile. In the CMS system, lines that have [A] cytoplasm must have a dominant allele present in the nuclear genome to restore male fertility (Table II). If the line lacks the dominant allele for fertility restoration, the plant will be male sterile. The genetic factors for each CMS system are inherited independently of each other. It is possible for a single line to restore more than one CMS system. The frequency of lines capable of restoring fertility varies among with speciﬁc race and method of restoration. The most commonly used CMS system is the A1 system. This was the original CMS system identiﬁed and characterized by Table II Genotypes and Corresponding Phenotypes for A-, B-, and R-Lines in the A1 Cytoplasmic–Genetic Male Sterility System in Sorghum Line

Cytoplasma

Genotypeb

Phenotype

A-line B-line R-line Hybrid

[A] [N] [A] or [N] [A]

rf rf rf rf RF RF RF rf

Male sterile Male fertile Male fertile Male fertile

a b

Cytoplasm types: [A], sterility-inducing cytoplasm type; [N], normal cytoplasm. Genotype: RF, dominant allele for fertility restoration; rf, recessive allele for fertility restoration.

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Stephens and Holland (1954) and further characterized by Maunder and Pickett (1959). The vast majority of commercial hybrid seed production uses A1 cytoplasm. Of the remaining systems, only a limited amount of hybrid seed has been produced using the A2 system. Hybrid seed production requires maintenance of the A-, B-, and R-lines (Fig. 2). Seed of a male-sterile A-line is increased by pollination using the complementary B-line. The sole purpose of the B-line (also known as a maintainer) is to perpetuate or maintain the A-line. The A-line and B-line are

Figure 2 Schematic of the sorghum hybrid seed production process utilizing cytoplasmic– genetic male sterility. The A-line parent is increased utilizing pollen from the B-line. The F1 hybrid is produced by pollinating the A-line with an R-line pollinator. That seed is sold to producers for grain production. Both the B-line and R-line are maintained through self-pollination. The inset picture is of a hybrid seed production ﬁeld in which the red parent is the female and the white parent is the pollinator.

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isocytoplasmic, meaning that these two lines are genetically identical except that the A-line has a sterility-inducing cytoplasm while the B-line has normal fertile cytoplasm. Thus, A-line plants that are male sterile can be pollinated with pollen from B-line plants to regenerate seed of the A-line which is 100% male sterile. To produce hybrid seed, the male-sterile A-line is pollinated with pollen from the male-fertile R-line plants. The R-line (also known as a restorer line) is genetically very different than the A-line and it carries the dominant fertility restoration alleles needed to restore fertility in the progeny of the A-line. The seed that is produced on the A-line from this pollination is the seed that is planted by the producer for commercial grain production. In the early stages of hybrid development, these crosses usually are made using pollination bags and hand transfer of pollen, and fertile lines are maintained by bagging to ensure self-pollination (Fig. 3). Upon the commercialization of a hybrid, seed increases are made in environments that are conducive to seed production and quality. Typically, 12– 18 rows of the male-sterile A-line are planted with 2 –6 rows of the pollinator, R-line, interspersed between sets of A-line rows (Fig. 2). The exact ratios of female to male rows vary depending on the company and producer preferences. The male rows may be harvested early or they may be cut down to eliminate contamination prior to harvest of the crop. In addition, the rows are typically rouged several times to eliminate off-type plants in the seed increase. At maturity, the seed on the A-line is harvested, cleaned, treated, and bagged for commercial sale to producers.

V. IMPROVEMENT METHODOLOGY As mentioned previously, efforts to improve sorghum have led to signiﬁcant changes in the types of sorghum that are currently grown. In the early 20th century, sorghum improvement switched from farmer selection to trained plant breeders. Until 1956, sorghum breeders selected and developed pure-line cultivars that were grown by producers. Therefore, sorghum breeders followed the breeding procedures developed for self-pollinated crops. After 20 years of development, hybrid sorghums were ﬁrst marketed in 1956 and adoption of hybrids in the US was nearly 100% only 5 years later. In most of the developed world, hybrid sorghums comprise the vast majority of production and in these regions breeders switched their emphasis to hybrid development. In this situation, many of the techniques developed for corn breeding were now applicable to sorghum hybrid-breeding programs. Alternatively, in many of the less developed regions of the world, sorghum producers still rely on pure-line cultivars. Therefore, sorghum breeders must use methodology appropriate for the development of either pure lines or hybrids.

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Figure 3 Self-pollinated (A) and experimental testcross hybrid (B) increases of sorghum in a breeding program. Self-pollination ensures the purity of the parental lines as some level of outcrossing occurs naturally. In the development of experimental testcross hybrids, pollen is moved from the R-line row directly onto the A-line row to the right. Testcrosses are identiﬁed by the striped bags.

Because of the variation in types of sorghums grown throughout the world, numerous breeding procedures have been developed and adopted by sorghum breeders. Since sorghum is a self-pollinated species, most of the breeding methodologies (both cultivar and hybrid) are based on the production of segregating populations which is followed by selection in segregating populations. The selections are usually allowed to self-pollinate during selection to produce homozygous uniform lines. Where pure-line cultivars are grown, the

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potential of these new lines is evaluated, while in hybrid-breeding programs these lines will be testcrossed to measure their value as a parental line. Population improvement program efforts are also being undertaken in a few sorghumbreeding programs. This type of approach is facilitated by access to usable sources of genetic male sterility. The commonly employed methodologies for sorghum improvement are described in more detail.

A. POPULATION IMPROVEMENT The goal of most population improvement programs is to accumulate favorable alleles for the traits of interest while maintaining as much genetic diversity as possible. These recurrent selection methods usually require signiﬁcant amounts of hybridization. In the past, there was relatively little effort in population improvement in sorghum due to the fact that sorghum is a self-pollinated species. The integration of genetic male sterility into adapted germplasm has facilitated their adoption into some sorghum-breeding programs and for speciﬁc applications. While population improvement programs are not the dominant method of sorghum breeding, they are useful as a source of genetic variation and improved traits. In a population improvement program, genetic male sterility eliminates the need for emasculation. The most commonly used genetic male sterile is ms3 (Rattunde et al., 1997). The method of selection in population improvement programs in sorghum ranges from mass selection to family-based selection. In mass selection, breeders select individual plants expressing the trait of interest and then all the seed from the selected plants are bulked. To maintain segregation and recombination in the next cycle, sorghum breeders must hybridize selections or select from male-sterile plants. In family-based population improvement programs, families are created and then these families are evaluated in replicated testing to identify the most suitable parental genotype. The breeder uses this information to select the parental lines used to create that family. Like mass selection, the breeder must intermate the selections using controlled pollination or genetic male sterility to produce the populations for the next cycle. Various family types are evaluated ranging from half-sib, full-sib or S1 families (Hallauer and Miranda, 1981). Sorghum-breeding programs have used population improvement for a wide variety of traits including drought tolerance, increased yield, wide adaptation, improved quality, and pest resistance. Signiﬁcant improvements in yield have been reported using this methodology, but the transfer of the gains made in population improvement programs to hybrids has been difﬁcult (Rattunde et al., 1997). Typically, germplasm from population improvement programs must be self-pollinated to produce inbred and uniform lines that are acceptable for hybrid production. If genetic male sterility was used in the population improvement

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program, then the breeder must take care to ensure that the recessive alleles that cause male sterility are eliminated from the derived lines. Once uniform, all materials from a population improvement program must pass through an inbred and pure-line development program to be used in commercial production.

B. CULTIVAR AND INBRED LINE DEVELOPMENT Regardless of the breeding goals, sorghum-breeding programs, must at some point, produce inbred or pure lines. The speciﬁc approaches vary and may include breeding methods such as pedigree, bulk, and single-seed descent, but they all allow for self-pollination. In general, speciﬁc crosses are made using the methodology described previously between lines for the speciﬁc improvement of certain traits. F1 progeny are self-pollinated to produce an F2 population. From the F2 generation until uniform lines (usually between the F4 and F6 generation) are produced, sorghum breeders use various methods of selection to improve the agronomic, disease, and stress characteristics of these lines (Fig. 4). Each program has standards for evaluating these lines for speciﬁc characteristics at deﬁned generations. The appropriate generation for the selection of speciﬁc traits is dependent on the heritability of the trait and the environments in which the sorghum breeder is selecting. In general, traits with higher heritability (maturity, height, grain color, etc.) are selected in the early generations while traits with lower heritability are selected in more advanced generation (yield, drought tolerance, disease, and insect resistance). These more complexly inherited traits must also be screened in speciﬁc environments, where they may or may not be expressed in any given year. As the lines approach phenotypic uniformity, pureline breeding programs begin replicated evaluation and agronomic testing. In hybrid programs, the new lines are testcrossed to conﬁrm whether the line restores or maintains fertility to measure their general combining ability and suitability as a parent in hybrids.

C. HYBRID DEVELOPMENT Long before sorghum hybrids were a reality, sorghum breeders were aware of the potential yield increases offered in hybrids. Stephens and Quinby (1952) documented the yield advantages of hybrid sorghums using hybrids produced by hot-water emasculation techniques. The limiting factor to hybrid production was the lack of an economically feasible method of producing hybrid seed. The development of the CMS system eliminated this problem and sorghum hybrids were adopted immediately upon their commercial release in the late 1950s. Quinby et al. (1958) reported yield increases of 58 and 22% over the best parent under dryland and irrigated conditions, respectively. In a study of 391 locations in four countries, Doggett (1969) found that hybrid yields were

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Figure 4 Pedigree breeding scheme used by the Texas Agricultural Experiment Station sorghumbreeding program at College Station, Texas. This scheme is used for the development of new B- and R-line germplasm. Initial crosses are made using either plastic bag crosses or hand emasculations. Open-pollinated selections are made in each generation until the F5 where the plot is self-pollinated and used to make testcross hybrids. At the F5 generation, new B-lines enter sterilization and testcrossing while new R-lines are evaluated in testcrosses.

consistently higher than that of the best parent and the advantages of hybrids were accentuated in dryland environments. Sorghum breeders have been able to improve the productivity of hybrids as well. Miller and Kebede (1984) reported a 40% yield increase in new hybrids over the original sorghum hybrids of the late 1950s. While hybrid productivity was increasing, it is critical to note that the productivity of the inbred parents increased as well (Miller and Kebede, 1984). These trends clearly indicate the importance of both additive and dominant gene action in sorghum.

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Most hybrid sorghum-breeding programs divide their inbred development program into two distinct groups. One group is devoted to the development of female inbred lines (A/B-lines) and the second is devoted to the development of male inbred lines (R-lines). The two groups of inbred lines are developed using the methodologies described above and they are kept separate to maximize heterosis between parents of the two groups. While heterotic groups are not as well deﬁned in sorghum as they are in corn, sorghum breeders have typically used males (R-lines) and females (A/B-lines) as sorghum heterotic groups. In sorghum hybrid evaluation, testcrossing is critical to identify those parental lines which will produce the highest yielding hybrids. Identifying the best parental lines for hybrids based on line per se performance is simply not efﬁcient. Moran (2003) reported a correlation of r ¼ 0:19 between lines and hybrid of a recombinant inbred line population. Sanchez-Gomez (2002) and Aydin (2003) found similar results. While all of these correlations were statistically signiﬁcant, the correlations are too low to be of any practical value. Therefore, testcross evaluation is crucial for effective selection of parental lines. While it varies among programs, each program has a standard generation in which testcrossing is initiated. Most programs begin testcrossing in the F4 generation, but some begin as early as the F3 or as late as the F6 generation. Whenever testcrossing begins, it follows a standard pattern; new R-lines are testcrossed to a standard A-line tester to measure their general combining ability and their ability to restore fertility to the hybrids. Lines that produce high yielding hybrids with appropriate agronomic parameters are advanced for additional testing. These R-lines are hybridized to several potential A-lines to identify hybrids with speciﬁc combining ability. The breeder is looking for the precise combination that will produce a hybrid with high yield, good stability, good agronomic characteristics (height, maturity, etc.), and acceptable abiotic and biotic stress tolerances. Prior to testcrossing, the new B-lines that are derived from the breeding program must be male-sterilized. In most sorghum-breeding programs, new B-lines are sterilized via a backcrossing program in which a standard A-line with a similar pedigree to the new B-line is used as the source of the male-sterile cytoplasm (Fig. 5). The B-line is then used as a recurrent parent to produce an A-line that is isocytoplasmic to the B-line. In each generation of backcrossing the breeder selects plants and progenies that are fully male sterile and are the most similar to the B-line. The sterilization process usually requires a minimum of ﬁve backcrosses and most sorghum-breeding programs utilize winter nurseries to reduce the amount of time required for sterilization. Because sterilization of B-lines is lengthy process and relatively few new A/Blines will ever be used in the production of new hybrids, several plant-breeding programs have developed methods to test the general combining ability of new B-lines prior to sterilization. Most of these methods use A3 cytoplasm malesterile versions of commonly used R-lines for commercial hybrid production

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Figure 5 The advanced testing and sterilization scheme used by the Texas Agricultural Experiment Station sorghum-breeding program at College Station, Texas. From the F5 generation (Fig. 4), testcross hybrids and BC0 sterilization lines are grown for evaluation and continued backcrossing. If testcross performance is acceptable, sterilization is continued through backcrossing until the A-line is identical in phenotype to the B-line. As the A-line becomes available, additional hybrid evaluation is performed to conﬁrm heterosis and line acceptability (similar to the R-line testcross evaluation).

(Lee et al., 1992). Several other programs make preliminary testcrosses midway through the sterilization process to eliminate the least desirable A/B-lines. Whether testing new B- or R-lines, the choice of testers is critical for the correct identiﬁcation of the best new parental lines. During initial hybrid testing, a limited number of testers can be hybridized to each new line. Consequently, the speciﬁc testers used must be carefully selected to identify the best new parental lines correctly and efﬁciently. While poor performing genotypes often make the best testers, most sorghum-breeding programs use elite testers that are applied across all new parental lines. In most cases, at least two testcross hybrids are made to determine the general combining ability of the experimental line. Based on this evaluation, advanced lines will be hybridized with additional parental

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lines to identify speciﬁc combinations that are particularly productive and have commercial potential. Equally important when evaluating potential parental lines is their performance with regard to seed production. Seed producers must be able to consistently coordinate ﬂowering of the A-line and the R-line. If the two lines have a poor “nick,” the A-line will have extremely low seed set and, consequently, poor yields of hybrid seed. The pollinator line should start shedding pollen prior to the emergence of stigmas in the A-line and the R-line must continue to shed pollen throughout the ﬂowering of the A-line. In addition, the pollen shed from the R-line should be consistent and relatively unaffected by normal environmental conditions. Obviously, the R-line must consistently restore fertility to the hybrid. The A-line parent must produce seed yields high enough to justify seed production costs. The stigmas of the A-line parents must be receptive to wind-blown pollen and the duration of stigma receptivity should be long enough to minimize minor nicking problems. A-lines that have low tillering capacity or lines in which tillers mature at the same time the main panicle matures are desirable. In addition, the A-lines with larger seed size, strong seedling vigor, and good panicle exsertion ensure high seed quality, vigor and easier harvesting of the seed crop. The hybrid testing program is used to evaluate a signiﬁcant number of new hybrids annually. The program must also be efﬁcient at progressively eliminating any undesirable hybrids so that a select few hybrids with superior agronomic characteristics will be advanced to the ﬁnal stages of testing and possibly, commercial release. The initial phases of hybrid testing are limited to a small number of environments with limited replication due to the large number of experimental hybrids that are being evaluated. As hybrids advance through the testing phases, the number of environments and the testing becomes more precise. These tests are conducted in a wide range of environments that allow the breeder to identify the adaptation range of each hybrid. In addition, the hybrids are evaluated for their reaction to diseases, pests, and abiotic stresses such as drought and fertility. By the time a hybrid is considered for release, it is being evaluated in over 30 replicated tests annually. It is also grown in “strip tests” which are long, multirow plots in ﬁeld that allow producers to evaluate potential new releases. Experimental hybrids are compared to the performance of currently available check hybrids. In addition, seed production personnel evaluate the inbred to ensure that adequate quantities of seed can be produced. All of this information is critically reviewed prior to the commercial release of the hybrid.

D. USE OF EXOTIC GERMPLASM— SORGHUM CONVERSION S. bicolor is an incredibly diverse species with variation for most every trait of importance to sorghum-breeding programs. To regions of the world where sorghum is an introduced crop, this genetic variation is especially important in

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providing the genetics for further improvement and adaptation of the crop. Duncan et al. (1991a) estimated that almost every sorghum hybrid currently grown in the US has some portion of its genome derived from the sorghum accessions introduced into breeding programs in the past 20 years. Because of its importance, extensive efforts have been made to collect and preserve the diversity present within the species. Two major sorghum germplasm collections exist today. The International Center for Research in the Semi-Arid Tropics (ICRISAT) in India holds over 30,000 accessions. The second collection is maintained by the USDA and they hold over 40,000 accessions. These resources represent collection efforts that began in the early 20th century and still continue. Passport information on entries in both collections is accessible on-line and in some cases additional data pertaining to any unique characteristics that a speciﬁc line may possess is available. One limitation to the utilization of most of the accessions in either collection is the presence of photoperiod sensitivity. Most sorghum accessions are photoperiod sensitive because of the selective advantages photoperiod sensitivity provided to the crop as it was grown in its center of diversity. In a recent growout of almost 30,000 accessions from USDA collection, over 25,000 were photoperiod sensitive. Photoperiod sensitivity limits the use of germplasm in temperate sorghum-breeding programs because photoperiod-sensitive sorghums will not reach anthesis until it is late in the growing season, if at all. The sorghum conversion program was developed in the late 1950s to produce germplasm not limited by photoperiod sensitivity (Stephens et al., 1967). It evolved into a cooperative effort among research personnel in the Texas Agricultural Experiment Stations at Lubbock and Chilicothe, Texas and the USDA-ARS in Mayaguez, Puerto Rico. The purpose of this program is to convert tall and late photoperiod-sensitive sorghum genotypes to short and early photoperiod insensitive sorghum. The goal of this project is to make germplasm with valuable genetic diversity available for use in all sorghumbreeding programs. Photoperiod-sensitive sorghum genotypes selected for conversion are used as recurrent parents in a backcrossing program in which an extremely short and early parent is used as a donor parent of short and early alleles (Fig. 6). The crosses and F1s are made and grown in Puerto Rico. Segregating populations from this cross are then grown in Chilicothe, Texas where short and early segregants are self-pollinated and selected. These selections are then backcrossed to the exotic parent and the process is repeated until the “converted” lines are identical to the exotic line for all traits except height and maturity. This typically requires ﬁve backcrosses. This program has resulted in two different sets of germplasm that have been useful in sorghum improvement programs. First, partially converted bulks are released for use in breeding programs. The term partially converted indicates that

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Figure 6 (a) Schematic of the approach used in the sorghum conversion program to convert exotic sorghum accessions that are late and tall in temperate environments to exotic sorghum accessions that are short and early in temperate environment. (b) An example of the “converted” line (IS12553C) and the original exotic (IS12553) when grown in College Station, Texas. (Fig. 6A is adapted and modiﬁed from Johnson et al., 1971.)

this material is in the early stages of backcrossing. It is still segregating for various traits, but it is all short and early. In addition, it maintains a higher frequency of the adapted donor parent germplasm which makes transition of the germplasm into applied programs more efﬁcient. Second, fully converted lines have been used directly in breeding programs and for research on the diversity and variation in sorghum for an array of traits ranging from disease resistance to grain quality and yield. Since the conversion program began approximately 40 years ago, over 700 photoperiod-sensitive sorghum accessions have been fully converted and released from the program (Rosenow et al., 1997a,b; Dahlberg et al., 1998). The impact of this program has been tremendous. From the program, numerous converted and partially converted lines have been used by sorghum-breeding programs to enhance a wide array of traits in modern sorghum cultivars and hybrids. The speciﬁc lines from this program that have had an impact for each speciﬁc trait are provided in the trait-based breeding sections that are provided in the next section. However, a number of lines have proven very unique and have found wide use for numerous traits (Table III). They also appear in the pedigrees of germplasm and parental lines released and used for production today. The program continues to be important to sorghum breeding and it continues today, with

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Table III Converted Sorghum Accessions Designated with an “X” Possess Resistance to Grain Mold, Downy Mildew, Head Smut and/or Anthracnose of Sorghum SC designationa SC103-11E SC120 SC137 SC155 SC166 SC277 SC325 SC414 SC423 SC425 SC575 SC630-11E SC647 SC650-11E SC689 SC719-11E SC748-5

IS designation

Grain moldb

IS2403C IS2816C IS12628C IS12646C IS12657C IS7377C IS2462C IS2508C IS2579C IS3579C IS3553C IS1269C IS2395C IS2856C IS2729C IS7013C IS3552C

X

Downy mildewc

Head smutd

X X X X X X

X X X X X X X X X X X

Anthracnosee

X X X X X

X X

X

X

X

X X X

X

a SC No. is the sorghum conversion number assigned to each line entered in the TAES–USDA sorghum conversion program. b Sorghums with grain mold resistance had grain mold ratings that averaged less than 2.0 [on a scale of 1 (resistant) to 5.0 (susceptible)] over multiple environments. c Sorghums had less than 1% systemic infection and no local lesion infection caused by pathotypes 1 and 3 of P. sorghi (Weston and Uppal) C. G. Shaw over several environments. d Sorghums had less than a 5.0 mean percent systemic head smut infection caused by Sporisorium reilianum (Kuhn) Langdon and Fullerton over several environments. e Sorghums with anthracnose disease rating of less than 1.2 are included in this listing. The rating scale is 1.0 (no disease) to 5.0 (plant death).

attempts to integrate molecular genetic tools into the process to make it more efﬁcient.

VI. TRAIT-BASED BREEDING EFFORTS While the relative importance of speciﬁc traits varies from region to region, there are several traits that are consistently required in sorghum varieties or hybrids. First, producers demand hybrids that are adapted, high yielding, and stable in their production potential across years. Second, because the speciﬁc stress may vary from year to year, producers require tolerance to abiotic and biotic stresses that may occur in their production region. Genetic tolerance to these stresses provides a defensive mechanism to protect yield potential. They are

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the basis for consistent production. Finally, producers and consumers require a certain quality of grain, which varies widely depending on the use of the grain and environment in which it is grown. To address these issues, breeders have used germplasm in the sorghum collections. In the temperate sorghum regions, it has been germplasm derived from the conversion program that has been extremely useful in improving the crop. The speciﬁc genetic mechanisms and inheritance are contingent on the trait under study, the environment in which it is grown and the genotype providing the trait of interest. In this section, a summary of the useful germplasm and improvements in sorghum are highlighted for each trait of signiﬁcant importance in breeding programs.

A. YIELD AND ADAPTATION In every sorghum improvement program for hybrids, yield and adaptation are the primary objective for improvement. Producers purchase grain sorghum hybrids with high yield potential that is stable across variable environments. However, genotypes that meet these criteria are difﬁcult, if not impossible to identify. Therefore, breeders have focused on the development of hybrids for speciﬁc adaptation regimes. One way to deﬁne these regimes is by the relative maturity of the hybrid. The relationship between yield, maturity, and height is critical to understand prior to deﬁning the types of hybrids and their adaptation. The relationship between maturity and grain yield is dependent on the type of environment in which the hybrids are grown (Fig. 7). If the environment is not limited by stress, then grain yield is strongly correlated with increased maturity. However, if stresses such as drought are encountered during the growing season, the positive relationship between yield and maturity is eliminated and in most cases earlier maturity hybrids will outyield later hybrids. This switch is primarily due to the ability of earlier maturity hybrids to escape the effects of lack of water later in the season. Finally, if the growing season is limited by temperature, late or full season hybrids will not be able to reach physiological maturity and yields are signiﬁcantly reduced. For these reasons, hybrids are classiﬁed by maturity into three primary classes; early, mid-season and full season hybrids. The range in maturity between early and full season hybrids is approximately 2 weeks. Most variation in the maturity of these groups is due to variation in the days from emergence to anthesis because the number of days from anthesis to physiological maturity is not as variable in sorghum hybrid germplasm (Barten et al., 1999). Several studies are underway to identify variation for grain ﬁll duration as a mechanism to increase adaptation and yield and the initial results are promising, but additional research is needed to incorporate this work into the breeding material (Kofoid et al., 1999).

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Figure 7 Scatter plot depicting the relationship between plant height and grain yield and days to mid-anthesis and grain yield in sorghum. In each ﬁgure, data points are entry means from the combined analysis of a replicated evaluation of commercial and experimental hybrids grown in ﬁve environments in both 2000 and 2001. The environments varied widely in their production practices (both irrigated and dryland) and growing environments.

Early maturity hybrids are speciﬁcally targeted at dryland environments where drought stress and/or short growing seasons are consistently encountered. These hybrids have lower but stable yield potential. Mid-season hybrids are the largest group of hybrids and they are predominant in sorghum-growing regions where additional moisture is expected or available. These hybrids have a higher yield potential and can respond if moisture is available. Finally, full season hybrids are grown in environments where moisture is not limiting or irrigation is available.

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These hybrids have very high yield potential, but if stress is encountered yields will be signiﬁcantly reduced. While variation in height is encountered in each maturity classiﬁcation, later maturity hybrids are typically taller. A second consideration in adaptation is the tropical or sub-tropical adaptation versus temperate adaptation. Certain parental lines and hybrids are better adapted to more temperate environments while others are more suitable for tropical climates. While some of this is due to differences in disease and pest reaction, there is a deﬁnite response to the temperature and daylength observed in temperate and tropical environments. Therefore, breeders must utilize and develop hybrids suitable for speciﬁc environment. In rare cases, hybrids have been identiﬁed that have adaptation across both types of environments. The relationship between plant height and grain yield is much more consistent. In most every type of environment, there is a positive relationship between plant height and grain yield (Fig. 7). In favorable environments, this relationship is strong and consistent. In stress environments, relationship remains positive, but not as consistent, primarily because in severe stress environments taller hybrids are more prone to lodging. While the relationship between height and yield is solid, most producers expect hybrids of moderate height because of their concerns regarding lodging. The development and adoption of hybrids led to rapid increases in productivity, based primarily on capturing heterosis in a hybrid combination. After the rapid adoption of hybrids, genetic improvements in yield continued to be made. The introduction of new germplasm was the basis for improved yield potential in both parental lines and hybrids. Between 1960 and 1980, Miller and Kebede (1984) estimated that genetic gains in yield averaged 1 – 2% annually. Today, yield has improved but the rate of improvement is slowing primarily due to a shift in production environments and a reduced number of breeding programs devoted to sorghum improvements. Further improvements will rely on the utilization of new technology to more effectively identify sources of enhanced yield performance and incorporation of these genetics into elite sorghum germplasm. Mapping of genetic loci for grain yield and its components should provide a basis for future improvements in yield and productivity. In recent years, several QTL maps for grain yield have been completed and are available for use in sorghum breeding and enhancement (Tuinstra et al., 1997; Sanchez-Gomez, 2002; Moran, 2003). In addition, Wooten (2001) identiﬁed favorable yield QTL derived from S. propinquum, indicating that favorable alleles for yield exist in low yielding exotic germplasm. Efforts to introgress these alleles into elite sorghum germplasm are currently underway. Recovering favorable alleles from exotic germplasm may prove to enhance yield through these QTLs per se and through increased heterosis. Even though sorghum has been grown as a hybrid crop for over 40 years, heterotic groups have never been clearly deﬁned. Because of the requirement to classify germplasm as either a maintainer or restorer of fertility to A1 cytoplasm,

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these two groups became, by default, the standard heterotic group even though there was no sound scientiﬁc justiﬁcation for it. Sorghum breeders have known that lines within these groups show different levels of heterosis. While this information is extremely important, previous attempts to deﬁne heterotic groups have had little to no success. Gilbert (1994) used RFLP analysis in combination with testcross hybrid evaluation to identify heterotic groups, but the limited coverage of the genome provided by RFLPs precluded any clear patterns. Menz et al. (in press) completed an extensive screening of the sorghum genome using SSR and AFLP markers and using cluster analysis, determined that elite sorghum genotypes were grouped based on phenotypic classiﬁcation and fertility restoration status. Efforts to conﬁrm this hypothesis in ﬁeld-testing are currently underway. If these groups are legitimate, they will be extremely useful in the selection of parents for breeding populations as well as the selection of appropriate testers for combining ability analysis. Jordan et al. (2003) used 113 RFLP markers to classify 70 inbred lines and then determine if heterosis could be predicted based on genetic distance. While a correlation between hybrid yield and genetic distance was detected, it was too low to be of practical value. However, the use of speciﬁc regions of the genome associated with heterosis was more predictive and may be useful in identifying heterotic hybrids.

B. BIOTIC STRESS Sorghum serves as a host to over 100 pathogen and insect pests, some of which cause signiﬁcant economic impact on an annual basis. While cultural and chemical control may be used in certain speciﬁc environments, breeding for disease and insect resistance is the only consistent mechanism that can be utilized throughout the world. In addition, genetic resistance is often the only economically feasible mechanism of controlling these biotic stresses. While it is not feasible to discuss all diseases and pests that affect sorghum production, breeding approaches for major diseases and pests are described in this section. 1. Diseases Diseases, either individually or in combination, cause signiﬁcant amounts of economic loss on an annual basis (Thakur et al., 1997). However, not all of the diseases described by Thakur et al. (1997) are emphasized in sorghum disease resistance breeding. For a breeding program to address the biotic stress of interest, it must have heritable sources of resistance, an effective mechanism and environment for screening, and an effective plan to produce resistant germplasm of sufﬁcient performance to justify its development. The economically important diseases that affect sorghum can be roughly divided by the tissue that they infect. Panicle diseases such as grain mold and

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head smut infect the panicle and/or grain, thus reducing yield and quality. Foliar diseases such as sooty stripe infect the leaves and leaf sheaths. These diseases are especially damaging in forage sorghums. Stalk diseases such as charcoal rot destroy the culm and roots, resulting in lodging, which can drastically reduce yield in a very short time period. Finally, there are some diseases such as sorghum downy mildew which infect the whole plant. Breeding for disease resistance depends on the economic importance of the disease, the availability of genetic resistance and its inheritance and effective screening mechanisms for the disease. The economic importance of diseases varies from location to location but several diseases are consistently damaging and most breeding programs have an emphasis on breeding for resistance. Lodging and stalk rot resistance. Breeding resistance to stalk rots and stalk lodging is probably one of the most important disease resistance breeding efforts. Charcoal rot (caused by Macrophomena phaseolina) is common in many sorghum-growing regions while Fusarium (caused by Fusarium moniliforme sensu lato) and Anthracnose (caused by Colletotrichum graminicola) stalk rots are not as common but important in speciﬁc regions. Because charcoal rot is closely related to drought susceptibility, breeders have found that breeding for staygreen (post-ﬂowering drought tolerance) also enhances resistance to charcoal rot. Consequently, breeding for charcoal rot follows the same breeding methodology that is used for drought resistance breeding. Other forms of stalk rot are not associated with drought conditions and screening methodologies have been developed that involve pathogen inoculation and visual evaluation of infection. For all stalk lodging diseases, good sources of resistance have been identiﬁed and utilized in breeding programs (Bramel-Cox et al., 1988; Tessa et al., 2004). Bramel-Cox et al. (1988) indicated that both charcoal and Fusarium stalk rot were both complexly inherited traits with multiple gene action. Tessa et al. (2004) recently compared several accessions reported to have resistance to charcoal rot for their level of resistance to Fusarium stalk rot. Only one of the four accessions evaluated had resistance to Fusarium stalk rot. In most cases the inheritance of this resistance is quantitative, indicating a need to conduct screening and selection in an advanced generation. Anthracnose. Anthracnose, caused by the fungus C. graminicola (Ces.) Wils., is one of the most destructive diseases of sorghum in warm, humid regions. The pathogen can infect above-ground portions of the plant including stalk, foliage, panicle, and grain, and individual genotypes often differ in their reaction to foliar, stalk or panicle infection (Hess et al., 2001). Yield losses may be as high as 50% in susceptible cultivars (Harris et al., 1964), and are greatest during extended periods of cloudy, warm, humid, and wet weather, especially when it occurs in the early grain ﬁlling phase (Thakur and Mathur, 2001). Sorghum researchers from Asia, Africa, and the Americas have identiﬁed sources of resistance to many important sorghum diseases. Accessions from the ICRISAT world collection, the USDA collection in Grifﬁn, Georgia, and the

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USDA/TAES sorghum conversion program have been scored for resistance to numerous sorghum diseases (Table III). Sorghum breeders have incorporated anthracnose resistance from some of the identiﬁed resistance sources into their breeding programs, but many other sources of resistance have not been utilized. Given the highly variable nature of the anthracnose pathogen, sorghum breeders need to have access to as many different sources of resistance as possible. These different resistance sources can be used to pyramid resistance genes to make durable varieties or to respond to changes in the composition of the fungal population (Casela et al., 1992). Wiltse (1998) intercrossed 13 different sources of anthracnose resistance to establish genetic relationships between the germplasms. Segregation in the progeny of resistant £ resistant crosses indicates that different genes control anthracnose resistance in the parents. At least ﬁve different sources of resistance were present among 13 sorghum conversion lines with anthracnose resistance. More recently, Mehta et al. (2000) evaluated progeny from additional resistant £ resistant crosses and expanded the number of unique sources of anthracnose resistance to at least six. Sorghum cultivars can react differently to anthracnose in different locations around the world (Rosenow and Frederiksen, 1982). Variation within C. graminicola is well documented (Ali and Warren, 1987; Cardwell et al., 1989; Hazra et al., 1999). In addition to genetic variation in the species, Rosewich et al. (1998) found that asexual reproduction at a location may result in a predominance of one race that is only occasionally inﬂuenced by genetic drift and gene ﬂow. Regardless of the mechanism generating the variation within C. graminicola, breeders must screen potentially resistant germplasm in as many environments as possible to ensure that the resistance incorporated into their hybrids and cultivars is stable across environment and pathogen race. Rooney et al. (2002) identiﬁed several sources of resistance with consistent resistance across numerous environments. The heritability of anthracnose resistance in sorghum has been studied for over 50 years. In most cases, researchers identify resistance genes that protect against a single isolate of the pathogen, and have described several different simply inherited forms of genetic resistance to anthracnose (Rosenow, 1992; Thakur et al., 1997; Rooney et al., 2002). Mehta (2002) identiﬁed three dominant and three recessive sources of resistance. Regardless of whether resistance is dominant or recessive, the simple inheritance of these resistance genes suggests that breeding to improve anthracnose resistance will succeed, although the stability and durability of the resulting resistance will be subject to changes in the dominant pathogen race(s). Parental lines and improved sorghum germplasm with enhanced anthracnose resistance have been released (Duncan et al., 1990; Miller et al., 1992a,b). Some of these lines are used directly in commercial hybrid or cultivar production, while others are used as breeding sources, but most were developed by using traditional breeding methodology. To facilitate selection for anthracnose resistance,

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Tenkouano et al. (1993) proposed selection at the seedling stage based on phytoalexin content. More recently, markers linked to anthracnose resistance have been reported (Boora et al., 1998; Mehta, 2002). It is hoped that the availability of markers linked to these resistance sources will help in the introgression of these genes into elite sorghum germplasm. In programs where anthracnose resistance is an important breeding objective, new germplasm is evaluated in multiple environments that have consistent anthracnose infection. In some cases, trials are inoculated to increase the amount of disease pressure. Combining the anthracnose reactions from typical production environments provides the best method to identify lines and hybrids with adequate levels of anthracnose resistance. Grain mold. Grain mold of sorghum is a major problem when grain development coincides with warm, wet weather. Grain mold describes the diseased appearance of sorghum grain resulting from infection by one or more parasitic fungi (Williams and Rao, 1981). Although many species cause grain mold, the pathogens most commonly associated with the disease are F. moniliforme sensu lato (Leslie and Marasas, 2002) and Curvularia lunata (Walker) Boedjin (Castor and Frederiksen, 1980). An array of screening and selection methodologies have been developed for grain mold, and signiﬁcant information has been published on the identiﬁcation of resistance, the mechanism of resistance, the inheritance of resistance, and the potential for future improvements. Numerous sources of grain mold resistance in sorghum have been identiﬁed and described (Gray et al., 1971; Glueck and Rooney, 1976; Castor and Frederiksen, 1980; Bandyopadhyay and Mughogho, 1988). Numerous traits have been associated with increased grain mold resistance, but none of them confer complete resistance to the disease. Initially, resistant sorghums usually had a testa layer and were high in tannins; however, additional and more thorough screening revealed that many guinea-type sorghums and zerazera sorghums had high levels of grain mold resistance without a testa (Hahn and Rooney, 1986). Corneous endosperm types are more resistant to grain mold than ﬂoury endosperm types (Glueck and Rooney, 1980), and grain hardness also is associated with grain mold resistance (Jambunathan et al., 1992). Other structural-based kernel traits that have been reported to improve grain mold resistance are a thin mesocarp, the presence of surface wax, red pericarp color (due to the presence of ﬂavan-4-ols), and an open panicle structure (Glueck and Rooney, 1980; Esele et al., 1993; Menkir et al., 1996). With all of the reported associations, however, there are numerous exceptions. All of these mechanisms and the sources of resistance have been very important in the development of new germplasm with even better grain mold resistance. The biochemical basis for grain mold resistance has yet to be identiﬁed. Most cultivars with a testa resist weathering, and they produce ﬂavan-3-ols and high

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levels of other phenolic compounds that may enhance mold resistance (Waniska et al., 1992). Esele et al. (1993) found that red pericarp genotypes were more resistant to weathering than genetically similar white pericarp genotypes. Genotypes with a red pericarp typically contain higher levels of ﬂavan-4-ols, and this compound may increase grain mold resistance (Waniska et al., 1992). Seetharaman et al. (1996) identiﬁed four proteins from sorghum endosperm with antifungal activity, but they found no relationship between antifungal protein content and grain mold incidence. Rodriguez-Herrera et al. (1999) reported a positive correlation between antifungal protein content and grain mold resistance. These results imply that antifungal proteins per se do not confer grain mold resistance but that they do have an important role in grain mold resistance. Many of the traits described in the previous section are qualitatively inherited (Rooney, 2001), but their contributions to total grain mold resistance usually accounts for only a portion of the total variation for the trait. Thus, grain mold resistance commonly is considered a quantitative trait, and general and speciﬁc combining ability components of variation for grain mold resistance are highly signiﬁcant (Dabholkar and Baghel, 1980). Murty and House (1984) used generation means analysis to evaluate resistance to sorghum grain mold, and found that the F1 hybrid was more resistant to C. lunata and F. moniliforme sensu lato than the mid-parent, suggesting that grain mold resistance has dominance effects. More recently, Rodriguez-Herrera et al. (2000) conducted a generation means analysis of a Sureno £ Tx430 population for grain mold resistance in eight Texas environments, and detected additive effects in all eight environments, and dominance effects in seven of the eight. Epistatic effects were detected in only two of the eight environments, but a combined analysis indicated that higher order interactions were important when evaluated across environments. Rodriguez-Herrera et al. (2000) concluded that selection in speciﬁc environments is useful for enhancing resistance to mold in those environments, but that it may not provide grain mold resistance across a wide range of environments. Further improvements in grain mold resistance are needed to maintain sorghum yield and grain quality. Several quantitative trait loci (QTLs) that inﬂuence grain quality and grain mold incidence in West Africa (Rami et al., 1998) and Texas (Klein et al., 2001a) have been identiﬁed. In the study of Klein et al. (2001a), each QTL individually accounted for 10 –24% of the phenotypic variation, and collectively these QTLs account for approximately 45% of the phenotypic variation for grain mold. These QTLs were not consistently expressed in every environment, however, and genotype £ environment interaction is critical in grain mold resistance. These results are consistent with prior observations that breeding for stable resistance to grain mold is difﬁcult, and they support the conclusions of previous researchers that grain mold resistance mechanisms differ by environment and that these resistance mechanisms are controlled by different genetic loci.

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Although the mechanism(s) underlying grain mold resistance require more research, sorghum-breeding programs have made substantial improvements in grain mold resistance by using the currently available information. The traditional strategy of hybridizing two parents with high levels of grain mold resistance (but due to different resistance mechanisms) to produce derived lines and hybrids with higher levels of grain mold resistance has been successful. The use of overhead sprinkler systems to ensure high levels of disease pressure for screening also has made scoring grain mold resistance more reliable. Continued use of these technologies should further increase grain mold resistance. Combining molecular genetic technology with sorghum breeding will probably yield the most substantial improvements in grain mold resistance. For example, the ﬁrst association of grain mold resistance and antifungal proteins in sorghum was in a grain mold QTL mapping population (Rodriguez-Herrera et al., 1999). In an attempt to use marker-assisted selection (MAS) for grain mold resistance, Franks (2003) used the QTL detected by Klein et al. (2001a) in ﬁve different sorghum-breeding populations. All ﬁve populations used a common source of grain mold resistance. While MAS was efﬁcient in the same population in which the trait was originally mapped, it was ineffective in any of the other four populations. This indicates that additional research is required to efﬁciently identify markers that are applicable across populations. While not yet technically feasible, grain mold resistance may be best addressed using genetic transformation with transgenes which would enhance mold resistance in the caryopsis. Downy mildew. Downy mildew, caused by the fungus Peronosclerospora sorghii, is found throughout the world. This fungus infects sorghum systemically and/or through local lesions. Outbreaks of sorghum downy mildew are sporadic and depend upon inoculum load and environmental conditions. Downy mildew outbreaks have been signiﬁcantly reduced in Mexico and the United States by the use of metalaxyl seed treatments (Craig and Odvody, 1992). Resistance to downy mildew has been identiﬁed from several sources (Sifuentes and Frederiksen, 1988; Craig and Odvody, 1992; Table IV). These sources are race speciﬁc and changes in race often result in lines losing their resistance (Craig and Odvody, 1992). In the United States, the development of the metalaxyl seed treatment fungicides has minimized the incidence of sorghum downy mildew and probably reduced the overall effort in breeding for downy mildew resistance. Recently, a metalaxyl resistant biotype of downy mildew has been identiﬁed in Texas. The emergence of this chemical resistance has reemphasized the importance of breeding for genetic resistance to downy mildew. The chemical resistance biotype is classiﬁed genetically as a pathotype 3. Therefore, screening programs have continued to screen for resistance and incorporate that resistance into elite sorghum parental lines. While sources of resistance are small in number, the resistance that they possess is quite high, stable across environments and highly heritable. The key to the identiﬁcation of

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Table IV Differentials Used to Identify Speciﬁc Races of Sorghum Down Mildew (Caused by P. sorghi (Weston and Uppal) C. G. Shaw Race Pedigree

P1

P2

P3

P4

P5

QL3 (India) RTx7078 SC170-6-17 SC155 82BDM499 SC414-12E RTx2536 RTx430 CS3541

R S R R R R S R R

R S S R R R S R S

R S S R R R S S S

R U R U U R S R U

R U R U R S S S S

S, susceptible; R, resistant; U, unknown reaction.

resistant types is through the use of downy mildew nurseries in which susceptible hybrids or cultivars are grown to maintain high inoculum levels. The inheritance of downy mildew resistance in sorghum is dependent on the environment and the source of resistance used. In most cases, the inheritance of downy mildew resistance has been oligogenic and the number of genes involved varies from 1 to 6 (Bhat et al., 1982; Rana et al., 1982; Craig and Schertz, 1985; Sifuentes and Frederiksen, 1988; Reddy et al., 1992). In some cases, molecular markers have been linked to downy mildew resistance, but none of these markers have yet been placed on a map of the sorghum genome (Xu et al., 1994; Gowda et al., 1995; Oh et al., 1996). Head smut. Of the four smuts that infect sorghum, head smut (caused by Sporisorium relianum) is the most economically damaging. Because chemical control is not available, crop rotation and the deployment of genetic resistance are critical to minimize outbreaks of the disease. Races of head smut have been identiﬁed and characterized using several genotypes with speciﬁc responses to the different races (Table V). From systematic screenings for head smut, several sources of resistance to head smut have been identiﬁed, characterized, and utilized in breeding programs. Inheritance of resistance to head smut is variable, and the dominance or recessive nature of the trait depends on the source of resistance and the environment in which the resistance is evaluated. Cao et al. (1988) reported that resistance to head smut was quantitatively inherited with additive, dominant or epistatic effects. Magill et al. (1997) reported that head smut resistance in BTx635 was controlled by two genes. Efforts to map the genetic factors conditioning resistance to head smut are in progress and molecular markers linked to head smut resistance should be available in the near future.

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W. L. ROONEY Table V Differential Accessions Used to Identify Different Races of Sorghum Head Smut (Caused by S. reilianum) Race

Designation

1

2

3

4

Tx7078 Early Hegari Tx414 SC170-6-17 BTx635

S R R R R

S S R R R

S R S R R

S R S S R

S, susceptible; R, resistant.

Most sorghum-breeding programs to reduce head smut still utilize ﬁeld evaluation and select against progeny that show any level of infection. Screening for head smut is done in ﬁelds with high inoculum levels, and rows of highly susceptible checks are included in a systematic pattern as controls and to ensure suitable levels of inoculum for the following year’s evaluation. Ergot. There are no reports of sorghum germplasm with complete resistance to ergot, caused by Claviceps africana (Moran et al., 2002). Therefore, when cool and wet weather is encountered during anthesis, sorghum ergot is capable of infecting all types of sorghum. However, due to the nature of ergot infection, male-sterile lines are uniquely susceptible to signiﬁcant infection by the disease. Since hybrid sorghum seed production relies exclusively on cytoplasmic malesterile lines, the sorghum seed industry is extremely interested in any mechanism to reduce the susceptibility of sorghum to ergot. In hybrid seed production, ergot infection directly reduces yield by preventing seed production. Ergot speciﬁcally threatens male-sterile lines because the disease only infects unfertilized ovaries. Once the ovary is fertilized, the developing zygote shows resistance to the pathogens’ attempts at infection. When the ovary is infected, fungal hyphae develop into spore fungal masses. In addition to the reduction in seed yield, losses in seed quality occur when honeydew from infected ﬂorets contaminates surrounding grains. This contamination makes harvest, cleaning and the distribution of the seed difﬁcult, even impossible. Moran et al. (2002), evaluating commercially important sorghum R-, A-, and B-lines, concluded that ergot occurred in all genotypes at four different locations. In Rwanda, six resistant lines were identiﬁed (Mukuru, 1999). Musabyimana et al. (1995) identiﬁed 12 lines with greater tolerance to ergot, with disease severity below 10%. Dahlberg et al. (2001) evaluated 100 accessions from the USDA germplasm collection and found that IS8525 was the most tolerant line. IS8525 not only had the lowest ergot ranking among all lines tested

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in Isabela, Puerto Rico, but also showed some ergot tolerance in a male-sterile testcross hybrid. Reed et al. (2002) evaluated 18 genetically diverse sorghum lines, including cultivated landraces and wild accessions, as well as in potential alternate hosts, including S. halepense (L) Pers. for resistance to C. africana. They concluded that only Sorghum spp. were susceptible to ergot; however, within the sorghum germplasm pool, two wild accessions were resistant to ergot. Based on the low levels of infection observed in male-sterile hybrids produced using IS14131 and IS14257, Reed et al. (2002) concluded that tolerance expressed in these accessions appeared to be physiological and not pollen mediated. Based on the results of Dahlberg et al. (2001) and Reed et al. (2002), IS8525, IS14131, and IS14257 are the only known germplasm sources with physiological resistance to ergot in male-sterile genetic backgrounds. Mateo (2003) obtained heritability estimates for ergot tolerance ranging from 0 to 60% from IS8525 depending on the environment. These estimates dropped even more when sterile testcrosses were evaluated. The large amount of genotype £ environment variability indicates that improvement of ergot tolerance across environments will be very difﬁcult. Striga. Striga ssp. are parasitic weeds which are endemic throughout most of Africa and cause signiﬁcant economic damage to many of the most important crops grown on that continent. In many regions of Africa, Striga hermonthica is a consistent pathogen of sorghum. The problem is so severe that a multidisciplinary approach is needed for control. Cultural, biological, and genetic resistance mechanisms are recommended for the control of Striga. Butler et al. (1997) summarized the life cycle of Striga and different mechanisms of control. While genetic variation for resistance to Striga is limited, breeders have utilized this resistance to develop cultivars with improved tolerance to Striga (Ejeta et al., 1997). Inheritance of Striga resistance has been reported as both quantitative and qualitative. Several studies reported that resistance was quantitatively inherited but gene action was primarily non-additive (Obilana, 1984; Kulkarni and Shinde, 1985). Recently, Hausmann et al. (2001) reported both GCA and SCA effects were signiﬁcant in a diallel study of Striga resistance in sorghum cultivars in Africa. Alternatively, Hess and Ejeta (1992) reported that resistance in SRN39 was inherited as a recessive trait controlled by one or two genes. The apparent inconsistency in genetic inheritance has been clariﬁed with a better understanding of the mechanisms of genetic resistance. One mechanism of resistance is the identiﬁcation of genotypes that do not produce or release compounds that induce Striga germination. Once methods of assessing the production of those compounds were developed, qualitative inheritance of resistance was clearly demonstrated in several cases where resistance had appeared quantitative in previous studies (Ejeta et al., 1997).

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While several mechanisms of Striga resistance are known to exist, the best characterized and most commonly used form of resistance is the elimination of compounds known to elicit Striga germination. This method is highly effective at reducing Striga infection, and screening methods are easily completed in lab conditions and the results are much more reliable than phenotypic screening under ﬁeld conditions. This method is commonly found and has been exploited in several sorghum cultivars (Ejeta et al., 1997). However, even simply inherited traits in the laboratory are more complex in the ﬁeld, as several researchers have reported signiﬁcant genotype £ environment interactions in reaction to Striga (Hausmann et al., 2001). This interaction and quarantine restrictions in the movement of both sorghum and Striga ensure that both laboratory and ﬁeld screening methodology will be used in the improvement of Striga resistance.

2.

Insect Pests

Numerous pests and insects are capable of inﬂicting signiﬁcant economic damage to sorghum. To manage these pests, integrated pest management systems rely on an array of different control approaches. In cases where cultural control mechanisms are not always effective and/or chemical control is either not available or economically feasible, breeding for resistance to insect pests of sorghum remains an important objective. The particular insect that causes economic damage depends on the type of environment and the speciﬁc area of production. For example, the sorghum shoot ﬂy (Atherigona soccata) is a major pest of sorghum in Asia, but it is not common in other sorghum production regions. For sorghum breeders in India, signiﬁcant resources are devoted to identiﬁcation and introgression of resistance to this pest (Peterson et al., 1997). In West Africa, damage from paniclefeeding bugs causes major reductions in both yield and quality, but breeders have been effective at identifying and selecting genotypes with enhanced resistance to these two species (Henzell et al., 1997). Other insects that cause signiﬁcant damage, but are localized to speciﬁc geographic regions include yellow sugarcane aphid [(Sipha ﬂava (Forbes)], chinch bug [Blissus luecopterus (Say)], the fall armyworm [Spodoptera frugiperda (J. E. Smith)], and the corn leaf aphid [(Rhopalsiphum maidis (Fitch)]. Peterson et al. (1997) reviewed and summarized current efforts to improve the tolerance to these pests. In addition to the pests described previously, there are two particularly important and economically damaging pests, sorghum midge and greenbug, that remain the focus of many sorghum-breeding programs. Breeding efforts on these two insects are described in more detail. Sorghum midge. The sorghum midge [(Stenodiplosis sorghicola (Coliquett)] is the most damaging and prevalent insect pest of sorghum in the world. Midge

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lay eggs in the sorghum ﬂower at anthesis. The eggs hatch and the larvae consume the developing caryopsis. Combined with a short life cycle, sorghum midge populations can increase exponentially in matter of weeks. Slight infestations of sorghum midge result in signiﬁcant economic damage; heavy infestations at anthesis can result in complete loss of grain yield. Worldwide, yield losses due to sorghum midge are estimated at over $300 million annually (Henzell et al., 1997). Methods of control vary widely from region to region, but cultural, chemical, and genetic control mechanisms are all critical components in an integrated midge management plan. Field-based screening nurseries have identiﬁed several different sources of genetic resistance (Sharma, 1985; Peterson et al., 1994). Among these sources of resistance, several different mechanisms of midge resistance are documented to exist, ranging from antixenosis (non-preference) to antibiosis and tolerance. Regardless of the mechanism of resistance, these sources of resistance have been used as the basis of the midge resistance breeding programs in the United States, Australia, and ICRISAT (Henzell et al., 1997). Most studies to determine the heritability of midge resistance indicate that it is a quantitatively inherited trait with speciﬁc control contingent on the genotypes and environments which were under evaluation. Several studies indicated that midge resistance is controlled by an unknown number of partially dominant genes and that midge resistance in hybrid combinations was predominantly recessive, meaning that both parents must possess the trait for the hybrid to express it (Teetes and Johnson, 1978; Widstrom et al., 1984; Agrawal et al., 1988). Others reported that general and speciﬁc combining ability effects were important (Patil and Thombre, 1985; Agrawal et al., 1988) while Sharma et al. (1996) reported that gene action for midge resistance was primarily additive. Tao et al. (2003) conducted QTL analysis of progeny from a cross of two sources of midge resistance that used different mechanisms of resistance. They found a single QTL from ICSV745 conditioning antibiosis and several QTLs from 90562 conditioning antixenosis. Given that these studies were conducted in a wide range of environments with several different sources of resistance, it is not surprising that variation in the results was observed. Based on these results, it is best to conclude that midge resistance is heritable, but that expression of resistance is contingent on environment and speciﬁc sources of resistance that are utilized. Utilizing these sources and this information, breeding programs in the US, Australia, and ICRISAT speciﬁcally devoted to improving midge resistance have been successful at improving the level of tolerance to midge in sorghum hybrids. Henzell et al. (1994) summarized the methods of each program. In each program the methods were distinct and the objectives slightly different, but all have been successful in improving the level of resistance available in locally adapted germplasm. In all cases, screening for tolerance or resistance must be conducted under ﬁeld conditions as sorghum midge will not reproduce in greenhouse or laboratory

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environments. Consequently, screening must occur when midge levels are high enough to result in damage. In some environments such as Australia, midge pressure remains constant throughout the growing season. Therefore, evaluation under all production environments and all generations of a breeding program can be accomplished. This has allowed the DPI-Queensland program to develop several important parental lines with very good agronomic adaptability with midge resistance (Tao et al., 2003). In Texas, midge populations ﬂuctuate widely ranging from none early and increasing exponentially to excessively high pressure in late season plantings. Consequently, screening for midge resistance requires late planting and identiﬁes genotypes with a very high level of midge resistance. The shift in planting changes the environment during the development of the crop resulting in a genotype that is not as well adapted to the normal planting date as germplasm selected under normal planting dates. This paradox has made selection for both adaptation and midge resistance more challenging in the US. Nevertheless, the improvements in midge resistance in all programs have reduced (but certainly not eliminated) the effect of midge on sorghum production throughout the world. Greenbug. Greenbug [Schizaphis graminum (Rondani)] is a relatively new pest of sorghum in the Western hemisphere. The greenbug, a pest of small grain crops, caused widespread damage to sorghum in the late 1960s (Harvey and Hackerott, 1969). The original biotype to attack sorghum was biotype C and since then an additional 10 biotypes have been detected, with three of these (E, I, and K) causing damage in sorghum (Peterson et al., 1997). At the onset of the problem, sources of resistance were quickly identiﬁed and the goal of every sorghum-breeding program was to introgress that resistance into elite parental lines and hybrids. Depending on the source, resistance to biotype C was dominant or partially dominant and simply inherited (Johnson and Teetes, 1972; Weibel et al., 1972a). Because they were simply inherited and dominant, transfer of the trait to one parent resulted in the production of resistant hybrids. Resistant germplasm and hybrids were widely available and commonly grown by the mid-1970s (Peterson et al., 1994). The increase and identiﬁcation of new biotypes refocused emphasis on breeding for greenbug resistance in sorghum in the late 1970s. Biotype E was reported in 1979 and most sources of resistance to biotype C were ineffective on biotype E (Porter et al., 1982; Peterson et al., 1997). Sources of resistance to biotype E were identiﬁed and in most cases resistance was simply inherited and dominant (Johnson et al., 1981). The discovery of biotype I greenbug followed in 1990 and soon after several sources of resistance were identiﬁed (Harvey et al., 1991; Andrews et al., 1993). Resistance in these lines appeared to be controlled via epistatic interaction at two genetic loci. More recently, biotype K was identiﬁed (Harvey et al., 1997). Katsar et al. (2002) conducted an extensive study to identify genetic loci associated with greenbug resistance in sorghum. They evaluated four

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different populations segregating for resistance to biotypes C, E, I, and K, and found that at least nine loci conditioned resistance in various forms and in certain populations to one or more of the biotypes. They concluded that resistance to different biotypes of greenbugs is not necessarily a simply inherited trait, but it is a function of the allelic variations found at numerous loci. Agrama et al. (2002) found nine QTLs associated with resistance to greenbug biotypes I and K, again indicating that resistance to these biotypes is controlled by multiple loci. Most sorghum-breeding programs utilize greenhouses for screening of germplasm for greenbug resistance. Because resistance to most biotypes is highly heritable and conditions for ﬁeld evaluation can be inconsistent, screening of breeding material occurs at the seedling stage in early to mid generations of breeding. Lines are planted in ﬂats and seedlings in ﬂats are exposed to high numbers of greenbugs to identify those lines with resistance to the pest. Lines that are resistant are further evaluated in subsequent generations in both greenhouse and ﬁeld evaluation.

C. ABIOTIC STRESS The maximum recorded yield for sorghum is 21 t ha21, but the average yield worldwide is only 1.28 t ha21 (Wittwer, 1980; FAO, 2001). Even in the USA, average yields are only 3.2 t ha21 (FAO, 2001). Based on these numbers, sorghum, in a typical production environment, averages between 5 and 15% of its recorded maximum yield potential. While biotic stresses reduce yield potential in speciﬁc environments, most of the reduction in sorghum yield is attributed to abiotic stress, primarily drought stress (Kramer and Boyer, 1995). Important abiotic stresses in sorghum are broadly deﬁned into three categories: water, temperature, and nutritional. As sorghum is commonly grown in water-limited environments, drought stress is likely the single most limiting factor in sorghum production. Temperature extremes, both heat and cold stress, are encountered in many regions of the world that produce sorghum. Finally, nutritional stresses, which include major nutrients such as nitrogen, phosphorous and potassium as well as micronutrients such as aluminum and iron, are encountered in many regions of the world and can be extremely severe in certain environments. 1.

Drought Stress

Even though sorghum possesses excellent drought resistance compared to most other crops, drought stress is the primary factor that reduces sorghum production worldwide (Rosenow et al., 1997c). The crop is commonly grown in regions of the world where water is limiting and, therefore, the crop commonly experiences periods of extreme water stress at some point within the growing

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season. Sorghum improvement programs have long realized that enhancing the drought tolerance of sorghum would improve and stabilize yield and productivity of the crop. Because genotypes respond differently to different types of drought stress, several general types of drought resistance mechanisms in sorghum must be considered. Early research in sorghum indicated that the most effective way to reduce loss due to water stress was through the use of early maturing genotypes to avoid late season water stress (Blum, 1979). While technically not a drought resistance mechanism, sorghum production and its growth as a crop in the Midwest US was based on the development of early maturing genotypes that avoided late season drought stress (Smith and Frederiksen, 2001). In many regions of the world, the use of speciﬁc maturity types to utilize seasonal rainfall is still a common practice and an important mechanism for controlling losses due to water stress. While drought escape is a desirable method of controlling losses due to water stress, it is not a feasible method in many areas of the world because of inconsistent weather patterns or the fact that unacceptable yield potential may be lost to avoid drought stress (Dalton, 1967). In these situations, the plant must have the morphological or genetic capability to tolerate the water stress. A signiﬁcant effort to identify these characteristics, their expression and their genetic control has been undertaken so that the drought tolerance of the crop is further improved (Blum, 1979; Howarth et al., 1997; Rosenow et al., 1997a –c). Drought stress response in sorghum is dependent on the stage of growth in which the drought stress occurs. Pauli et al. (1964) divided sorghum growth into three stages. Growth stage 1 (GS1) is the vegetative stage that begins with germination and ends at panicle differentiation. Growth stage 2 (GS2) is the preﬂowering or reproductive phase of growth ranging from panicle differentiation until the cessation of anthesis. Growth stage 3 (GS3) is the post-ﬂowering or grain ﬁll phase that begins immediately after anthesis and continues until physiological maturity of the grain. This division of growth stages is particularly useful in classifying drought reaction, as in each stage the drought resistance reaction is controlled by different genetic mechanisms (Rosenow et al., 1997a –c). Drought stress tolerance in GS1 is an important trait especially in the harsher production environments and the interaction between genotype and environment begins at planting with the germination process. Sorghum germination is inﬂuenced by the amount of available soil water and the genotype of the seedling and the environment in which the seed was produced (Evans and Stickler, 1961; Howarth et al., 1997). In most production environments, soil moisture levels are suitable to initiate germination (Soman, 1990). When germination and emergence are reduced, seed quality, which is determined by the seed production environment, and storage conditions and soil temperature during germination are the important factors (Mortlock and Vanderlip, 1989).

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There have been relatively few reports on variation within sorghum for seedling drought tolerance. Smith et al. (1989) indicated that differences in germination and emergence among genotypes were observed at different levels of soil water stress. Gurmu and Naylor (1991) also detected differences in germination and emergence between two sorghum cultivars at differing soil water stress levels. Wenzel (1991) reported that additive effects controlled variation for seedling drought tolerance and that the trait was highly heritable. However, the relative magnitude of this effect was minimal compared to the variation observed for soil temperature effects. Signiﬁcant differences among hybrid genotypes for seedling survival have not been reported in the US (Rosenow et al., 1997a – c). For these reasons, research to improve germination and seedling emergence has focused on tolerance to temperature extremes. In later stages of growth, two distinct types of water stress reaction have been deﬁned and characterized. Both reactions are based on growth stage and have distinct and different phenotypic expressions (Rosenow and Clark, 1981; Rosenow et al., 1983; Fig. 8). The pre-ﬂowering stress response occurs when the plant encounters signiﬁcant drought stress during GS2 prior to anthesis. Postﬂowering water stress results from drought stress that is encountered at GS3 during grain ﬁll. Sorghum susceptible to pre-ﬂowering drought stress will exhibit symptoms such as leaf rolling, leaf tip burn, delayed ﬂowering, poor panicle exsertion, panicle blasting, and reduced panicle size (Rosenow et al., 1997a – c). In a breeding nursery, pre-ﬂowering susceptibility is evident when a characteristic “saddle effect” is observed where panicle development occurs only at the ends of a plot (presumably due to additional soil moisture available in the alleys between plots). Because pre-ﬂowering stress occurs during panicle development, it affects yield potential by inﬂuencing panicle size and seed number. Because of the importance of the trait and its impact on yield, sorghum improvement programs have identiﬁed and successfully used numerous sources of resistance to pre-ﬂowering drought stress (Table VI). These sources of resistance have been utilized by breeders to develop inbred lines, hybrids, and cultivars that have excellent pre-ﬂowering drought stress. While the physiological basis of pre-ﬂowering drought stress is not well known, the genetics of pre-ﬂowering drought stress have been evaluated. Because the evaluation of pre-ﬂowering drought stress is primarily subjective and is related to numerous phenotypic characteristics, there has been relatively little research to determine the inheritance of the trait (Rosenow et al., 1997a – c). More recently, the development of molecular marker technology has allowed sorghum breeders to dissect the inheritance of pre-ﬂowering drought tolerance. Tuinstra et al. (1996) evaluated a recombinant inbred line population from the cross of Tx7078 £ B35 and found six distinct genomic regions that were speciﬁcally associated with pre-ﬂowering drought tolerance. These loci accounted for approximately 40% of the total phenotypic variation for yield

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Figure 8 Different types of drought stress responses seen in sorghum. (A) Pre-ﬂowering stress susceptible hybrid (left) and tolerant hybrid (right). (B) Post-ﬂowering drought stress tolerant genotype (left) and susceptible (right).

under drought stress and most of these regions were detectable across environments. Kebede et al. (2001) reported that the pre-ﬂowering drought resistance in the cross of SC56 £ Tx7000 had a high broad-sense heritability and they identiﬁed four QTLs that controlled pre-ﬂowering drought tolerance in sorghum. While Tx7000 was identiﬁed by the authors the source of preﬂowering drought tolerance, approximately half of the favorable QTL alleles were derived from SC56, which was designated as pre-ﬂowering drought susceptible. In addition, none of the QTLs identiﬁed by Kebede et al. (2001) were consistent across all environments. They also noted a strong relationship between QTL for pre-ﬂowering drought resistance and days to ﬂowering.

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Table VI Lines Released and Reported by Rosenow et al. (1997a– c) to have Pre-ﬂowering Drought Resistance, Post-ﬂowering Drought Resistance or Both Traits Pre-ﬂowering tolerant IS2403C (SC103-14)a Tx7000 (Caprock)c Tx7078c TAM422c Tx430c BTx623c BTx3197c Tx2536c Tx2737c Tx432c SC414-12E (IS2508 der.)a Ajabsido (from Sudan)d Koro Kollo (from Sudan)d Segaolane (from Botswana)d El Mota (from Niger)d

Post-ﬂowering tolerant

Both pre- and post-ﬂowering tolerant

IS12553C (SC33-14)a IS12555C (SC35-14)a IS12558C (SC38-14)a IS12568C (SC56-14)a IS3071C (SC237-14)a IS6705C (SC265-14)a IS8263C (SC328-14)a IS17459C (SC599-14, Rio)a IS11549C (SC1017-14)a KS19e BTx642c Tx2908c QL36f QL41f

P898012b P954035 (SC33-9 der.)b IS12543C (SC23-14)a IS3462C (SC701-14)a CSM-63 (from Mali)d

a

Cooperative release from the TAES/USDA sorghum conversion program. Releases from Purdue University. c Releases from the Texas Agricultural Experiment Station, Texas A&M University. d Cultivars from the country listed in parentheses. e Release from the Kansas Agricultural Experiment Station, Kansas State University. f Releases from DPI, Queensland, Australia. b

Water stress encountered during GS3 can also result in signiﬁcant reduction in yield as the plant is unable to completely ﬁll the grain. Sorghum susceptible to post-ﬂowering drought stress will exhibit symptoms such as reduced kernel size, signiﬁcant leaf and stem death and lodging (Rosenow et al., 1997a – c). The increase in lodging is due to the plant remobilizing carbohydrate from the stem in an attempt to complete the grain ﬁll process. Once the stem is weakened, charcoal rot (caused by M. phaseolina) invades and further weakens the plant, resulting in signiﬁcant lodging. Sources of resistance to post-ﬂowering drought stress are less common than those found for pre-ﬂowering drought stress, but breeders have been successful in identifying genetic resistance to post-ﬂowering drought stress (Table VI). Because sources of post-ﬂowering drought resistance remain green while susceptible types do not, the resistance to post-ﬂowering drought stress is known as staygreen drought tolerance (Rosenow et al., 1983). Staygreen genotypes are less susceptible to lodging, more resistant to charcoal rot, and they retain greater green leaf area and higher levels of stem carbohydrates than non-staygreen genotypes (Mahalakshmi and Bidinger, 2002).

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While sources of post-ﬂowering drought stress are more limited than those for pre-ﬂowering drought stress, there has been substantially more research on the heritability and physiology of post-ﬂowering drought resistance. Upon the description of non-senescent sorghum genotypes (Duncan et al., 1981), independent reports on the genetic control of non-senescence in sorghum described the trait as both dominant and recessive in terms of inheritance (Duncan, 1984; Rosenow et al., 1997a –c). Tenkouano et al. (1994) determined that non-senescence was regulated by dominant and recessive epistatic interactions between two loci controlling non-senescence. Walulu et al. (1994) reported that the staygreen reaction in line B35 was controlled by a major gene that exhibits varied levels of dominant gene action depending on the environment. In a diallel analysis, Van Oosterum et al. (1996) also found that staygreen was moderately heritable with dominant gene action. Tuinstra et al. (1997) identiﬁed 13 regions of the genome associated with at least one measure of post-ﬂowering drought tolerance, but only two of these QTLs were stable across environments with major effects on staygreen and yield. Crasta et al. (1999) identiﬁed seven genomic regions associated with staygreen in line B35, but only three of these QTLs were stable across environments. These three QTLs also accounted for 42% of the total phenotypic variability for staygreen. Xu et al. (2000) also identiﬁed several genomic regions with major effects for staygreen. Tao et al. (2000) identiﬁed two genomic regions that were consistently associated with staygreen response in Australia. These reports consistently indicate that at least two loci account for a signiﬁcant amount of the variability associated with staygreen, but there is no way to know if the genomic regions were consistent across studies. Kebede et al. (2001) identiﬁed genomic regions controlling staygreen from SC56 and then compared them to previous results. Three of the genomic regions that they had identiﬁed were consistent genomic regions identiﬁed in previous studies (Tuinstra et al., 1997; Crasta et al., 1999; Xu et al., 2000). In addition, several of these regions were syntenic with regions of the corn genome associated with staygreen in that species. However, no congruency was observed for QTL with minor effects, indicating that these smaller QTLs were likely environment speciﬁc (Kebede et al., 2001). Therefore, it appears that these associations with several major effect QTL could be especially valuable for use in MAS programs (Nguyen and Jordan, personal communication). The phenotypic manifestation of pre- or post-ﬂowering drought tolerance is the result of several phenotypic and physiological traits that have been identiﬁed and characterized by sorghum physiologists. Traits that have been associated with drought resistance include heat tolerance (discussed later in this chapter), osmotic adjustment (Basnayake et al., 1995), transpiration efﬁciency (Muchow et al., 1996), rooting depth and patterns (Jordan and Miller, 1980), and epicuticular wax (Maiti et al., 1984). The physiological basis of these and other traits associated with drought tolerance has been reviewed by Kreig (1993) and

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Ludlow (1993). While all of these traits have been associated with drought tolerance in sorghum, most have not been of any practical use in improvement programs because of the difﬁculty in evaluation and/or selection. Drought stress screening is a challenging and inconsistent process. Rather than rely on rainout shelters, which are expensive and space limited, most programs utilize low-rainfall environments that are conducive to drought. Germplasm is evaluated in several different water stress regimes including dryland (no supplemental irrigation), pre-ﬂowering stress (supplemental irrigation after anthesis) and post-ﬂowering stress (supplemental irrigation prior to anthesis). In most situations, these nurseries are replicated in multiple environments to ensure appropriate and adequate stress in at least one environment. Breeding approaches to screening for drought tolerance were thoroughly reviewed by Rosenow et al. (1997a – c). Because of the difﬁculty and lack of consistent association of a single trait with drought resistance in the whole plant, pre-ﬂowering stress is generally rated as described by Rosenow et al. (1997a –c). While many factors inﬂuence postﬂowering drought stress, Wanous et al. (1991) showed that leaf death ratings (or green leaf percentage) are strongly related to post-ﬂowering drought stress rating. Borrell et al. (2000a) demonstrated that the presence of the staygreen trait consistently enhanced yield in trials where post-ﬂowering drought stress was encountered and had no effect on yield in more favorable environments. Selection for staygreen has resulted in the identiﬁcation and development of inbred lines, cultivars and hybrids with improved drought resistance throughout the world (Henzell et al., 1992; Rosenow et al., 1997a – c; Mahalakshmi and Bidinger, 2002). The evaluation of sorghum germplasm for staygreen has proven to be the most effective empirical approach for enhancing drought tolerance in sorghum. Due to the limitations of ﬁeld evaluation (Rosenow et al., 1997a –c), further improvements in drought stress tolerance will require the integration of molecular genetic and physiological technology into sorghum improvement programs. Molecular marker analysis has revealed genomic regions that inﬂuence both pre- and post-ﬂowering drought tolerance. These QTLs are now being used in MAS programs to introgress these regions into elite but drought susceptible parental lines, with limited success (Nguyen et al., 1997; Borrell et al., 2000b; Coulibaly, 2002). In addition to the use of these QTLs for MAS, the eventual goal of this research must be to identify the genes in this genomic region responsible for drought tolerance. Once the functional genomics of the regions is elucidated, then the physiologic reactions can be related to genetic loci and selectable markers. All this work will require close collaborations between the molecular geneticist, physiologist and breeder, but identifying the physiology and functionality of the genetic regions inﬂuencing drought resistance is imperative for further improvements in drought tolerance.

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2.

Temperature Stress

As sorghum is a warm season cereal grain, it shows a higher tolerance for warm temperatures than most other cereal grain crops. Because the crop is often grown in very hot and dry regions of the world, there has been continual interest in identifying variation within the species for tolerance to extreme temperatures at various phases of growth. In contrast, sorghum possesses less tolerance to cold growing conditions. More recently, in more temperate sorghum-growing regions, the increase in minimum tillage systems has made early season cold tolerance an important trait. In many regions of the world, sorghum production encounters heat and drought stress concurrently; thus, initial efforts in breeding for temperature stress emphasized heat tolerance. Jordan and Sullivan (1982) screened a series of genotypes for both heat and desiccation tolerance. While heat and desiccation tolerance commonly occurred together, heat and desiccation tolerance of individual genotypes was not signiﬁcantly correlated. These results indicate that heat and drought tolerance are unique and independent traits. Genetic variability for heat tolerance has been detected in several studies. Wilson et al. (1982) conducted both greenhouse and ﬁeld trials in which they modiﬁed soil temperatures by adjusting the color of the soil. Their results indicated that variability among genotypes existed for seedling emergence at high temperatures. Researchers, using several different techniques, have documented variation for heat tolerance past the seedling stage. Sullivan (1972) developed the leaf disc method to measure heat tolerance and Sullivan and Ross (1979) used this methodology to demonstrate a positive correlation between heat tolerance and higher grain yield. Jordan and Sullivan (1982) used the leaf disc method to screen approximately 130 lines and their hybrids for heat tolerance; they found signiﬁcant variation existed among the lines and concluded that breeding for heat tolerance is a viable objective. ICRISAT breeders used “leaf ﬁring” ratings as a measure of a plant’s inability to survive exposure to high temperature; however, the repeatability and consistency of screening was highly dependent on environmental conditions (Peacock, 1982). Smillie (1979), when measuring chlorophyll ﬂuorescence, found that chlorophyll ﬂuorescence increases at speciﬁc temperatures. The temperature at which this increase occurs can be correlated with irreversible damage to photosynthetic membranes. The use of this screening tool was proposed by Peacock (1982), but there have been no reports of its application. Selection for heat tolerance has had limited success because (1) laboratory techniques to screen for heat tolerance have not been effective in improving heat tolerance in ﬁeld studies and (2) ﬁeld screening for heat tolerance is difﬁcult to manage and is often confounded with drought tolerance. Sorghum improvement programs have traditionally emphasized drought tolerance and any improvement in heat tolerance has been selected in concurrence with drought tolerance.

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In recent years, there has been signiﬁcant interest in enhancing the cold tolerance in sorghum. As a species native to subtropical or tropical climes, sorghum is not tolerant of cool temperatures. At germination, temperatures below 158C reduce germination and emergence in most sorghum genotypes and temperatures below 78C stop the germination process. During early season growth, cool temperatures depress chlorophyll synthesis and growth (McWilliams et al., 1979). Prior to and during anthesis, cool temperature can result in pollen abortion and sterility, which causes signiﬁcant reductions in seed set and yield. Sources to cold tolerance have been identiﬁed in several breeding programs around the world. Singh (1985) evaluated sorghum cultivars grown at high elevations in Africa and from that screening developed a collection of germplasm tolerant to cold temperatures from anthesis to seed maturation. This germplasm has been extremely useful for the development of sorghum cultivars and hybrids in the highland production regions of Mexico, Eastern and Southern Africa (Peacock, 1982). In more temperate environments, tolerance to cold temperatures at germination and emergence is more important than cold tolerance at anthesis. The need for seedling cold tolerance has become a higher priority as minimum tillage systems have become more popular (Yu and Tuinstra, 2001). Early work to identify sources of seedling cold tolerance in sorghum was successful (Nordquist, 1971; Soujeole and Miller, 1984; Singh, 1985), but the best sources were unadapted or undesirable for grain production (Stickler et al., 1962), meaning that breeding efforts would be necessary to transfer the cold tolerance to adapted germplasm. Seedling cold tolerance is manifested as variation in germination, emergence, seedling vigor under cool temperatures. Mann et al. (1985) developed a technique to evaluate sorghum genotypes for their base temperature, which is the lowest temperature at which seed will germinate. While this evaluation resulted in some very interesting results regarding variation among genotypes for base temperature, the inheritance of the trait was either very low or complex, involving cytoplasmic and nuclear factors (Abdalla, 1982; Wenzel, 1988). Bacon et al. (1986) completed four cycles of recurrent selection for seedling cold tolerance using genetic male sterility to facilitate genetic recombination. In this study, seedling emergence under cool temperatures improved 2.8% per cycle. During selection, maturity was decreased, yield was increased, and plant height remained unchanged. The results indicate that selection for improved cold tolerance is feasible. Yu and Tuinstra (2001) evaluated parental lines varying for seedling cold tolerance and their hybrids in a Design II mating scheme to determine the relative importance of general and speciﬁc combining ability. They found that general combining ability is more important than speciﬁc combining ability for seedling vigor in sorghum. The ﬁndings of the latter two studies indicate that selection for seedling cold tolerance should result in substantial

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improvements in the tolerance expressed in commercial production using traditional approaches where the environment is appropriate for selection. 3. Nutrient Stress Because sorghum is commonly grown in soils that induce some form of nutrient stress, sorghum geneticists have worked to characterize the response of sorghum to soil nutrient stress. Depending on the location and soil type, several nutrient deﬁciencies or problems are encountered by sorghum. These include phosphorous deﬁciency, aluminum toxicity in acid soils, salinity toxicity, and iron chlorosis on alkaline soils. A signiﬁcant amount of research has been completed to characterize the basic response of the sorghum plant to these stresses. Sorghum has been characterized as a species that is moderately tolerant to saline soils (Francois et al., 1984). In their study, Francois et al. (1984) determined that sorghum was more tolerant to soil salinity at germination than at any other stage of growth. In addition, vegetative growth was less affected by increases in soil salinity than was grain yield. Yang et al. (1990) reported that S. halepense was more tolerant than S. bicolor and that S. halepense may be useful for increasing the salinity tolerance of grain sorghum. Variation exists within sorghum for tolerance to soil salinity, but relatively little effort has been placed on improving this trait in grain sorghum. Taylor et al. (1975) screened 48 sorghum genotypes for variation in tolerance to salinity at germination and seedling growth. They found signiﬁcant variation in seedling growth due to genotype, indicating that genotypes possess differing levels of tolerance to soil salinity. Ratanadilok et al. (1978) reported that the inheritance of seedling salt tolerance was a quantitatively inherited trait with a moderate to high broad-sense heritability estimate. Further efforts to improve salinity tolerance have not been pursued as the trait was not viewed by plant breeders as a high priority trait. Aluminum toxicity was identiﬁed as a serious constraint to sorghum production in sorghum many years ago (Sanchez and Logan, 1992). Plant physiologists and soil fertility experts quickly realized that it would be more feasible to develop sorghum genotypes that are more tolerant of acid soils (Clark and Gourley, 1987). To accomplish this goal, a signiﬁcant amount of effort is required to develop screening protocols and use these methods to determine if variation exists among genotypes. Several methods of screening genotypes for tolerance to Al toxicity have been developed. Most of these methods involve the evaluation of germplasm under greenhouse conditions (Furlani and Clark, 1981; Malavolta et al., 1981) and they are summarized by Borgonovi et al. (1987). Field screening for Al tolerance has been restricted due to the lack of uniform experimental areas with respect to Al

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saturation in addition to other nutrient levels. While these problems exist, Borgonovi et al. (1987) recognized the importance of ﬁeld evaluations and made recommendations for ﬁeld site preparation and evaluation. Duncan et al. (1983) found that the correlations between Al-toxicity tolerance as measured in greenhouse and ﬁeld experiments were generally good, but there were some signiﬁcant exceptions to the correlations. The authors thought that the exceptions (lines tolerant in greenhouse but susceptible to ﬁeld conditions) were due to variations in the exact cause of the acid soils. The results indicate that lab assays are useful for initial screening, but that ﬁnal evaluations must be completed in multiple environments with differing soil types to ensure that Al tolerance is expressed in ﬁeld conditions. Signiﬁcant variability exists among sorghum genotypes for Al-toxicity tolerance. Several studies detected genetic variation for seedling growth in low pH and Al toxicity in greenhouse conditions (Bastos and Gourley, 1982; Furlani et al., 1983; Boye-Goni and Marcarian, 1985). Other reports detected variation among genotypes for Al-toxicity tolerance in ﬁeld experiments (Duncan, 1981; Duncan et al., 1983; Gourley, 1983). From these studies, several sorghum genotypes were identiﬁed that had consistently high levels of tolerance to Al toxicity (Duncan et al., 1983). The inheritance of Al-toxicity tolerance appears to be complex, quantitative, and dependent on the speciﬁc source of resistance. Pitta et al. (1979), evaluating ﬁve parental lines and their hybrids under ﬁeld conditions, suggested that control of Al-toxicity tolerance was heritable and controlled by low number of dominant genetic loci. Borgonovi et al. (1987), evaluating a different set of parental lines and hybrids, concluded that speciﬁc combining ability effects were predominant, which indicates a more complex and quantitative pattern of inheritance. Boye-Goni and Marcarian (1985) evaluated six sorghum parental lines in a half-diallel for combining ability effects for Al-toxicity tolerance. They reported that general combining ability was much more important than speciﬁc combining ability, indicating that genetic control is predominantly additive. The relatively high heritability estimates indicated that while the number of genetic loci inﬂuencing this trait is not known, selection for Al tolerance should be effective. Selection programs to develop sorghum tolerant to Al toxicity were initiated in the 1980s and these projects were quite successful. Duncan et al. (1983) described a program that used multiple environments in different generations of selection to identify genotypes with agronomic performance and tolerance to a wide array of acid soil tolerances. From this program, two sets of germplasm were released which had increased tolerance to acid soils (Duncan, 1981, 1984). Gourley (1987) used genetic male sterility to develop random mating sorghum populations for selection of acid soil tolerant germplasm. Enhanced tolerance to Al toxicity was detected in sorghum lines regenerated from tissue culture (Waskom et al., 1990). The lines were derived from Tx430 and Hegari and in each case, the regenerated lines showed signiﬁcantly higher levels of Al-toxicity

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tolerance than the original inbred line (Miller et al., 1992b). The variation was attributed to somaclonal variation induced via tissue culture and regeneration. From this research, several lines were released as germplasm (Duncan et al., 1991b). While signiﬁcant progress has been made in the development of acid soil tolerant germplasm, little to no research has been completed to determine the genomic location of Al-toxicity tolerance. Information regarding these issues will enhance further efforts to improve sorghum.

D. GRAIN QUALITY Sorghum grain is processed and utilized in many different ways. Approximately 40% of the world production is used as a food grain to make a wide array of products ranging from unleavened ﬂat breads to fermented traditional beverages (Hulse et al., 1980). Another 45% is utilized as a feed grain for poultry, pork and beef production. When used as feed grain, it is processed in an array of different ways. Industrial applications, such as alcohol or starch extraction, use approximately 5% of worldwide production. In each situation, quality is deﬁned by different parameters which can often change in different regions of the world. Therefore, it is impossible to clearly deﬁne a set of attributes that deﬁne highquality grain. Numerous kernel characteristics control the basic physical appearance of sorghum grain and its chemical composition. Simply inherited genetic loci control pericarp color, mesocarp thickness, presence of a pigmented testa, and endosperm color have a direct effect on the basic appearance of the grain (Rooney, 2001; Table VII). Additional factors such as the environment, insects, and grain molds interact with the genotype to produce the phenotypic characteristics observed in the harvested grain. In addition to the physical appearance of the grain, variation in the chemical composition of the grain affects the nutritional and processing characteristics of sorghum grain. Variation for endosperm color, protein content and digestability, starch content and digestability, and fat content is under genetic control and can be manipulated to produce different types of grain sorghum. There is often a perception that the feed and nutritional value of sorghum is drastically lower than that of corn and other cereal grains. However, that perception is reality only when the grain possesses a pigmented testa. The pigmented testa layer is high in polyphenols commonly known as tannins. While tannins provide protection to the grain from insects, birds, and weathering, they are also associated with signiﬁcant reduction in the nutritional value and palatability of both feed and food quality sorghums which possess them. Due to these strong antinutritional factors, tannin sorghums are grown only in a few regions where the pest pressure precludes the production of non-tannin sorghum. In all other areas, sorghum breeding programs any breeding lines with a

Table VII Genotypes at Loci that Inﬂuence Grain Color and Quality, their Corresponding Phenotype, and a Speciﬁc Released Line Possessing that Speciﬁc Genotype and Phenotype Genotype

Phenotype

Pericarp

Mesocarp

Testa

Spreader

Plant

Grain

Tannins

Example

PPQQ PPQQ PPQQ PPQQ PPQQ PPQQ PPQQ PPQQ PPQQ PPqrqr PPQQ PPQQ PPqq ppQQ ppQQ ppQQ ppQQ

RRYYII RRyyII RRyyii RRyy– – RRYYii RRYYII RRyyii RRyyii RRYYII rryyII RRYYII RRYYii RRyyii RRyyii RRyyii RRyyii RRyyii

zz ZZ zz zz zz zz zz zz ZZ ZZ ZZ ZZ zz ZZ ZZ ZZ ZZ

b1b1B2B2 b1b1b2b2 b1b1B2B2 B1B1B2B2 B1B1B2B2 B1B1B2B2 B1B1B2B2 B1B1B2B2 B1B1B2B2 b1b1B2B2 b1b1B2B2 b1b1B2B2 b1b1B2B2 b1b1b2b2 – – –

SS SS SS ss ss SS SS ss ss ss SS SS SS – – – –

Purple Purple Purple Purple Purple purple Purple Purple Purple Red Purple Purple Tan Tan Tan Tan Tan

Red, chalky White, pearly, yellow endosperm White, chalky White pearly, purple testa Red, chalky Dark brown-red, chalky Brown, chalky White, chalky Dark brown-red, pearly White, pearly Dark red, pearly Light red, pearly White, chalky White White, pearly, food grade sorghum Waxy endosperm Waxy endosperm

No No No Yes Yes Yes Yes Yes Yes No No No No No No No No

BTx378 RTx430 BTx3197 SC109-14E SC719-11E SC103-12E DOBBS HEGARI RTAM2566 RTAM428 SC630-11EII SC630-11Eii 77CS5 RTx436 BTx635 BTxARG-1 Tx2907

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Plant

A summary of the genetics inﬂuencing grain color is provided by Rooney (2001).

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pigmented testa or parental lines that produce hybrids with a pigmented testa are eliminated from the program. This ensures sorghum grain with good feed and nutritional values. Grain and plant color inﬂuences the potential end uses of the grain. In India where sorghum is used as a food grain, essentially all the grain sorghum produced has a white pericarp. In the western hemisphere where sorghum is used predominantly as a feed grain, grain color is not a factor and most hybrids have a red pericarp because it tends to tolerate grain weathering slightly better than white pericarp sorghums. Furthermore, plant color can affect grain quality. Neither white nor red grain produced on a sorghum plant with red or purple plant color is acceptable for use in food products because the pigments often stain the grain, resulting in products with an off-color and possibly off-ﬂavors. In most regions, sorghum with good food quality is clean, free of molds, weathering or insect damage. The grain has a white pericarp, which is easily removed from the endosperm during milling, and it is produced on a plant with tan plant color and light or straw-colored glumes. These traits ensure that the sorghum grain will produce ﬂours that have light color and bland ﬂavor. These same characteristics are also desirable for animal feeding. The dual-purpose nature of these grain types has lead to a signiﬁcant breeding effort to produce these types of grain sorghum hybrids. Utilizing germplasm from the sorghum conversion program and sorghum cultivars from regions of the world where food sorghum is important, the Texas Agricultural Experiment Station has released several germplasm and parental lines that have good food grain quality and in the right combinations, these lines produce hybrids with good grain quality (Miller et al., 1992a). The food-quality hybrids now available are generally full season and competitive with traditional sorghum hybrids in high input environments where water stress is limited. Breeding efforts in some sorghum programs are now focused on the development of early and mid-season hybrids with strong drought tolerance and good grain quality. Grain produced from these hybrids would have greater and more diverse market potential than grain sorghum from traditional hybrids. Soon after the development of hybrid sorghum, there was signiﬁcant interest in the development of yellow endosperm sorghum hybrids and the assumption that these hybrids were of greater nutritional value. While yellow endosperm sorghum does contain higher levels of carotenoid pigments, the basic nutritional value of yellow endosperm sorghum is not greater than other sorghums. However, several important and good parental lines with yellow endosperm were developed and utilized by the industry to produce a series of “bronze” and “cream” sorghum hybrids. A bronze hybrid has a red pericarp and yellow endosperm while a cream hybrid has a white pericarp and yellow endosperm. Currently, breeding programs do not focus on yellow endosperm as a major trait of importance but neither do they select against it.

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Variation in the relative amount of amylopectin in the endosperm is controlled by a single gene, Wx (Karper, 1933). In normal sorghum grain (WxWx), the endosperm contains 20 – 30% amylose and 70 –80% amylopectin. In grain from waxy sorghum (wxwx), the starch in the endosperm is amylopectin and no amylose is present. Waxy endosperm sorghums are reported to be more digestible and easier to process than normal endosperm sorghums. While waxy parental lines have been used to produce both waxy and heterowaxy hybrids (grain from a heterowaxy hybrid will be segregating for waxy endosperm), these hybrids have not found widespread production because yields of them are slightly lower than other hybrids (Aydin, 2003). Whether this is due to waxy endosperm or other factors are yet to be determined, but there is little effort to improve the yield potential of waxy endosperm sorghum. In the US, a greater proportion of grain sorghum is used in ethanol production. Ethanol conversion efﬁciency is increased when carbohydrate content is increased. Efforts to develop sorghum with higher carbohydrate have been initiated, but initial screening of a limited set of germplasm has revealed that genetic variation for percentage of carbohydrate content is limited; most variation is due to environmental conditions. A better approach for increasing carbohydrate content may be to increase total grain yield, which would result in an increase in carbohydrate production, although the relative percentage will not be increased. Variation for protein content in grain sorghum is also strongly inﬂuenced by environmental conditions. Protein content in the grain typically decreases in high yielding environments and increases in low yielding environments. In each case, these changes are primarily due to the increase and reduction of starch synthesis, respectively. Genetic variation for protein content and quality are known, but these increases in content or quality are accompanied by severe reductions in agronomic and yield characteristics. Consequently, sorghum-breeding programs have been reluctant to directly incorporate them into their programs and there has been no long-term approach (analogous to the QPM for corn at CIMMYT) to improve the agronomic potential of high protein content and quality sorghum. While the digestability of protein in sorghum grain is strongly affected by the method of processing, signiﬁcant variation for the inherent potential for protein digestability is known to exist (Weaver et al., 1998). Initially, increased protein digestability was associated with softer endosperm types, which are typically unacceptable because of their susceptibility to grain mold and weathering. However, the two traits appear to be inherited independently indicating that there is the potential to incorporate this trait into sorghum with no loss in weathering resistance.

E. FORAGE SORGHUM Besides its value as a cereal grain, there is signiﬁcant use of S. bicolor genotypes speciﬁcally for forage. While no speciﬁc statistics are kept, seed sales

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of forage sorghum indicate that planted forage sorghum acreage may be near 10 million acres. In addition, the range of production is much wider than that of grain sorghum. Until recently, forage sorghum breeding was usually a minor component of grain sorghum-breeding programs but this has changed with the renewed interest in silage sorghums and brown midrib sorghums. Kalton (1988) lists numerous forage sorghum-breeding objectives, but these can be condensed to increased yield, increased quality, and pest resistance. Several types of forage sorghum cultivars and hybrids are produced. Forage sorghums for silage are usually medium to large stemmed hybrids with high grain and forage yield potential on a plant that will grow to 2 –3 m in height. These hybrids combine large forage yields with yields comparable to full season grain sorghum hybrids shorter in height. These silage-type hybrids produce silage yields similar to that of corn, using approximately 1/3 less water than is required by corn (Pedersen and Rooney, 2004). Forage sorghums for hay or grazing are typically pure-line sudangrass cultivars or sorghum – sudangrass hybrids. Sorghum – sudangrass hybrids are created using a male-sterile grain female hybridized to a sudangrass pollinator. This crop is managed to produce thinner culms, extensive tillering and high amounts of leaf material and regrowth after harvest for multiple cuts is required. The production of hybrid forage sorghums uses methodology similar to that of grain sorghum with several modiﬁcations. Seed production is based on the cytoplasmic male sterility system, and forage sorghum hybrids can be produced by simply replacing the grain sorghum pollinator with a forage sorghum pollinator. However, it is also common to use a grain type, male-sterile F1(A£B) as a female to increase the seed yield in production. Gorz et al. (1984) reported 45 – 82% more sorghum –sudangrass seed production using F1 sorghum hybrids as seed parents compared to using lines as seed parents. Because these are threeway hybrids, they will segregate for traits that differ between the two parents used to produce the F1 seed parent. However, in forage sorghum segregation is relatively unimportant because the whole plant is harvested, the crop is tall and any noticeable variation is hidden. Harvey (1977) reported that 15% of all sorghum – sudangrass hybrids were three-way hybrids; that number is undoubtedly higher today. For both types of forage sorghums, yield and quality are complex and quantitatively inherited traits. Numerous studies (Blum, 1968; Dangi and Paroda, 1978a,b; Pedersen et al., 1982) have shown general combining ability to be of primary importance for both forage yield and quality indicating that forage sorghum improvement should be made by direct selection for these traits. Because of these factors, breeding for improved forage sorghums follows methodology very similar to breeding for grain sorghum hybrids, with several notable exceptions. In silage sorghum, emphasis is placed on both forage quality and grain yield. For sorghum – sudangrass, emphasis is placed on the development of pollinator lines with good combining ability for leaﬁness, forage

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quality, regrowth capability, and high tillering capacity. Grain yield in the sudangrass pollinators is important only to ensure enough production for pollinator seed. Because forage quality is critical, resistance to foliar diseases is even more important in forage sorghums than in grain sorghums. Commonly used females are combine height (typically 3-dwarf) with recessive alleles ﬁxed at three loci (usually dw1 Dw2 dw3 dw4). Therefore, pollinators must have dominant alleles at the appropriate genetic loci to produce 2-dwarf or 3-dwarf hybrids for commercial forage production. In addition to height, the pollinator parents must provide characteristics suitable for forage production, such as tillering ability, regrowth capability, and general forage quality and yield. Combinations that are selected for production are based on combining ability, seed production potential and several speciﬁc phenotypic traits, such as photoperiod sensitivity and brown midrib. The incorporation of photoperiod sensitivity into sorghum – sudangrass hybrids improves forage quality by expanding the management window. Photoperiod-sensitive hybrids will not initiate reproductive growth until daylengths shorten to near 12 h. In the US, these hybrids will not ﬂower until October, long after the growing season has ﬁnished. Because forage quality is tightly linked to anthesis and maturity, the lack of anthesis allows these hybrids to maintain forage quality for a longer period of time. This trait is controlled by two genes which interact in duplicate-dominant epistasis to produce the photoperiod-sensitive phenotype (Rooney and Aydin, 1999). This unique system allows photoperiod-sensitive hybrids to be produced using two photoperiodinsensitive parents (Fig. 9). Brown midrib is a phenotypic characteristic originally described in sorghum by Porter et al. (1978). In most every case, brown midrib phenotype is controlled by a single recessive gene and it is the result of a recessive mutation in the lignin biosynthesis pathway (Bout and Vermerris, 2003). Numerous brown midrib mutants have been described, each with its own speciﬁc level of expression in the midrib and on lignin content (Porter et al., 1978). Of the numerous loci characterized, three, bmr6, bmr12, and bmr18, have been incorporated into forage sorghum pollinators. Each inﬂuences lignin content in slightly different ways, but all express brown midrib. Consequently, brown midrib is consistently associated with improved forage quality, although that is not necessarily a correct association. Brown midrib sorghums have consistently shown improved palatability and digestability in feeding trials, presumably due to reduced lignin levels in the plants. Variation for forage quality among the different brown midrib mutants exists and breeding programs must balance the needs of agronomic production with forage quality when deciding which mutants to use (Fritz et al., 1981). In addition, because these traits are recessive, the same allele must be incorporated in both parental lines. If two parents are hybridized that are both brown midrib but controlled by different loci, the resulting hybrid will not be brown midrib.

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Figure 9 Schematic of the genetic control and phenotype for photoperiod sensitivity in forage sorghum–sudangrass hybrids and silage hybrids (based on genetics described by Rooney and Aydin, 1999).

Therefore, parental combinations for bmr hybrids must be matched based on the bmr allele each parent possesses. Brown midrib reduces lignin content and lignin content is critical to maintaining the structural integrity of the plant. Therefore, it was not surprising to discover that brown midrib sorghums are very susceptible to lodging. Logically, the lowest lignin lines (consequently highest forage quality) were also the most susceptible to lodging. Breeding to improve lodging in brown midrib has reduced lodging and maintained most of the forage quality attributes of brown midrib sorghum. Further improvements should make brown midribs even more widely grown.

F. SWEET SORGHUM FOR SYRUP Sweet sorghums were some of the ﬁrst sorghums to come to North America. They were originally grown as a source of sweetening prior to the availability of other sweeteners. In recent years, sorghum syrup production has rebounded with acreage estimated at over 3000 acres in Kentucky in the mid-1990s (Bitzer, 1994). It continues to be a high value crop grown on small acreage throughout the Southeastern US. Sweet sorghums are members of S. bicolor, but these speciﬁc genotypes possess juicy stems with a high concentration of sugar. Sweet sorghum cultivars have been bred using traditional pedigree breeding approaches for high juice extraction, high soluble solid contents (high brix), and high total yield. Sweet sorghum varieties are tall, resist lodging and must have resistance to anthracnose (both foliar and stalk), and maize dwarf mosaic virus. Breeding for sweet sorghums was conducted by the

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USDA-ARS and Mississippi State for many years (Freeman et al., 1986). This program released cultivars (such as Brandes, Theis and Dale) that are still grown today (Coleman and Broadhead, 1968; Broadhead et al., 1970, 1974). While this breeding program has been discontinued, sweet sorghum cultivars have been recently released from breeding programs in the Southeastern US (Harrison and Miller, 1993; Day et al., 1995). In addition to their use in syrup production, there has been occasional interest in the use of this type of cultivar for ethanol production as well (Schaffert and Gourley, 1982). With the renewed interest in biofuels, it is likely that opportunities to develop sorghums for ethanol production will become available. Sorghums for ethanol production would have different selection criteria that those of sweet sorghums for syrup, but the sweet sorghum and silage sorghum genotypes would form the basis of a breeding program for ethanol production.

G. BROOMCORN Broomcorns are phenotypically unique members of S. bicolor that produce extremely long, highly branched and loose panicles. These types of sorghum are grown speciﬁcally for their panicle which is harvested, the seed removed and the panicles (or brushes) are used to produce brooms. In past years, signiﬁcant acreages of broomcorn were grown throughout the world to manufacture brooms for that particular region. More recently, broomcorn production tends to be localized to geographic regions of the world where environmental conditions are favorable and inexpensive labor is readily available. Broomcorn is grown as a cultivar. Weibel et al. (1972b) successfully produced broomcorn hybrids, but hybrid vigor did not enhance the yield or quality of the brush, effectively ending any interest in broomcorn hybrids. Since that time, there has been relatively little effort in the systematic improvement of broomcorn cultivars. Many of the early cultivars are still in production today. Traits of importance to broomcorn production are brush yield and quality. Broomcorn varieties are typically susceptible to anthracnose and maize dwarf mosaic virus and due to the lack of breeding; the diseases are avoided by producing broomcorn in environments not conducive to either disease. When used breeding approaches mirror those of grain and forage sorghum-breeding programs.

VII. BIOTECHNOLOGY IN SORGHUM IMPROVEMENT As with most agronomically important crop species, sorghum genomics is an area of active research. In the past 10 years, over 11 genetic linkage maps

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of sorghum have been published (Subudhi and Nyugen, 2000; McIntyre et al., 2001). Of these 11, two are highly saturated and have formed the basis for future genomic analysis (Menz et al., 2002; Bowers et al., 2003). These two maps have been used to create BAC libraries, isolate genes, correlate genetic and physical maps and provide robust molecular markers for QTL mapping efforts (Subudhi and Nyugen, 2000; McIntyre et al., 2001; Islam-Faridi et al., 2002). From this work, numerous studies to identify QTL for agronomically important traits have been conducted and QTLs have been identiﬁed for a wide array of important traits (Table VIII). This work has been important in understanding the genetic inheritance of speciﬁc traits and the best breeding approaches. In some cases, they have led to the cloning of the gene responsible for a speciﬁc phenotype, and in others, the linkages are used for potential MAS. Adoption of other molecular technology is important and is being tested. Markers detected for simply inherited traits such as maturity, height, and fertility restoration have been identiﬁed and tested for the applicability to MAS schemes. These tests have had varying degrees of success. QTLs have been identiﬁed for drought stress (pre- and post-ﬂowering), grain mold, grain yield, and grain quality.

Table VIII Summary of Qualitative and Quantitative Trait Loci Identiﬁed in Sorghum Trait Drought tolerance (pre- and post-anthesis)

Anthracnose Rust Head smut Downy mildew Maturity Height Yield and components

Grain quality and mold resistance Leaf blight resistance Fertility restoration Pre-harvest sprouting resistance Greenbug Midge Tillering Seed size and dispersal

Reference Tuinstra et al. (1996, 1997), Crasta et al. (1999), Subudhi et al. (2000), Tao et al. (2000), Xu et al. (2000), and Coulibaly (2002) Boora et al. (1998) and Mehta (2002) Tao et al. (1998) Oh et al. (1994) Gowda et al. (1995) and Oh et al. (1996) Lin et al. (1995) and Childs et al. (1997) Lin et al. (1995) and Pereira and Lee (1995) Pereira et al. (1995), Tuinstra et al. (1997), Rami et al. (1998), Sanchez-Gomez (2002), and Moran (2003) Rami et al. (1998), Klein et al. (2001a), and Franks (2003) Boora et al. (1999) Klein et al. (2001b) Lijavetzky et al. (2000) Katsar et al. (2002) Tao et al. (2003) Paterson et al. (1995) Paterson et al. (1995)

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Coulibaly (2002) was unsuccessful in using QTL markers to introgress postﬂowering drought stress from the donor to several elite inbreds. Franks (2003) had limited success in using QTL for grain mold as markers to enhance grain mold resistance: they were effective in progenies with the exact same pedigree in which the QTLs were mapped, but they were not effective in any other population. The potential remains for the use of markers for simply inherited traits for introgression or pyramiding of traits, but there have been no reports published to document their use in sorghum. Sorghum transformation has been reported and repeated by several research groups, both public and private and it is now possible to create transgenic sorghum (Jeoung et al., 2002). Although the technology to produce transgenic sorghums is now available, several factors have limited its potential use. Because S. bicolor hybridizes at low frequencies with johnsongrass (S. halepense) (Arriola and Ellstrand, 1996), any gene that is incorporated into cultivated sorghum will likely be transferred to wild, weedy species. This potential for gene transfer limits transformation for any trait that would give a weedy species a selective advantage. This would certainly include herbicide resistance traits, and possibly insect resistance traits as well. At this point, most of the transgenes currently available for incorporation are related to herbicide and insect resistance. As additional transgenes for traits that are selectively neutral in natural populations become available, then sorghum would be potential candidate for transformation. These include grain quality, disease resistance and possibly some forms of insect resistance (Gray et al., 2001; Izquierdo et al., 2001; Zhao et al., 2003).

VIII. CONCLUSION In the past century, sorghum has been transformed into a major grain crop grown throughout the tropical and sub-tropical regions of the world. Sorghum researchers in different parts of the world have developed improved genotypes with speciﬁc characteristics to its end use. The types of end uses of sorghum vary widely, from forage to food, feed and industrial uses of the grain. The types of cultivars of sorghum also vary widely, from traditional landraces to pure-line cultivars and commercial hybrids. These developments have helped to maintain the role of sorghum in agricultural production. Sorghum will remain particularly important in regions of the world where drought stress is common. Given that water is becoming more and more limiting for agricultural production, sorghum will continue to play a major role in agricultural production systems throughout the world. To meet future needs, sorghum researchers are faced with signiﬁcant challenges. The number of public and private agencies conducting sorghum

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improvement research has dropped in the past 15 years and resources for the remaining programs are limited. Reductions in the number of research programs reduce the ability of sorghum community to make steady and consistent improvement in the performance and quality of the crop. In contrast to the reduction in sorghum research program, research in sorghum genomics has increased and developed to the point that is providing useful information on the inheritance of traits, the locations of genes controlling these traits, and in some cases, cloning of the gene responsible for a trait. In addition, characterization of genetic diversity will allow more efﬁcient exploitation of the genetic diversity available in sorghum. Finally, sorghum transformation can be used to enhance quality and agronomic traits of value to the people who produce and utilize sorghum. Given these factors, the challenge to sorghum researchers is to utilize the existing resources to integrate new technology with traditional approaches as efﬁciently as possible to achieve steady improvements. There is little doubt that this effort is being and will continue to be accomplished.

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Rosenow, D. T. (1992). Using germplasm from the world collection in breeding for disease resistance. In “Sorghum and Millets Diseases: A Second World Review” (W. A. J. de Milliano, R. A. Frederiksen, and G. D. Bengston, Eds.), pp. 319– 324. ICRISAT, Patancheru (A.P.), India. Rosenow, D. T., and Clark, L. E. (1981). Drought tolerance in sorghum. “Proceedings of the 36th Annual Corn and Sorghum Research Conference, 9–11 December 1981, Chicago, IL”, pp. 18– 30. Rosenow, D. T., and Frederiksen, R. A. (1982). Breeding for disease resistance in sorghum. “Sorghum in the Eighties” (L. R. House, L. K. Mughogho, and J. M. Peacock, Eds.), Vol. 1, pp. 447– 455. ICRISAT, Patancheru (A.P.), India. Rosenow, D. T., Quisenberry, J. E., Wendt, C. W., and Clark, L. E. (1983). Drought tolerant sorghum and cotton germplasm. Agric. Water Manag. 7, 207–222. Rosenow, D. T., Dahlberg, J. A., Peterson, G. C., Clark, L. E., Miller, F. R., Sotomayor-Rios, A., Hamburger, A. J., Madera-Torres, P., Quiles-Belen, A., and Woodﬁn, C. A. (1997a). Registration of ﬁfty converted sorghums from the sorghum conversion program. Crop Sci. 37, 1397–1398. Rosenow, D. T., Dahlberg, J. A., Stephens, J. C., Miller, F. R., Barnes, D. K., Peterson, G. C., Johnson, J. W., and Schertz, K. F. (1997b). Registration of 63 converted sorghum germplasm lines from the sorghum conversion program. Crop Sci. 37, 1399– 1400. Rosenow, D. T., Ejeta, G., Clark, L. E., Gilbert, M. L., Henzell, R. G., Borrell, A. K., and Muchow, R. C. (1997). Breeding for pre- and post-ﬂowering drought stress resistance in sorghum. “Proceedings of the International Conference on Genetic Improvement of Sorghum and Pearl Millet”. INTSORMIL, Lincoln, NE, pp. 400–411. Rosewich, U. L., Pettway, R. E., McDonald, B. A., Duncan, R. R., and Frederiksen, R. A. (1998). Genetic structure and temporal dynamics of a Colletotrichum graminicola population in a sorghum disease nursery. Phytopathology 88, 1087–1093. Sanchez, P. A., and Logan, T. J. (1992). Myths and science about the chemistry and fertility of soils in the tropics. In “Myths and Science of Soils in the Tropics”. “SSSA Spec. Publ. 29” (R. Lal and P. A. Sanchez, Eds.), pp. 35–46. SSSA, Madison, WI. Sanchez-Gomez, A. (2002). “Identiﬁcation of quantitative trait loci for grain yield in a recombinant inbred B-line population in sorghum”. Ph.D. Dissertation. Texas A&M University, College Station, TX. Schaffert, R. E., and Gourley, L. M. (1982). “Sorghum as an energy source”. “Sorghum in the Eighties: Proceedings of the International Symposium on Sorghum, 2 –7 November 1981”. ICRISAT, Patancheru (A.P.), India, pp. 605–623. Schertz, K. F., and Clark, L. E. (1967). Controlling dehiscence with plastic bags for handcrosses in sorghum. Crop Sci. 7, 540 –542. Schertz, K. F., and Dalton, L. G. (1980). Sorghum. In “Hybridization of Crop Plants” (W. R. Fehr and H. H. Hadley, Eds.), pp. 577–588. American Society of Agronomy, Madison, WI. Seetharaman, K. E., Waniska, R. D., and Rooney, L. W. (1996). Physiological changes in sorghum antifungal proteins. J. Agric. Food Chem. 44, 2435–2441. Sharma, H. C. (1985). Screening for sorghum midge resistance and resistance mechanisms. “Proceedings of the International Sorghum Entomology Workshop, ICRISAT”, pp. 275 –292. Sharma, H. C., Abraham, C. V., Bidyasagar, P., and Stenhouse, J. W. (1996). Gene action for resistance to sorghum midge, Contarinia sorghicola. Crop Sci. 36, 259– 265. Sifuentes, J., and Frederiksen, R. A. (1988). Inheritance of resistance to pathotypes 1, 2, and 3 of Peronosclerospora sorghi in sorghum. Plant Dis. 72, 332 –333. Singh, S. P. (1985). Sources of cold tolerance in grain sorghum. Can. J. Plant Sci. 65, 251–257. Smillie, R. M. (1979). Coloured components of chloroplast membranes as intrinsic membrane probes for monitoring the development of heat injury in intact tissues. Aust. J. Plant Physiol. 6, 121–133. Smith, C. W., and Frederiksen, R. A. (2001). History of cultivar development in the United States: from “Memoirs of A. B. Maunder—Sorghum Breeder”. In “Sorghum: Origin, History,

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CRITICAL REVIEW OF THE SCIENCE AND OPTIONS FOR REDUCING CADMIUM IN TOBACCO (NICOTIANA TABACUM L. ) AND OTHER PLANTS N. Lugon-Moulin,1 M. Zhang,2 F. Gadani,2 L. Rossi,1 D. Koller,2 M. Krauss2 and G. J. Wagner3 1

Philip Morris International R&D, c/o Philip Morris Products SA, 2000 Neuchaˆtel, Switzerland 2 Philip Morris USA RD&E Department, P.O. Box 26583, Richmond, Virginia 23261, USA 3 Agronomy Department, N212 ASCN, University of Kentucky, Lexington, Kentucky 40546-0091, USA

I. Introduction II. Cadmium in the Environment III. Cadmium in the Tobacco Plant A. Cadmium Tolerance in Tobacco B. Root-to-shoot Transport C. Root and Shoot Distribution D. Cadmium in Field-grown Tobacco Leaves E. Stalk Position Versus Cadmium Accumulation F. Developmental Stage Versus Cadmium Accumulation G. Variation Within the Leaf H. Sub-cellular Localization I. Differences Between Varieties J. Summary IV. External Factors Affecting Cadmium Concentration in Tobacco Leaves A. Soil Characteristics B. Agronomic Practices C. Additional Factors D. Summary V. Options to Reduce the Cadmium Content in Tobacco Leaves A. Molecular and Biochemical Approaches B. Breeding Strategies to Reduce Cadmium C. Soil Cadmium Remediation VI. Conclusion Acknowledgments References

111 Advances in Agronomy, Volume 83 Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved. DOI 10.1016/S0065-2113(04)83003-7

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N. LUGON-MOULIN ET AL. Cadmium (Cd) accumulation in crop plants such as tobacco can lead to human exposure to this carcinogenic metal. Therefore, efforts should be made to minimize the Cd content of soils and crops. We review the options for reducing Cd content of plants, with emphasis on tobacco, a plant that can accumulate relatively high levels of this metal in its leaves. Many studies aimed at understanding Cd biology in plants do not reﬂect ﬁeld conditions, because, often of necessity, non-ﬁeld-like conditions were used. Thus, further study is needed to understand which processes govern Cd uptake, accumulation, etc., under ﬁeld conditions. Numerous factors, such as soil characteristics, agronomic practices, and environmental conditions, impact the uptake of Cd by plants, including tobacco. Identifying anthropogenic sources of Cd and controlling application may limit Cd accumulation in agricultural ﬁelds. Soil remediation strategies may be envisaged to reduce Cd availability to the plant (e.g., soil amendments) or to extract Cd from the soil (e.g., phytoextraction). Another approach for reducing Cd in crops involves genetic modiﬁcation of the plant to reduce Cd uptake or to change its partitioning in the plant. Indeed, more knowledge has been gained in recent decades regarding the mechanisms governing the transport, accumulation, and compartmentalization of Cd by tobacco and other plants. Several types of genes can be considered for genetic engineering to affect these processes. Although no single remedy appears to exist that might drastically reduce the Cd content of crops, including tobacco, an integrated approach may prove useful. q 2004 Elsevier Inc.

I. INTRODUCTION Cadmium (Cd) is classiﬁed as a known human carcinogen (Class 1) by the International Agency for Research on Cancer (IARC, 1993). Since Cd contamination of agricultural products including tobacco (Nicotiana tabacum L.) is primarily responsible for human exposure to this metal, efforts should be made to minimize the Cd content of crop plants. The purpose of this paper is to provide a review of the published data concerning Cd in tobacco and to discuss the options available for reducing it in tobacco and other plants. Other pertinent reviews of studies relating to Cd have been published, including the environmental aspects (WHO/FAO, 1992b), Cd in soils and plants (McLaughlin and Singh, 1999), Cd in the food chain, the health consequences of Cd intake in humans (Ryan et al., 1982; Gairola et al., 1992; WHO/FAO, 1992a), and Cd in mainstream cigarette smoke (Smith et al., 1997). In this review, Cd concentrations are presented in different units [parts per million (ppm), mg/l, mM, etc.] depending on the original work. We did not turn all concentrations into standardized units, because it was not always clear

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whether the original concentrations were calculated on a Cd atom basis, or a Cd(X) molecular basis.

II. CADMIUM IN THE ENVIRONMENT Cadmium, a so-called heavy metal (Nieboer and Richardson, 1980) that was ﬁrst isolated in 1817, is predominantly found in the divalent form (Cd2þ). It occurs naturally in the Earth’s crust and most waters. Water usually contains small amounts of Cd: sea water has an average Cd concentration of about 0.1 mg/l or less, and river water contains dissolved Cd concentrations up to about 0.5 mg/l, although higher values have been reported under certain conditions (Jensen and Bro-Rasmussen, 1992; WHO/FAO, 1992a,b). The concentration and distribution of Cd in the atmosphere depend on the source of emissions (mostly anthropogenic, but also natural, e.g., volcanic eruptions) and other parameters, such as wind direction (Alloway and Steinnes, 1999; Ragosta et al., 2002). Atmospheric concentrations of Cd usually range up to 5 ng/m3 in rural areas, 5– 15 ng/m3 in urban areas, and up to 60 ng/m3 in industrial areas, although higher values can occur (Jensen and Bro-Rasmussen, 1992; WHO/FAO, 1992a,b). Data from several European countries indicate that atmospheric deposition is 2 –7 g Cd/ha/year (Jensen and Bro-Rasmussen, 1992; Alloway and Steinnes, 1999). In the lithosphere, an average Cd concentration of 0.2 ppm has been estimated (Lindsay, 1979). In non-polluted soils, Cd concentration is usually less than 1 ppm (Kabata-Pendias and Pendias, 1992; Traina, 1999), although it can differ between different soil types. Sedimentary rocks generally contain the highest concentrations of Cd, ranging from less than 0.3 to more than 15 ppm (WHO/FAO, 1992b; Baize et al., 1999; Traina, 1999). Anthropogenic factors are the principal sources of Cd in agricultural soils. Cadmium contamination of agricultural soils is mainly due to the use of Cdcontaining phosphate fertilizers (McLaughlin et al., 1996) and sewage sludges, and, to a lesser extent and especially in industrialized areas, from atmospheric deposition (Alloway and Steinnes, 1999). Once in the soil, Cd is highly persistent and may become available for uptake by plants. Various processes (adsorption/desorption, precipitation/dissolution, and Cd –ligand complex formation) determine the behavior of Cd in the soil solid phase and the soil solution. Numerous components, such as metal oxides, silicates, calcite, hydroxyapatite, microorganisms, and organic matter, which are present in different quantities in different soils, are able to adsorb or bind Cd (Christensen and Haung, 1999). As a soft Lewis acid, Cd can form strong complexes with sulfur compounds (S22, HS2, organic sulﬁdes, and thiols) and with halide ions (e.g., Cl2) (Traina, 1999). In most soils, the majority of Cd is

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found in the solid phase, with only a small fraction in the soil solution (Christensen and Haung, 1999). The latter fraction is thought to represent the main source of Cd that is available for plant uptake. To estimate the bioavailable fraction of Cd in soils, chemical extractants, such as diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), or sodium nitrate (NaNO3), have commonly been used. But these methods of quantitation may not take into account the biological processes occurring in the rhizosphere (e.g., root exudation), which may increase the actual bioavailable Cd around the roots. Moreover, a certain fraction of the adsorbed Cd may be desorbed and thus become available for plant uptake or leaching (Christensen and Haung, 1999). Tobacco root cultures have been used to estimate Cd availability in sludges, but this approach does not account for species-speciﬁc differences (Metzger et al., 1992). In the soil solution, Cd can be found in a free hydrated form, or complexed with organic or inorganic ligands, or in association with humic substances. Wagner (1993) estimated that solutions of non-polluted soils would contain 40 –320 nM Cd at a neutral pH, and about twice this level at a lower pH. Helmke (1999) reported values ranging from about 3 to 200 nM Cd in the solutions from various soils, with free hydrated Cd concentrations ranging from 0.1 to greater than 100 nM. In a recent study on 64 soils (urban, forest, and agricultural soils) containing various levels of Cd contamination, free dissolved Cd concentrations ranged from 0.1 to 2000 nM (Sauve´ et al., 2000).

III. CADMIUM IN THE TOBACCO PLANT Tobacco and other plants require several mineral nutrients, including some metals, for their primary metabolism (Reid, 2001). However, most metal micronutrients are toxic to plants when present at high levels. Moreover, metals such as Cd, mercury (Hg), and lead (Pb) are not thought to be required for plant metabolism. Cadmium may induce oxidative stress and proteolytic degradation, and can have numerous adverse effects on plant physiology, particularly at high concentrations, including chlorosis, necrosis, vein reddening, and root growth retardation (Sanita` di Toppi and Gabbrielli, 1999). To resist Cd toxicity, plants can either exclude this metal or tolerate it (metal sequestered without toxic effects). Metal tolerance is typical for hyperaccumulator plants, which are deﬁned as plants that are able to accumulate a metal at levels that are 100-fold greater than those typically found in non-accumulator plants (Baker et al., 2000). However, natural Cd hyperaccumulators (e.g., Thlaspi caerulescens and Cardaminopsis halleri) are uncommon.

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A. CADMIUM TOLERANCE IN TOBACCO Compared to other crops, tobacco (Nicotiana tabacum) can accumulate relatively high levels of Cd as shown by the results of a pot experiment using soil containing 69 ppm Cd: tobacco leaf concentrations were three times higher than the highest value reported among all other crops tested (Davis, 1984). However, at relatively low Cd exposure, tobacco might not accumulate more Cd than leafy crops, such as spinach, Swiss chard, or lettuce (Wagner, 1993). Various studies have been performed under controlled conditions to study Cd uptake, phytotoxicity, and tolerance in tobacco. However, the Cd exposures used in many of the reported studies were much higher than those found in a ﬁeld situation, and therefore the results may not be directly applicable for understanding the ﬁeld response of tobacco to Cd. Tobacco grown hydroponically with 1.5 mg/l cadmium chloride (CdCl2) developed normally and generally without visible symptoms of toxicity, despite the accumulation of up to 226 ppm Cd in the lower leaves of the plant. In a separate experiment, necrotic spots were observed on lower leaves, which contained a mean Cd concentration of 560 ppm (Phu-Lich et al., 1990). Nicotiana rustica (cv. Pavonii) seedlings grown hydroponically with 5 –40 mM Cd did not show signs of Cd toxicity, and reduced root growth was only observed with 40 mM Cd (Vo¨geli-Lange and Wagner, 1996). We have obtained comparable results in our experimentation (Fig. 1). Exposure to hydroponic levels up to 100 mM CdCl2 was phytotoxic to varying degrees, depending on the Nicotiana species; and exposure to levels closer to 10 mM yielded leaf-lamina Cd concentrations around 200 ppb. Tobaccos (cv. Samsun NN and cv. Samsun) grown in a solution containing 2 mg/l CdCl2 contained up to 255 and 352 ppm Cd in their leaves, respectively, without showing visible symptoms of toxicity, although the yield of cv. Samsun NN was signiﬁcantly reduced by 18.2% in dry weight (Harkov and Brennan, 1981; see also Clarke and Brennan, 1983). Similarly, Clarke and Brennan (1989) did not observe visible symptoms of toxicity in tobacco that contained up to 383 ppm Cd in its leaves, but the overall plant growth was affected. Interestingly, after a 30-day treatment with 0.25 ppm Cd, shoot height signiﬁcantly increased by 10 –40%, and internode length, leaf number and generally fresh weight also increased, while leaf and root dry weight generally decreased, compared with control plants (Clarke and Brennan, 1989). Others have noted an apparent stimulatory effect of low Cd on the growth of plants and algae (Wagner, 1993). A stimulatory effect was also described for tobacco callus tissue and tobacco cell suspension cultures at low to moderate Cd concentrations (Maroti and Bognar, 1985; Hirt et al., 1989, 1990) while high Cd inhibited growth. Hence, while relatively low Cd levels can stimulate some growth parameters in tobacco, such as shoot height, very high Cd concentrations are toxic to the plant. In tobacco ﬁelds, exposure to Cd is not expected to cause visible symptoms

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Figure 1 Demonstration of phytotoxic effects of CdCl2 during hydroponic greenhouse growth of Nicotiana species. N. tabacum (A, B, C), N. rustica (D, E, F), and Nicotiana sylvestris (G, H, I) were grown hydroponically and exposed to 0 mM (A, D, G), 10 mM (B, E, H), and 100 mM (C, F, I) of CdCl2. The plants were ﬂooded and then drained with growth solution (Hoagland’s solution plus Cd) three times daily (i.e., the plants were not grown in a continuous state of ﬂooding). The reservoirs feeding the hydroponic ﬁelds were replaced weekly or as needed. The photographs show that these three species have different sensitivities to Cd exposure. N. sylvestris was nearly killed by the 100 mM dose, and N. tabacum was clearly stunted under the same condition, while N. rustica appeared to thrive.

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of Cd toxicity, since ﬁeld Cd levels are typically low and also because of tobacco’s relative tolerance to Cd. Various effects of high to very high Cd exposure on tobacco physiology have been reported; they include retarded growth, yellowing of leaves, decreased biomass, decreased photosynthetic rate, decreased leaf chlorophyll a and b content, decreased chlorophyll a ﬂuorescence, inhibition of photochemical activity of photosystem II (but not that of photosystem I), rotting of roots, leaf chlorosis, and small rust-like circular spots on leaves (Jiang and Li, 1989; Li et al., 1990, 1992; Phu-Lich et al., 1990; Min et al., 1997; Guadagnini, 2000; Choi et al., 2001). Increasing the Cd concentrations up to 150 ppm in the growth medium led to an increase in the protein content of mid-stalk position leaves (cv. Dajinyuan) (Li et al., 1990, 1992). It is noted that Cd may also affect tobacco – virus interactions (Citovsky et al., 1998; Ueki and Citovsky, 2002). For example, Harkov and Brennan (1981) found that increasing Cd levels from 0 to 2 mg/l in the growth medium increased the sensitivity of tobacco (cv. Samsun NN) to tobacco mosaic virus (TMV). Unlike this TMV-hypersensitive cultivar, no evidence could be found for increased sensitivity to TMV in the systemic host cv. Samsun. Ex vivo studies (tobacco hairy root, cell and tissue cultures) also reﬂect upon Cd phytotoxicity, tolerance and accumulation patterns in tobaccos (Reese and Roberts, 1984; Piqueras et al., 1999; Fojtova and Kovarik, 2000; Guadagnini, 2000; Nedelkoska and Doran, 2000b; Fojtova et al., 2002). Senescence in BY-2 (tobacco bright yellow) cultured cells required a 10-fold higher Cd concentration, compared with that inducing animal cell death, suggesting that there are mechanisms by which tobacco is able to cope with limited exposure to high levels of this metal (Fojtova and Kovarik, 2000). Different Cd concentrations in the culture medium can have quite different effects on cell morphology, viability, and DNA integrity of BY-2 suspension cells (Fojtova and Kovarik, 2000). However, tolerance to Cd can be increased by progressively elevating Cd levels in the culture medium (Huang and Goldsbrough, 1988; Domazlicka and Opatrny, 1989). Differences in Cd uptake and tolerance reported among studies may be due to different initial Cd concentrations in the medium, different exposure time, different species, or other variables.

B. ROOT-TO- SHOOT TRANSPORT Cadmium primarily enters the tobacco plant through the roots. Once in the roots, it can either be stored or exported to the shoot. In N. tabacum, the proportion of Cd translocated from root to shoot is higher than in N. rustica, which efﬁciently retains more Cd in its roots (Fig. 2; Wagner and Yeargan, 1986). Cadmium translocation to the shoot appears to be rapid. In N. rustica exposed to

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Figure 2 Cd distribution in hydroponic, greenhouse-grown Nicotiana species. Six species of Nicotiana were grown hydroponically in the greenhouse and exposed to 10 mM CdCl2. The plants were ﬂooded with growth solution (Hoagland’s solution plus Cd) and drained three times daily. The plants were grown to ﬂowering and then harvested. The different plant organs were separated. The respective organs from three plants were pooled together to create one sample. All samples were lyophilized, digested via microwave in concentrated nitric acid, and analyzed for Cd by inductively coupled plasma-mass spectrometry (ICP-MS). The data showed that N. rustica distinguished itself by having higher Cd concentration in the roots and lower concentration in the leaf lamina, compared with the other ﬁve species.

40 mM Cd, the Cd was detected in the leaves within 3 h (Vo¨geli-Lange and Wagner, 1996). Grafting experiments suggest that N. tabacum roots control the amount of Cd translocated to the shoots and, therefore, the amount of metal accumulated in the leaf (Wagner et al., 1988). Indeed, Cd treatment, added in the nutrient solution or in the soil of stem grafts of N. rustica (cv. Pavonii) shoots on N. tabacum (cv. KY14) roots, resulted in leaf Cd accumulation typical of N. tabacum, and vice versa. Transport to the shoot primarily takes place through the xylem. Xylem loading is likely facilitated by yet-unidentiﬁed membrane-transport processes. Cadmium can certainly reach the xylem via symplastic transport, but probably also through the extracellular spaces (apoplastic transport), at least under high-level exposure. Apoplastic transport has been suggested for zinc (Zn) in T. caerulescens grown with high external Zn concentrations (White et al., 2002). In the xylem, transpiration-driven mass ﬂow may play an important role in the movement of Cd, probably in the form of non-cationic, organic acid complexes, such as Cdcitrate (Senden et al., 1995). Theoretical studies predict that the majority of the iron (Fe) and Zn (the latter being chemically similar to Cd) in xylem sap should be chelated by citrate. In contrast, phytochelatins (PCs) and other

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thiol-containing ligands do not seem to play a direct role in Cd transport in the xylem (Salt et al., 1998), although results obtained by Larsson et al. (2002) suggest that, on prior exposure to high Cd, the presence of PCs in Arabidopsis thaliana may increase Cd uptake and translocation from roots to shoot. Recently, Gong et al. (2003) demonstrated that PCs can be transported from roots to shoots and are indeed involved in Cd root-to-shoot transport in Cd-exposed (20 mM CdCl2) A. thaliana (see Section V.A.3 for a discussion of PCs). Once in the shoot, it is possible that some Cd may be redistributed in the plant via the phloem, as suggested by results of studies on Zn in wheat (Herren and Feller, 1994) and by data obtained with leaf-applied 109Cd (Cakmak et al., 2000). The transport mechanisms of Cd from root to shoot remains to be elucidated in tobacco and other plants, and it likely differs with Cd exposure level.

C. ROOTaAND SHOOT DISTRIBUTION The pattern of Cd accumulation may differ substantially within the genus Nicotiana (Fig. 2; Wagner and Yeargan, 1986). For example, N. tabacum appears as a leaf and root Cd accumulator, whereas N. rustica is primarily a root accumulator. Cd concentration in tobacco, which increases with increasing amounts of Cd in the growth medium, is usually higher in the leaves than in the stem (MacLean, 1976; Ruick and Schmidt, 1981; Clarke, 1983; Clarke and Brennan, 1989; Mench et al., 1989). Clarke and Brennan (1989) reported mean leaf and stem Cd concentrations of 0.7 and 0.3 ppm, respectively, in tobacco grown under controlled conditions, without exogenous Cd supply. Moreover, N. tabacum can accumulate higher Cd concentrations in its leaves than in its roots (MacLean, 1976; Clarke and Brennan, 1983, 1989; Logan and Chaney, 1983; Wagner and Yeargan, 1986; Mench et al., 1989; Keller et al., 2003). By estimating the rootto-leaf Cd ratio from average root and leaf Cd concentrations of 16 cultivars grown under greenhouse conditions without added Cd (Clarke and Brennan, 1989), ratios of less than 1.0 are obtained (from 0 to 0.75; Fig. 3). Ratios greater than 1.0 can also be found for tobaccos exposed to low exogenous Cd. The Brazilian tobacco cultivars, T28, T10, T63, T44, and T48 (Salviano et al., 2001), and “Taiwan tobacco #5” (Wen, 1983) were grown hydroponically, and higher Cd concentrations were reported in the roots than in the shoot/leaves (Fig. 3; Wen, 1983; Salviano et al., 2001). However, these authors found moderate Cd concentrations in the plants that were grown with no added Cd in the growth solution. This suggests Cd contamination either during plant growth or during sample preparation for metal analyses.

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Figure 3 Cd concentrations in leaves (shoot for cv. T28, T10, T63, T44, and T48) and roots of hydroponically or pot-grown tobacco not exposed to exogenous additions of Cd, with root-toleaf (or root-to-shoot) ratios estimated from these average Cd concentrations. Data are from MacLean (1976), Clarke and Brennan (1983, 1989), Wen (1983), Mench et al. (1989), and Salviano et al. (2001).

D. CADMIUM IN FIELD- GROWN TOBACCO LEAVES Several studies have reported Cd concentrations in ﬁeld-grown tobacco leaves. The Cd concentration is variable, depending on agro-climate, soil parameters (see Section IV), and cultivar (see Section III.I). Usually, the Cd concentration in ﬁeld-grown tobacco leaves (including midribs and veins) ranges from less than 0.5 to 5 ppm (Westcott and Spincer, 1974; Frank et al., 1977, 1980, 1987, 1991; Nwankwo et al., 1977; Schmidt et al., 1985; Murty et al., 1986; Oto and Duru, 1991; Saldivar et al., 1991; Tamboue et al., 1991; Bell et al., 1992; Yue, 1992; Cai et al., 1995; Gondola and Kadar, 1995; Shariat and Eddin, 1999; Matsi et al., 2002; Tsotsolis et al., 2002). However, the reports of these studies typically do not deﬁne the leaf stalk position, the tobacco maturity stage, the tobacco variety or the exact part of the leaves analyzed (e.g., with or without midribs). These factors, which are discussed in detail below, may all, to different extents, inﬂuence the reported Cd concentration of tobacco leaves. Furthermore, the reported Cd concentration measured in tobacco leaves also depends on sample preparation and the accuracy of the analytical methods used. Older studies may be more likely to have methodological errors, so care should be taken when comparing values across studies (Scherer and Barkemeyer, 1983).

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Variation in Cd concentration in tobacco leaves is found both at the micro- and macrogeographic scales. For example, in Turkey, Oto and Duru (1991) reported lower concentrations in tobacco from the Aegean region than that from four other regions investigated. Bell et al. (1992) studied metals in mid-stalk, air-cured leaves of Maryland tobacco over several years. Of the 402 samples analyzed, 5% had Cd levels in the range of 0.4– 0.8 ppm, 22.5% in the range of 0.8– 1.5 ppm, 48% in the range of 1.5 –3.2 ppm, 19% in the range of 3.2– 5.7 ppm, and 5.5% had values between 5.7 and 15.6 ppm. The wide range in Cd levels of the last group was attributed to growth in the presence of municipal sludge (can be contaminated with Cd) or inadequate liming (see Section IV.B). Tobacco grown in Cd-contaminated areas can accumulate high levels of Cd in the leaves. Grabuloski et al. (1985) found 35.8– 47.7 ppm Cd in tobacco grown in the vicinity of a Zn and Pb smelter in Macedonia, while values ranged 0.75– 1.82 ppm Cd in the usual tobacco-producing regions of Macedonia. In Cd-polluted areas of Dayu county in the Jiangxi province of China, high average Cd concentrations, ranging from 8.6 (Yue, 1992) to 17.4 ppm (Cai et al., 1995), have been reported in tobacco leaves. In comparison, tobacco leaves from non-polluted areas of the same province had average Cd levels nearly 10 times lower (1.86 ppm; Cai et al., 1995). Yue (1992) also reported a lower average Cd value (1.48 ppm) for tobacco from ﬁve main tobacco-producing areas of China, but there were differences among these areas. It has been noted that ﬁeld trials using metal-contaminated sludge indicate that Cd in tobacco can reach exceptionally high values (67.4 ppm in the leaf lamina, Gutenmann et al., 1982).

E. STALK POSITION VERSUS CADMIUM ACCUMULATION More Cd accumulates in lower leaves than in the medium and upper leaves, suggesting a permanent Cd accumulation mechanism (Westcott and Spincer, 1974; Frank et al., 1977, 1980, 1987, 1991; Wagner and Yeargan, 1986; King, 1988; King and Hajjar, 1990; Phu-Lich et al., 1990; Miele et al., 2002). Studies performed on cured tobacco leaves from Canadian farms located in the prime growing regions of the watersheds of Lake Erie and Lake Ontario indicate that cured sand (lower) leaves have a higher average Cd concentration than upper leaves (Fig. 4; Frank et al., 1977, 1980, 1987, 1991). In contrast, Ruick and Schmidt (1981) found higher Cd concentrations in the laminae of the top leaves of tobacco (cv. “S53”) grown in ﬁelds in Germany (soil Cd background level: 0.101 ppm), yet the same cultivar grown in the laboratory in normal garden soil (Cd background level: 0.77 ppm) tended to have somewhat higher Cd concentrations in the lower leaves.

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Figure 4 Tobacco leaf Cd concentration according to stalk position. (A) Tobacco grown hydroponically with or without addition of Cd to the growth medium (data from Phu-Lich et al., 1990). (B) Field-grown tobacco from Lake Erie and Lake Ontario watersheds in Canada, sampled from 1974 to 1976, and from Ontario sales warehouses (1976p) (data from Frank et al., 1977, 1980). (C) Same as B, but sampled in 1981– 1989 (data from Frank et al., 1987, 1991).

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F. DEVELOPMENTAL STAGE VERSUS CADMIUM ACCUMULATION Leaf Cd accumulation in N. tabacum increases with leaf age (Ruick and Schmidt, 1981; Wagner and Yeargan, 1986). The Cd contents of a mature and an immature leaf of mature N. tabacum grown with 30 mM cadmium sulfate (CdSO4) ranged 300 – 750 and 180 – 350 ppm, respectively (Wagner and Yeargan, 1986). Wagner and Yeargan (1986) reported that Cd was primarily found as soluble Cd in seedling roots of N. tabacum and N. rustica (soluble:insoluble ratio 9:1). In contrast, the ratio was 1:1 in mature roots. The soluble Cd found in roots (both mature and immature) is largely in the form of anionic, low-molecular-weight Cd-binding peptides (phytochelatins), due to the relatively high Cd exposure. In mature roots, the nature of the insoluble fraction is unknown.

G. VARIATION WITHINtTHE LEAF Wagner and Yeargan (1986) reported that Cd was almost uniformly distributed in 38-cm-long mature leaves and 22-cm-long immature leaves of a mature tobacco, with some enrichment in the center lower portion and the center upper portion of mature and immature leaves, respectively. Bache et al. (1985) observed that the leaf lamina contained higher Cd concentrations than did the midribs. Tobaccos (cv. Virginia 115) grown in pots (soil: 0.98 ppm Cd) contained 1.87 and 1.20 ppm Cd in the laminae and ribs, respectively. When 1% of a sludge containing 87.2 ppm Cd was added to the soil, the values were 5.33 and 4.90 ppm Cd, respectively. Similar results were obtained for experimentally ﬁeld-grown tobacco: 3.18 and 2.24 ppm Cd in the leaf laminae and ribs, respectively; and 67.4 and 36.3 ppm Cd, respectively, when 224 metric tons/ha of a Cdcontaminated sludge (84 ppm Cd) was applied to the soil (Gutenmann et al., 1982). Under high Cd exposure, we have also observed that the leaf lamina of six hydroponically grown Nicotiana species contained higher Cd concentrations than did the midribs (Fig. 2). As the Cd concentration is usually higher in the lower leaves than in the upper leaves (see Section III.E), the Cd concentrations in the lamina and in the midribs will also differ according to stalk position. An example for ﬁeld-grown tobacco is provided by Ruick and Schmidt (1981), although these authors did not ﬁnd the highest Cd concentrations in the lower leaves (see Section III.E). They reported 1.57 – 2.57 and 1.47– 1.75 ppm Cd in lower leaf laminae and lower leaf midribs, respectively, while in the mid-leaves, the corresponding values were 1.79– 1.96 and 1.01– 1.35 ppm (Ruick and Schmidt, 1981). The leaf epidermis of tobacco contains long and short trichomes that may act as a sink during metal detoxiﬁcation processes (Choi et al., 2001). Compared with untreated seedlings, tobacco (cv. Xanthi) that was exposed to 200 mM Cd

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for several weeks had up to twofold more trichomes, which secreted more than 10-fold larger crystals (ca. 150 mm long), with an estimated 2.2% Cd content (Choi et al., 2001). These results suggest that, under extremely high Cd exposure, trichomes play a role in a Cd storage and disposal mechanism in tobacco. However, the Cd content of trichomes has not been studied in tobacco grown under ﬁeld Cd concentrations.

H. SUB-CELLULAR LOCALIZATION Within the plant cell, Cd can be found in various cell components (e.g., cell wall, cytoplasm, chloroplast, nucleus, vacuoles; Ramos et al., 2002). Distribution may vary according to the species, organ, and tissue. In addition, differences among studies also depend on the Cd concentrations used. Cd can accumulate in the cell vacuoles of higher plants, which may serve to protect the cell from the toxic effects of Cd (Chardonnens et al., 1998). When grown hydroponically with 20 mM Cd for 1 week, N. rustica (cv. Pavonii) predominantly sequesters Cd within the vacuole sap, but not the tonoplast (Vo¨geli-Lange and Wagner, 1990). However, at ﬁeld-like Cd concentrations, the accumulation pattern may be different. Although most Cd is found in the cell wall fraction in lettuce leaves (Ramos et al., 2002), it is not yet clear to what extent Cd can bind to the cell wall in ﬁeldgrown tobacco. Based on data obtained with tobacco hairy roots cultures exposed to 20 ppm Cd, it appears that most Cd is not associated with the root cell walls, although the very high concentrations used in the study do not allow extrapolation to ﬁeld-grown tobacco (Nedelkoska and Doran, 2000a,b). Using Cd concentrations up to 7.6 mM, Wagner and Yeargan (1986) reported signiﬁcant amounts of Cd in the insoluble fractions containing the tobacco cell walls. In their study, the soluble:insoluble Cd ratio was usually greater than 1. However, some insoluble Cd may be due to binding after homogenization. It is still not clear how Cd is partitioned at the sub-cellular level in conventionally ﬁeld-grown N. tabacum.

I. DIFFERENCES BETWEEN VARIETIES Crop species have been divided into those capable of low, moderate, or high cadmium accumulation (Arthur et al., 2000). Moreover, Cd concentration can vary among cultivars/varieties, as was found for various plant species (McLaughlin et al., 1996). For example, the durum wheat lines, Nile, Biodur, and Hercules, consistently exhibit low Cd concentration (, 100 ppb) whereas the cultivar, Kyle, consistently exhibits high (greater than 200 ppb) Cd concentration (Penner et al., 1995).

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Hydroponic studies show that the response of different N. tabacum cultivars to Cd stress may differ and depend, for example, on Cd supply. Salviano et al. (2001) found differences among ﬁve Brazilian tobacco cultivars grown hydroponically for 30 days. When no Cd was added to the growth solution, shoot Cd concentrations of cv. T28, T10, T63, T44, and T48 were 1.8, 1.5, 2.2, 2.5, and 1.9 ppm, respectively, while corresponding root concentrations were 2.1, 3.0, 2.8, 4.8, and 5.2 ppm, respectively. These data suggest different root-to-shoot transport between these cultivars. In a greenhouse experiment, Clarke and Brennan (1989) investigated the differences in Cd concentration in 16 commercial cultivars of tobacco exposed for 30 days to one of the three Cd treatments: 0, 0.25, and 1 ppm Cd as CdCl2. For the 0 ppm Cd treatment, leaf and root Cd concentrations did not signiﬁcantly differ among the 16 cultivars; only the Cd concentration in the stem of cv. G28 was signiﬁcantly higher (0.7 ppm) than that of cv. NC-232 or McNair-944 (0.1 ppm each). At higher Cd exposures, various responses were reported. For example, the leaves of cv. NC-232 reached a signiﬁcantly higher Cd concentration (382.6 ppm) than those of cv. Coker-48 (243.4 ppm) at the 1 ppm Cd treatment, but at the 0.25 ppm Cd treatment, the opposite occurred. These results show that Cd concentration of a cultivar may vary according to Cd exposure, and hence, those results from high Cd exposures may not mimic real-ﬁeld conditions. This is further illustrated by results obtained by Wagner and Yeargan (1986). When grown hydroponically with 40 mM Cd, the leaves of cv. Beinhart 1-1000 contained 35% of the Cd concentration found in other cultivars (cv. KY14, KY151, KY L4-L8, NC 95, and Burley 21). However, at lower Cd concentrations, the difference was less, as cv. Beinhart 1-1000 contained 80% of the amount of Cd, compared with other N. tabacum cultivars. Moreover, the response of different tobacco cultivars to Cd exposure may also depend on other factors. For example, when infected with TMV, cv. Samsun (a systemic host) appears to be a better leaf Cd accumulator than cv. Samsun NN (hypersensitive host) at Cd treatments from 0 to 2 mg/l (Harkov and Brennan, 1981). When uninfected with this virus, cv. Samsun also accumulated more Cd than cv. Samsun NN at the 0 ppm Cd treatment, but a reverse pattern was observed at the 2 ppm Cd treatment (Clarke and Brennan, 1983). Published ﬁeld data also suggest that some differences exist between tobacco cultivars, although all or part of the differences observed may be due to other factors, such as differences in soil characteristics, or soil Cd heterogeneity. For instance, tobacco varieties cultivated in Germany accumulated Cd at different concentrations when grown in the same, highly Cd-contaminated soil conditions (Isermann et al., 1983). Schmidt et al. (1985) found higher amounts of Cd in a ﬂue-cured tobacco than in a Burley variety. Available published data indicate that differences in Cd uptake and root-toshoot translocation abilities can exist among tobacco cultivars, although most

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tobacco cultivars have not yet been tested. Greenhouse pot studies show that the variety-speciﬁc response is not uniform across the Cd treatments. It is still not clear whether varietal differences in Cd uptake are important, relative to individual plant variation within a cultivar. As the experimental conditions are not the same across studies, direct comparisons are difﬁcult. Moreover, results obtained in greenhouse experiments may not be applicable to ﬁeld situations, where the extent of the differences observed among cultivars may change with various parameters (e.g., soil type, bioavailable Cd concentration in the soil, competing ions) both in time and space. However, in terms of Cd accumulation in N. tabacum leaves, the impact of external factors (described in Section IV.B) appears more important than that of a genetic effect (i.e., a cultivar effect).

J. SUMMARY It is important to emphasize here that most studies involving the addition of exogenous Cd to soil or culture solution have used high or very high Cd concentrations, relative to the levels encountered in the typical ﬁeld environment. Sanita` di Toppi and Gabbrielli (1999) surveyed 62 experimental studies in which Cd exposure ranged from 0.1 to 3000 mM Cd. They found that 91% of these studies used exposures of greater than 1 mM and a mean exposure time of 5 days. As noted in Section II, estimates of available Cd in typical ﬁeld soils range from , 0.003 to 0.32 mM. Thus, caution should be applied when attempting to extrapolate from most laboratory and greenhouse studies to the typical ﬁeld situation regarding mechanisms of Cd uptake and accumulation, sequestration, tissue partitioning, impacts of competing ions, sub-cellular distribution, etc. Available published data indicate that (1) tobacco can accumulate high levels of Cd, (2) tobacco seems to be relatively tolerant to this metal, (3) N. rustica can retain Cd in its roots more efﬁciently than N. tabacum, whereas the latter can translocate Cd from roots to the shoot more efﬁciently, (4) N. tabacum usually accumulates more Cd in its leaves (typically between more than 0.5 and 5 mg Cd/kg dry weight in ﬁeld-grown tobacco) than in its stem and roots, (5) all N. tabacum cultivars may not show the same Cd leaf accumulation patterns, particularly at high Cd exposure, (6) based on stalk position, the lower leaves of N. tabacum accumulate more Cd than the upper ones, both in ﬁeld-grown tobacco and under experimental conditions, and (7) vacuoles represent Cd storage organelles at high Cd exposure. Careful ﬁeld-based studies are needed to clarify and explain the accumulation and distribution, as well as the effects of Cd in the ﬁeld. The many laboratory and greenhouse studies that have probed various aspects of Cd exposure in plants have provided much insight into the responses of tobacco and other plants to the metal. But, comparison of results between studies is often difﬁcult due to variation in conditions of growth, treatment, species, etc.

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Extrapolation of results to the typical ﬁeld case is often clouded by the fact that most pot and hydroponic studies have applied artiﬁcially high or very high concentrations of Cd.

IV. EXTERNAL FACTORS AFFECTING CADMIUM CONCENTRATION IN TOBACCO LEAVES Numerous factors, such as soil characteristics, agronomic practices, and environmental conditions, will impact the uptake of Cd by plants. Several of the factors inﬂuencing the response of plants to Cd are discussed below.

A. SOIL CHARACTERISTICS As tobacco takes up Cd primarily through its roots, the nature of the soil plays an important role in the uptake process. Schmidt et al. (1985) estimated that about 0.47% (range: 0.13– 1.06%) of soil Cd is transferred to the tobacco leaves. Their estimates were derived from a study with Burley tobacco and 0.1 ppm Cd in the soil, of which about 1.5 g/ha was taken up by the tobacco (leaf yield: 1000 kg/ha). Several studies reported different Cd accumulation in tobacco according to soil type. Murty et al. (1986) found more Cd in the leaf laminae of tobacco grown on Alﬁsols than on Vertisols. In a greenhouse study, King (1988) planted tobacco in six mineral soils and three organic soils from the southeastern United States, adding 2 kg Cd/ha in the form of CdCl2. He found a signiﬁcant effect of soil type on the Cd content of the lower four leaves: leaf Cd concentration ranged from 12 to 96 mg/kg (no lime) and from 10 to 28 mg/kg (lime added) according to soil type. However, the addition of Cd salts to a soil does not reﬂect natural soil conditions, because bound and free Cd are not in equilibrium, whereas in natural soils, these fractions are usually in equilibrium. Mench et al. (1989) showed, for example, that for tobacco (cv. PBD6), the bioavailability of Cd added in the form of cadmium nitrate [Cd(NO3)2] is twice that of Cd found in a natural acid sandyclay soil typical of the tobacco-cultivated in the Bergerac region of France.

1.

pH

Cd accumulation by tobacco can be affected by soil type. Some soil characteristics, such as pH, appear to be more important than others. Indeed, Cd adsorption to soil increases with increasing pH. Zachara et al. (1992) showed that

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almost all Cd was sorbed to iron- or aluminum-oxides and aluminosilicates when the pH was above 8. When the pH decreases, Cd adsorption by clay iron, manganese (Mn) oxides and organic matter decreases (Alloway, 1995). Soil pH is important in controlling Cd accumulation by tobacco (Mulchi et al., 1987a; Bell et al., 1988; Adamu et al., 1989; King and Hajjar, 1990; Khan et al., 1992; Gondola and Kadar, 1995; Tsadilas, 2000), although its effect depends on various factors (pH range, soil characteristics, agronomic practices, environmental factors, crop species; King and Hajjar, 1990; McLaughlin and Singh, 1999; McBride, 2002). In contrast to most studies, Schmidt et al. (1985), in a greenhouse experiment, did not ﬁnd a signiﬁcant effect between two pH treatments (5.5 and 7.3) on the Cd content of tobacco leaves.

2.

Cation Exchange Capacity, Cadmium Content of Soil, and Other Soil Properties

Various studies have attempted to predict Cd accumulation in tobacco leaves based on some soil properties. The differences reported between these studies may be due to various factors (e.g., different soil type used). Gondola and Kadar (1995) found a signiﬁcant positive correlation between leaf Cd content and clay content of soils. In a study done in southern Maryland over a ﬁve-county region, encompassing 11 different soils at 33 farms, a signiﬁcant positive correlation was found between leaf Cd and soil cation exchange capacity (CEC) (Adamu et al., 1989). However, there was no signiﬁcant correlation between the Cd concentration of tobacco leaves and the total or DTPA-extractable Cd in the soil (Adamu et al., 1989). Similarly, the latter fraction could not predict Cd accumulation by tobacco (cv. Samsun 53) grown in an acid Cd-contaminated soil (20 ppm CdCl2 added) having received various amounts of lime. But there was a strong correlation between potassium nitrate (KNO3)-extractable Cd and total Cd uptake by tobacco (Tsadilas, 2000). Matsi et al. (2002) analyzed ﬁve tobacco types (Burley, ﬂue-cured, Basma, Kabakulak, Samsun) in northern Italy and Greece and found a signiﬁcant correlation between the level of Cd in tobacco and soil characteristics (DTPA-extractable Cd, pH, clay content; correlation coefﬁcient ¼ 0.40, p , 0:001). However, the correlation coefﬁcients ðrÞ were rather low. The correlations improved when only considering soils with ﬁne and moderately ﬁne texture. Moreover, when each variety was analyzed separately, the importance of the soil variables in predicting the Cd concentration in tobacco showed some variation (Matsi et al., 2002). This further emphasizes the complexity of the relationship between many interacting factors. Using stepwise regression analysis, Miner et al. (1997) could explain the Cd content of tobacco (cv. K326) grown on sewage-sludge-treated ﬁelds as being inﬂuenced (R2 up to 0.96) by soil factors, namely the pH, CEC, and extractable Cd (either EDTA-, DTPA-, or Mehlich 3-extractable). King (1988)

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predicted the Cd concentration in tobacco leaves grown on limed (pH 5.7 – 7.0) mineral soils by considering two soil variables, namely ammonium oxalateextractable Fe and Cd from limed soil (regression model, R2 ¼ 0:99). Cadmium removal by tobacco was also accurately estimated by considering the same two variables and pH ðR2 ¼ 0:99Þ: Exogenous Cd was added as a salt in this study. In a study done in Hungary, the soil Cd content did not signiﬁcantly correlate with the Cd contents of cured tobacco leaves obtained from the same crop year (Gondola and Kadar, 1995). Upper and lower leaves can accumulate approximately four and eight times, respectively, the Cd concentration found in the soil (Frank et al., 1980). Interestingly, ratios for chromium (Cr), Hg, molybdenum (Mo), nickel (Ni), and Pb were all much lower than for Cd (Frank et al., 1980). Frank et al. (1987) found that the ratio of Cd in lower (sand) leaves to available Cd in soil was 29 and 77 for crop years 1981 and 1983, respectively. The ratio of available soil Cd to total soil Cd varied according to year (0.30, 0.52, and 0.14 in 1980, 1981, and 1983, respectively) and was much higher than that for Cr, Ni, or Pb.

3. Inﬂuence of Other Metals The divalent cations calcium (Ca), cobalt (Co), copper (Cu), Ni, and Pb can compete with, and hence retard, the sorption of Cd by soils (McLaughlin et al., 1996). Moreover, the interactions of Cd with other metals and nutrients can occur during both the uptake process and the subsequent transport within the plant, and these interactions can vary with plant tissue, genotypes, metal, nutrients, and their concentrations (Pendias and Pendias, 1992; Landberg and Greger, 1994; Carvalho et al., 2002; Kabata-Kim et al., 2002; Zhang et al., 2002). Important complex interactions occur during plant uptake between the chemically related Zn and Cd (Welch and Norvell, 1999). Several studies have been designed to better understand the interactions between some metals and Cd in tobacco. Wen (1983) supplied Cu, Zn, and Cd at various concentrations to tobacco (cv. “Taiwan tobacco #5”) grown in quartz pots (pH 6.0– 6.5). The reported results suggest antagonistic effects between Zn and Cd, but because most treatments used metal concentrations impacting plant growth, and because only two plants were used per treatment, deﬁnitive conclusions could not be drawn. However, data obtained in a study using wheat suggest that these two ions may compete with each other during the uptake process at the root plasma membrane (Hart et al., 2002). It was suggested that Zn may inhibit the movement of Cd from one organ to another via the phloem (Welch et al., 1999). Interestingly, Cd concentration in wheat grain could be decreased by up to 50%

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by adding 2.5– 5.0 kg Zn/ha to ﬁelds with low levels of available Zn (Oliver et al., 1994). However, the effectiveness of this treatment decreased over time. Results by Green et al. (2003) also suggest that Zn addition can reduce Cd translocation from roots to the shoot of “Grandin” hard red spring wheat. Interactions between Cd and Fe have also been studied in tobacco. After a 7-day Cd treatment, Yang and Kuboi (1991) found that cultured tobacco (cv. Xanthi NC and cv. Saman SR-1) cells accumulated more Fe and Ca. The Cd concentration of pot-grown tobacco (cv. Dajinyuan) mid-leaves increased with increasing Cd concentration in the growth medium, and this increase was greater when Fe (1000 ppm) was applied with the Cd treatments (Li et al., 1990). In contrast, increasing Cd exposure caused plants to take up less Fe (Duan et al., 1994). Antagonistic effects between Cd and Fe on the physiological traits of tobacco (cv. Dajinyuan) were reported by Li et al. (1992).

B. AGRONOMIC PRACTICES Agronomic practices can impact the uptake of Cd by crops, including tobacco. To better understand the fate of Cd in soils and its uptake by plants, empirical stochastic models considering both metal inputs (e.g., through fertilizers) and outputs (e.g., plant uptake, leaching) have been developed (Keller et al., 2001a, 2002).

1. Sludge Amendments The use of municipal and industrial sludges as soil amendments has both economic and ecological advantages. However, as they can contain Cd, they can also elevate the level of this metal in soils and the plants growing in the soils (Wagner, 1993; Smith, 1994; Keller et al., 2001b). The leaf laminae of tobacco (cv. Virginia 115) experimentally grown on a sludge-amended ﬁeld contained 20 times the Cd concentration of control plants (67.4 and 3.2 ppm, respectively; Gutenmann et al., 1982). In a greenhouse study, Bache et al. (1985) grew cv. Virginia 115 on a Peat-Lite mix soil. When 1% Cd-polluted sludge (87.2 ppm Cd) was added to the soil, the leaf laminae contained 5.3 ppm Cd, while controls had 1.9 ppm. In a subsequent publication, Bache et al. (1986) reported 3.6 ppm Cd in the laminae of cv. Virginia 115 grown in soil, and 62.9 ppm Cd when grown on a municipal sludge-amended soil (sludge Cd concentration: 84 ppm; application rate: 224 tons/ha). Increasing the application rate of a Cd-containing sludge usually leads to an increase in tobacco leaf Cd concentration (Mulchi et al., 1987a,b; Bell et al., 1988; King and Hajjar, 1990). In ﬁeld experiments, Mulchi et al. (1987a) found

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that Cd content of Maryland tobacco (cv. MD609) leaves increased with increasing sludge application rates for three of the ﬁve sludge sources examined. The plant response was not linear; rather, it was quadratic, with respect to sludge application rates (Mulchi et al., 1987a,b). Application of two limed sludges did not result in signiﬁcant changes in leaf Cd concentrations. In a pot study, the effect of increasing sludge application rate (0, 18, 27, and 81 mg/ha) was shown to be more pronounced at low pH, and diminished as pH increased (King and Hajjar, 1990). Residual soil Cd (and other metals) can be derived from soil amendments used on other crops or in previous growing seasons. Hence, past sludge applications may still impact the Cd concentration of plants grown several years later, as suggested by results obtained by Bell et al. (1988). They studied the long-term effect of a sludge applied in 1972 on the Cd content of Maryland tobacco (cv. MD 609) grown in 1983 and 1984, and they reported an increase in leaf Cd concentration with increasing sludge application rate. Frink and Hullar (1985) (cited by Bell et al., 1988) studied tobacco grown in 1976 on a loam soil amended in 1974 with sludges from several sources. They also found an increase in leaf Cd concentration with increasing sludge application rate. In a ﬁeld study, Baldwin and Shelton (1999) applied three composts (containing from 1.0 to 2.9 ppm Cd) at three application rates (25, 50, and 100 tons/ha) to a Dyke soil in North Carolina, USA. The amount of Cd added to the soil varied from 0.03 to 0.29 kg Cd/ha according to the compost and the rate of application. They studied the uptake of metals by Burley tobacco (cv. TN90) grown in 1994 and 1995. Only the rate of application of the co-composted municipal solid waste/wastewater biosolids compost had a linear relationship with the Cd content of cured tobacco leaves; however, it should be noted that in 1994, 21% of the mature cured leaf samples had Cd values below the detection limit (0.8 ppm) using inductively coupled plasma-emission spectrometry (ICP-ES) and in 1995, samples had, in general, values below detection limits. Using soil variables (total- or extractable-Cd, pH), Mulchi et al. (1992) tried to predict the Cd concentration in tobacco leaves grown on two sludge-amended soils. The prediction efﬁcacy differed for these soils (R2 ¼ 0:75 – 0:84 and 0.91– 0.97, respectively), which may have been due to the different sludges used (some being limed), different soil types and soil –sludge interactions at the two sites (Mulchi et al., 1992). These studies show that sludge application can impact the uptake of Cd by tobacco. Several factors must be considered, such as the metal content of the sludge, the origin of the sludge, its application rate, the way in which the sludge and its components will interact with the soil (e.g., effect of pH), and both short- and long-term effects. In general, tobacco should not be grown in soils that have been amended with metal (Cd)-contaminated sludges in the past.

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2.

Liming

As the solubility of Cd is increased in acidic soils, liming is commonly used to raise soil pH and thereby lower Cd uptake by plants (McLaughlin et al., 1996; Dousset et al., 1999). However, liming does not always lead to a reduction of the Cd concentration in crops (Mench et al., 1994b; Mench, 1998; Maier et al., 2002a,b). Liming strategies have been tested to reduce the Cd concentrations of tobacco leaves. It was suggested that lime be applied to highly acidic tobacco soils. In a ﬁeld experiment, Khan et al. (1992) found that Cd concentration in Maryland tobacco leaves signiﬁcantly decreased after dolomitic lime application to two sandy loam soils. In a pot experiment, calcium carbonate (CaCO3) addition resulted in a signiﬁcant decrease in the Cd concentration of tobacco (cv. PBD6) leaves when the soil pH increased from 6.0 to 7.3 (Phu-Lich et al., 1990). When further increasing the pH to 7.7, only the Cd concentrations of the lower leaves decreased signiﬁcantly. In a ﬁeld experiment, a signiﬁcant decrease of Cd was found in all leaves of Burley tobacco (cv. BB16) when the pH increased from 5.5 to 6.4 with different concentrations of Ca– Mg amendments (Tancogne et al., 1989; Phu-Lich et al., 1990). A further increase in pH resulted in only a slight decrease in leaf Cd concentration. When using CaCO3 to change the pH from the 5.8– 5.9 range to the 6.6 –6.7 range, leaf Cd concentration was signiﬁcantly reduced (calcium nitrate [Ca(NO3)2] or ammonia sulfate [(NH4)2SO4] as the nitrogen (N) source, Phu-Lich et al., 1990; see also Tancogne et al., 1988). Liming, however, did not always lead to a reduced Cd content of tobacco leaves grown in Cd-polluted soil. Mench et al. (1994b) applied lime to a metalpolluted sewage-sludge application ﬁeld trial and found that this treatment signiﬁcantly increased the content of Cd in tobacco leaves, compared with the unlimed treatment (170 and 120 ppm, respectively). Liming did not change the Cd content of tobacco grown in an agricultural area near a non-ferrous metal smelter in France. Thus, the efﬁciency of liming on tobacco Cd concentration appeared to depend on the type and rate of liming material added and the type of soil. Other environmental and genetic factors may also affect the response to liming. By reviewing the effect of lime application to sludge-amended ﬁelds, Dousset et al. (1999) noticed that the form of the Cd salt at the time of soil application can be important. Liming may increase the proportion of exchangeable Cd when it is in the form of CdSO4 (salt), but when the Cd originates from sludge or previous harvest residue, the proportion of exchangeable Cd may remain unchanged. Moreover, the effect of liming on other tobacco characteristics, such as yield and chemical composition, has not been extensively studied. Lime application increased tobacco (cv. BU21) yield without considerably affecting leaf quality (chemical and organoleptic characteristics; Lee et al., 1989). However, liming may lead to an undesirable reduction in Mn concentration in tobacco leaves (Mulchi et al., 1987a, 1991, 1992; Khan et al., 1992; Moustakas et al., 1999).

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3. Fertilizers (a) Cadmium in fertilizers. Phosphate fertilizers (P-fertilizers) can contain high to very high levels of Cd, as phosphate rocks used in their manufacturing may contain from 0.2 to about 340 ppm Cd (reviewed in McLaughlin et al., 1996; McLaughlin and Singh, 1999). Cadmium may accumulate to high levels in certain soils as a result of Cd-contaminated P-fertilizers application. Therefore, Cd in P-fertilizers is of great concern, and individual European Union Member States have recently begun to assess the risks arising from fertilizer-derived soil accumulation of Cd (Cupit et al., 2002; de Meeuˆs et al., 2002). In addition to the Cd concentration in P-fertilizers, other factors need to be considered, such as the total input of fertilizer to the soil and the source of the phosphorus (P) anion (Lee and Doolittle, 2002). Bielinska et al. (1999) studied the effect of fertilizer application on the Cd concentration of soil under tobacco cultivation in Poland. The topsoil (0 –5 cm) of a ﬁeld fertilized with 240 kg NPK fertilizer/ha had about the same Cd content as the topsoil of a ﬁeld fertilized with 700 kg Flovit/ha (0.264 and 0.237 ppm, respectively). However, the 5– 15 cm layer and the 15 – 20 cm layer of the ﬁeld fertilized with the NPK fertilizer had higher Cd concentration (0.282 and 0.204 ppm, respectively) than the corresponding layers of the ﬁeld that was fertilized with Flovit (0.077 and 0.069 ppm, respectively). Reducing the use of high Cd fertilizers may limit further Cd contamination of agricultural soils, and total P application rates may be reduced by applying fertilizer in a concentrated area (banding) instead of an even application across a ﬁeld (broadcast). In contrast, non-P-fertilizers generally contain low levels of Cd. However, their use can still lead to an increase in Cd concentrations in crops, possibly through soil acidiﬁcation, enhanced mass ﬂow, or desorption of Cd from the soil solid phase into the soil solution (Gray et al., 2002; Maier et al., 2002b). (b) Effects of fertilizers on cadmium uptake by tobacco. A few studies suggest that the use of P-fertilizers may impact Cd accumulation by tobacco, although the Cd concentrations of the fertilizers used were not given or unknown (Murty et al., 1986; Semu and Singh, 1996). In India, Murty et al. (1986) grew tobacco on two different soils (Vertisol and Alﬁsol), with or without superphosphate application (the amounts of N, K, P and irrigation waters were different for the two soils). Tobacco grown in Vertisol without P addition had a lower Cd concentration in the leaf laminae (range: 0.100 – 0.347 ppm; mean: 0.218 ppm) than the tobacco that had received superphosphate (range: 0.392– 0.501 ppm; mean: 0.455 ppm). However, when farmyard manure was added to the treatments, the opposite was observed (without P addition the mean Cd concentration was 0.432 ppm; with P addition it was 0.354 ppm). Cadmium accumulation in tobacco grown in Alﬁsol did not vary with P treatment. Although no regular trend was observed for the effect of P application on the Cd content of tobacco leaves, these results support other published data that suggest that superphosphate addition may play a role in Cd

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uptake by tobacco depending on soil characteristics. For example, in Tanzania, Semu and Singh (1996) compared the Cd concentrations in tobacco leaves grown in soils having received either low or high levels of P-fertilizers. The ﬁelds that received high levels of P-fertilizers had more total Cd and DTPA-extractable Cd than the ﬁelds that received low levels of P-fertilizers. The lower leaves of the tobacco grown in ﬁelds with low levels of P-fertilizers contained less Cd (but not signiﬁcant) than the lower leaves of the tobacco grown in ﬁelds with high Pfertilizers (means: 0.084 and 0.159 ppm, respectively; ranges: 0.055 – 0.112 and 0.072 – 0.388 ppm, respectively). In a recent experiment, Miele et al. (2002) found that increasing rates (up to 160 kg/ha) of superphosphate fertilizer application had only a slight effect on the Cd concentration of tobacco leaves in Greece (cv. KS82) and Italy (cv. NC55). Metal accumulation in the plant was dependent on site and year. They concluded that a wise choice of P-fertilizers and their application at the suggested rates may not represent a major input source of metals in Italian and Greek tobaccos. Besides P-fertilizers, nitrogen fertilizers (N-fertilizers) may also, to some extent, affect Cd accumulation by tobacco, as suggested by results obtained by Phu-Lich et al. (1990). Under hydroponic conditions, they found signiﬁcant differences in the Cd concentration of tobacco leaves when the N form in fertilizers was in the form of nitrate (NO3) versus NO3:NH4 (2:1). By using NO3:NH4, a two- to fourfold increase in Cd concentration in leaves occurred, depending on solution Cd concentration (0.05, 0.10, and 1.50 mg/l) and stalk position. Nitrate slightly raised the pH of the solution, while ammonium (NH4) slightly lowered it. This may explain the differences in Cd uptake between the two treatments. In a pot experiment (limon-sandy soil; pH 5.8– 5.9) using (NH4)2SO4 as the N source, a threefold increase in leaf Cd concentration was found (irrespective of the stalk position), compared with using Ca(NO3)2 as the N source. However, when CaCO3 was added, no further signiﬁcant differences were found between the two types of N-fertilizers. Further study is needed to assess the effects of different types of fertilizers on Cd accumulation in real ﬁeld conditions, in soils with different characteristics, using different agronomic practices, and various tobacco cultivars.

4. Irrigation Water Irrigating ﬁelds is necessary for optimal growth of crops and, in some locations, for tobacco cultivation. Salts in irrigation waters and groundwater affect soil salinity and can impact the Cd concentration in plants because, for example, chloride may reduce Cd sorption by soil and form phytoavailable chloro-Cd complexes (McLaughlin et al., 1994; Welch and Norvell, 1999). Wu et al. (2002) reported a 15-fold difference between the minimum and the

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maximum Cd concentration in grains of durum wheat grown in the same ﬁeld, while soil Cd content varied only about 2.5-fold in various areas of the ﬁeld. Interestingly, there was signiﬁcant correlation between grain Cd content and soil salinity, particularly with the natural logarithm (ln) of soil chloride ion (Cl2). High levels of Ca2þ in irrigation waters may compete with, and lead to, desorption of Cd from soil surfaces (McLaughlin et al., 1996). While unpolluted irrigation waters may impact plant uptake of Cd through salinization, Cd-polluted irrigation waters can sometimes represent another important source of Cd contamination to the soil, thereby impacting plant uptake of Cd. In China, about 11,000 ha of land may have been polluted with Cd by irrigation water. A survey done in Dayu county in the Jiangxi province of China showed that Cd pollution was due to irrigation water contaminated by waste waters of tungsten-ore dressing plants (Cai et al., 1995). Average Cd concentrations were 0.047 mg/l in the irrigation water from the tributaries of the Zhang river and 0.89 ppm in the irrigated soil (background Cd level: 0.09 ppm). The average Cd concentration of tobacco grown in the exposed area was about nine times higher (17.4 ppm) than that in the control area (1.86 ppm, Cai et al., 1995).

5.

Other Agronomic Practices

Other agronomic practices may impact Cd uptake by plants, including tobacco. In a pot experiment, Mench (1998) found that the Cd concentration in the tobacco shoot systematically and signiﬁcantly increased after a 1-year fallow, regardless of the soil type (four soils tested) or soil Cd content (from 0.14 to 10.7 ppm). However, it is unknown whether fallowing affects Cd accumulation by tobacco in ﬁeld conditions. The use of different crops on the same ﬁeld (crop rotation) may change soil parameters in the rhizosphere (due to different root exudates or different root systems) that may subsequently affect the Cd uptake of the next crop. Tillage or plant spacing may also affect soil parameters and hence, Cd uptake by plants and tobacco, although the effects of these factors have not been well studied (Mench, 1998).

C. ADDITIONAL FACTORS 1.

Climatic Conditions

The use of experimental plot covers illustrates the impact that environmental conditions can have on Cd uptake by plants. Indeed, plot covers can signiﬁcantly increase air- and root-zone temperatures and relative humidity, and they can decrease irradiance. Their use can lead to increased Cd uptake by plants,

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compared with uncovered plants (Baghour et al., 2001; Moreno et al., 2002). Factors such as high precipitation tend to increase Cd uptake in wheat (Andersson and Pettersson, 1981).

2.

Atmospheric Deposition on Leaves

As Cd can be found in the air, it may be deposited via atmospheric dust on the surface of the tobacco leaves as well as on the soil. In a pot trial, Hovmand et al. (1983) reported that atmospheric deposition on leaves accounted for 20 –60% of the total Cd in plants grown in a Danish agricultural farmland located more than 5 km from industrial sources. By applying 109Cd on the leaves of various Triticum spp., Cakmak et al. (2000) recently found important differences in foliar uptake and subsequent translocation of Cd to the shoot and roots according to genotypes. These results may be accounted for by different leaf characteristics, including different uptake abilities by the plasma membranes (e.g., by transporters, see Section V.A.2). However, the respective contribution of direct leaf interception and root uptake to the total Cd present in tobacco leaves is unknown, although the latter is thought to be more important.

3.

Variation with Crop Year

As several agro-climatic variables are known to inﬂuence the concentration of Cd in plants, and as soils evolve with time, it is expected that differences in the Cd concentration of crops will occur in different crop years, at least under certain circumstances. Cured tobacco leaves (mixed stalk position) from Ontario, Canada sampled in 1973, 1974, and 1975 contained average Cd concentration of 3.20, 2.25, and 2.73 ppm, respectively, and ranges from 2.20 to 4.04, 1.25 to 4.30, and 1.40 to 7.02 ppm, respectively. Soil collected from chief tobacco-producing counties in Ontario between 1970 and 1975 had Cd concentrations ranging from 0.15 to 0.78 ppm, with a mean of 0.36 ppm (Frank et al., 1977). Using regression analyses, Frank et al. (1991) did not ﬁnd a signiﬁcant decline in Cd concentrations in cured leaves from Ontario over a 12-year period (1976 – 1988). Similarly, Oto and Duru (1991) did not observe clear differences in the Cd concentrations of Turkish tobacco from the same regions, obtained during consecutive years. Gondola and Kadar (1995) found signiﬁcant differences between crop years (1990 and 1991) for leaf Pb concentrations, but not for Cd, which measured 1.07 ppm in 1990 and 1.15 ppm in 1991. While studying the long term effects of sludge application, Bell et al. (1988) did not ﬁnd signiﬁcant differences in leaf Cd concentration as a function of crop year for Maryland tobacco (cv. MD 609) grown in 1983 and

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1984 (10.61 and 10.91 ppm, respectively). The plant concentrations for all other metals investigated (Zn, Cu, Mn, Fe, Pb, Ni) changed signiﬁcantly. These 2 years had different levels of rainfall in June, July, and August. Bell et al. (1992) investigated metal contents of mid-stalk air-cured leaves of Maryland tobacco. The mean concentration of Cd in the 1980 crop year (1.85 ppm) differed signiﬁcantly from that in the other years studied (1981, 2.19 ppm; 1982, 2.56 ppm; 1983, 2.53 ppm). These data support the idea that different conditions in different crop years may, in certain situations, affect the concentration of Cd in the tobacco plant. However, the factors responsible for these differences were not identiﬁed and are probably related to the inﬂuences already detailed in the above sections.

D. SUMMARY The external factors that contribute to the Cd content of the plant are either related to the Cd source (natural and artiﬁcial) or to Cd bioavailability. Even though the experimental data are contradictory and ambiguous in many instances, some external factors appear to impact Cd accumulation by tobacco. It appears that Cd-contaminated P-fertilizers can be a signiﬁcant source of Cd contamination in the tobacco ﬁeld. This is a preventable situation, and controlling this source of Cd can limit future, additional Cd contamination of agricultural ﬁelds. Atmospheric deposition can be a signiﬁcant source of Cd in soils, particularly in industrialized areas. However, natural soil and other sources (e.g., manure) of Cd will need to be addressed as well. While clay content might be adjustable by soil amendment, pH control via lime application has emerged as a primary means to control Cd bioavailability. Other strategies that leverage Cd bioavailability exist (see Section V.C). A better understanding of speciﬁc agricultural practices, as they relate to the soil properties and Cd bioavailability may guide farming practices in the future.

V. OPTIONS TO REDUCE THE CADMIUM CONTENT IN TOBACCO LEAVES A. MOLECULARaAND BIOCHEMICAL APPROACHES Recent advances in the understanding on molecular and biochemical mechanisms of metal ion uptake, extrusion, transport, and sequestration suggest that molecular and biochemical approaches have the potential to yield higher

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percentage reduction of Cd in tobacco leaves than the other approaches. Research in many of these areas is still at relatively early stages and the available information is often limited to studies using other plants. Based on our current knowledge, it is reasonable to expect that most of the metal transport and sequestration mechanisms observed in other plant species are relevant for tobacco as well, and therefore have been included in the discussions in this section.

1.

Root Exudates

Root exudates containing, for example, low-molecular-weight organic acids (LMWOA), may induce changes in the physicochemical characteristics of the surrounding soil, such as pH, moisture, electrical conductivity, redox potential, oxygen availability, or microbial community (Hinsinger et al., 2003; Jones et al., 2003). Hence, they may affect the solubility of various soil components (e.g., Cd) and thus the availability of such components to plant roots. However root exudates do not necessarily explain differences in Cd accumulation between taxa (Zhao et al., 2001). In the rhizosphere, organo-Cd complexes may account for a signiﬁcant portion of the soil solution Cd (Jones et al., 1994). In particular, citrate can efﬁciently solubilize Cd (Naidu and Harter, 1998; Nigam et al., 2002) and its exudation may enhance Cd solubility in the rhizosphere. As LMWOA may play a role in Cd solubilization and accumulation in plants (Cieslinski et al., 1998), genes that facilitate their release could be introduced by genetic engineering to reduce or enhance Cd uptake (Ryan et al., 2003). The concept of phytoextraction is further discussed in Section V.C.1. Because root cells are mostly mature cells with large vacuoles, vacuolar chelation may predominate over cytosol mechanisms (Rauser, 1999). Wagner (1995) argued that, at the low levels of Cd found in agricultural soils, little or no PCs would be induced, and vacuolar citrate would effectively complex cellular Cd. In response to nutrient metal ion deﬁciencies, such as Fe, graminaceous plants secrete phytosiderophores (e.g., mugenic and avenic acids) to increase the bioavailability of soil metals and help to carry the metals into plant tissue (also see Section V.A.2(a), for a discussion of Cd transport under Fe-deﬁcient conditions). For example, mugenic acids may limit the binding of Cd by hydrous Fe-oxide (Mench et al., 1994b). Phytosiderophores can mobilize Cd from a solid phase even in the presence of the competing metals, Fe, Ca, and Mg, but their presence did not result in a signiﬁcant increase in Cd uptake by barley and wheat (Shenker et al., 2001). This suggests that the release of phytosiderophores may not increase Cd phytoextraction efﬁciency (see Section V.C.1 for a discussion of phytoremediation). In contrast, phytosiderophores may be able to reduce Cd uptake. When maize was exposed to Cd, in hydroponic culture, in the presence of

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root exudates containing 20 -deoxymugineic acid, a prime maize phytosiderophore, the plants accumulated less Cd than did the corresponding experimental controls (Hill et al., 2002). While this result may be due to increased production of phytosiderophores under Fe-limiting conditions, other root exudates may have played a role. These results need to be veriﬁed in conditions that better mimic the ﬁeld situation. The results of these studies suggest that organic acids that are present in the rhizosphere soil may play a role in the solubilization of particulate-bound Cd into soil solution and its subsequent accumulation in plants. However, it should be emphasized that the responses will depend on various factors (e.g., soil characteristics, plant species). Moreover, the sorption of organic acids to the mineral phase and the mineralization by soil microorganisms play a key role in determining the effectiveness of organic acids in most rhizosphere processes (Jones, 1998). Soluble root exudates of N. tabacum (cv. PBD6) extract more Cd than those of N. rustica (cv. Brasilia) and much more than exudates of Zea mays (Mench and Martin, 1991). The extent of Cd extraction by root exudates correlated with Cd bioavailability to these three plants when grown in soil. An increase in Cd solubility in the rhizosphere of apical root zones due to root exudates is likely to be an important cause of the relatively high Cd accumulation in Nicotiana spp. (Mench and Martin, 1991). Although the nature of these exudates was not identiﬁed, it is possible that organic acids were the major components responsible for the increased Cd, Mn, and Cu extraction by Nicotiana root exudates. Krotz et al. (1989) examined the possible involvement of vacuolar organic acids in the accumulation of Cd and Zn in cultured tobacco cells exposed to non-growth-inhibiting and growth-inhibiting levels of these metals and in the presence and absence of Cd-peptide (phytochelatin). They concluded that tobacco suspension cells can accumulate Cd and Zn in the form of vacuolar organic acid (mostly malate and citrate) metal complexes. To our knowledge no transgenic tobacco has been engineered to express a protein involved in the biosynthesis of organic acids, with the aim of reducing Cd levels in the leaves. Such manipulation might impact respiratory control by disrupting malate and citrate homeostasis.

2.

Cadmium Transporters

Nutrients needed by the plant are often present in the soil solution in low amounts (e.g., P, N). Therefore, plants must use high-afﬁnity transport systems to accumulate these ionic nutrients. Cadmium, as well as other non-essential trace metal(loid)s such as Cr, Hg, and arsenic (As), are most likely carried across plant membranes via transporters, which may represent the means by which to modify

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Cd transport. Although various enzymes are known to transport Cd (Guerinot, 2000; Theodoulou, 2000; Williams et al., 2000; Clemens, 2001), no Cd-speciﬁc transporters have been unambiguously identiﬁed to date. Indeed, few ion transporters are shown to be absolutely ion speciﬁc. In addition, the regulation of the genes encoding these transporters may be complex, with regard to transcriptional and post-translational regulation. A few transporters that may participate in Cd uptake, accumulation, and mobilization are discussed below and are presented in Fig. 5. Their sub-cellular location(s) have generally not been established and an understanding of the regulation of all transporters is in its infancy. Also, additional transporter types will undoubtedly be identiﬁed before long (Ma¨ser et al., 2001). For example, Gupta et al. (2002) recently deﬁned a new class of heavy-metal (e.g., Cd) binding, histidine-rich proteins called metallohistins. (a) ZIP family. The ZIP family of metal transporters takes its name from zinc regulated transporter- (Z RT) and iron regulated transporter- (I RT) like Proteins (Grotz et al., 1998) (reviewed in Guerinot, 2000; Ma¨ser et al., 2001). The yeast

Figure 5 Possible transport mechanisms for Cd uptake and accumulation in plant cells (the cell wall is not shown), including one non-plant transporter (denoted by p) that may be used for genetic engineering. Experimental evidence for most transporters depicted is limited, with the exception of tonoplast CAX and the yeast YCF1 transporting Cd –GSH. Similarly, sub-cellular localization of many of the possible mechanisms shown has not been established. Note that the YCF1 protein was tentatively located in the transgenic plant tonoplast. The relative importance of various mechanisms may depend on Cd exposure level, tissue, developmental stage, species, and other factors. Increasing evidence suggests that the regulation of transporters is complex and integrative. An understanding of which mechanisms primarily function in a particular plant and growth condition will undoubtedly be advanced by integrative studies using transcriptomics and proteomics.

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ZRT1 gene encodes a high afﬁnity transport system for Zn, which can also transport Cd (Guerinot, 2000; Gomes et al., 2002). Fifteen members of the ZIP family have been identiﬁed in Arabidopsis (Ma¨ser et al., 2001), some of which can transport Cd across the plasmalemma. In plant roots, the IRT1 gene is transcriptionally responsive to Fe deﬁciency, but once expressed it may be capable of transporting Cd, for example, in addition to Fe (Eide et al., 1996; Cohen et al., 1998), as occurs in yeast expressing A. thaliana IRT1 (Korshunova et al., 1999). Pea seedlings grown in Fe-deﬁcient hydroponic conditions with 0.2 mM Cd(NO3)2 for 2 days contained approximately twice the amount of Cd in the roots, but about three times less Cd in the shoot, compared to seedlings grown in Fe-sufﬁcient conditions (Cohen et al., 1998). Interestingly, under Fe-deﬁcient conditions, IRT1 appeared to be responsible for a signiﬁcant increase in Cd uptake by the ecotype Ganges of Thlapsi caerulescens, but not in the Prayon ecotype, which is less efﬁcient at hyperaccumulating Cd (Lombi et al., 2002). Regulation of IRT1 is both at the transcript and protein levels. Under Felimiting conditions, overexpression of this gene led to constitutive expression of the mRNA, but the protein was present only in the roots (Connolly et al., 2002). IRT1-transgenic Arabidopsis accumulated more Cd than wild-type plants (Connolly et al., 2002). Cadmium speciﬁcity of the IRT1 transporter may be enhanced by genetic engineering, as suggested by results obtained in studies of yeast by Rogers et al. (2000). They substituted amino acid residues in IRT1 that are conserved among ZIP family members and created yeast mutants limited in the ability to transport Cd. While this transporter may be an interesting means by which to modify Cd uptake by plants, little is currently known regarding its properties, e.g., mechanisms of regulation. While A. thaliana ZIP1, ZIP2, and ZIP3 (and soybean GmZIP1; Moreau et al., 2002) may all transport Cd to some extent, the inhibition of Zn uptake in yeast by Cd was the most severe for ZIP2 (Grotz et al., 1998). Other members of this family may transport Cd; T. caerulescens TcZNT1 has been shown to mediate low-afﬁnity Cd uptake (Pence et al., 2000). Recently, Assunc¸a˜o et al. (2001) have cloned a homolog of ZNT1 in T. caerulescens (TcZNT2). (b) ABC transporters. The ATP binding cassette (ABC) superfamily is a large, ubiquitous, and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters are MgATP-dependent, vanadateinhibited pumps that do not depend on the vacuolar proton gradient. The study of plant ABC transporters is relatively new, but it represents a growing ﬁeld of investigation (e.g., Rea, 1999; Theodoulou, 2000; Martinoia et al., 2002). Full-size ABC transporters have been best characterized, to date. The major groups of these full-size proteins are the multidrug-resistance proteins (MDR), or P-glycoproteins (PGP), the multidrug-resistance-related proteins (MRP) and protein products of the pleiotropic drug resistance genes (PDR) (Martinoia et al., 2002).

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In yeasts and plants, some ABC transporters were shown to confer Cd tolerance. HMT1 is a Schizosaccharomyces pombe gene that encodes a half-size ABC protein that transports apo-PCs and PC –Cd complexes across the tonoplast (Ortiz et al., 1995). There are apparently no authentic HMT1 homologues in Arabidopsis, implying that plants presumably use other ABC transporters for the vacuolar uptake of Cd (Sanchez-Fernandez et al., 2001). The yeast Cd factor (YCF1) belongs to the MRP family and can mediate Cd resistance (Szczypka et al., 1994; Wemmie et al., 1994; Tommasini et al., 1996). The YCF1 gene, regulated by the yAP1 transcription factor (Wemmie et al., 1994), encodes a vacuolar pump capable of transporting organic glutathione (GSH) conjugates and Cd – GSH complexes. The protein catalyzes the uptake and hence, the vacuolar sequestration, of the Cd – GSH complex, Cd2·GSH2 [bis(glutathionato)-Cd] (Li et al., 1997). The structure of Cd2·GSH2 is close to that of Cd2·PC2 (a PC – Cd complex). However, the latter is not transported by the YCF1 protein (YCF1p) (Li et al., 1997), although plant PCs are known to chelate Cd (see Section V.A.3). Interestingly, yeasts bearing the change Trp1225 to Cys in a transmembrane domain of the YCF1p, tolerated a Cd concentration ninefold higher than the wildtype cells (Falco´n-Pe´rez et al., 2001). YCF1-deﬁcient yeast cells (Dycf1) removed 9% of the initial Cd from the growth medium after 24 h, compared with 23% for the control strain (Gomes et al., 2002). The hydrophobic N-terminal extension (a characteristic found in many MRP proteins) of YCF1p contains a cytosolic linker region essential for Cd resistance (Mason and Michaelis, 2002). Tobacco (cv. SR1) has been transformed with the YCF1 gene, as well as with the PDR5 gene, but transcripts of both genes were shorter than expected (Grec et al., 2000). Recently however, this gene was successfully overexpressed in Arabidopsis (Song et al., 2003). Transgenics had increased tolerance to, and accumulated more, Cd and Pb, suggesting that this gene may be useful for Cd phytoextraction (see Section V.C.1). The yeast Bpt1p, an YCF1p homologue, appears to play a minor role in Cd transport (Klein et al., 2002; Sharma et al., 2002). A plant member of the MRP family, AtMRP3, is able to complement yeast YCF1-deletion mutants that are sensitive to Cd (Tommasini et al., 1998). In Arabidopsis, transcript levels of this gene are increased both in roots and shoots of 7-day-old plantlets exposed to Cd. In 4-week-old plants, it is only upregulated in the roots (Bovet et al., 2003). Therefore, the developmental stage appears to play an important role in the expression of this gene in this species. (c) Nramp family. The natural resistance-associated macrophage protein (Nramp) family deﬁnes a family of related proteins that are likely implicated in the transport of divalent metal cations across the plasmalemma. Homologues have been found in a wide range of living organisms, including higher plants (Williams et al., 2000). A. thaliana Nramp 1, 3, and 4 were able to complement yeast mutants defective in Mn and Fe uptake (Thomine et al., 2000; Williams et al., 2000). AtNramp1 mRNAs are preferentially expressed in Arabidopsis roots, whereas

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AtNramp3 and 4 are expressed in roots and aerial parts (Thomine et al., 2000). Interestingly, AtNramp1, 3, and 4 expression in yeast leads to an increased Cd sensitivity and Cd accumulation (Thomine et al., 2000). Arabidopsis seedlings grown on metal-replete medium and overexpressing AtNramp3 displayed Cd hypersensitivity, as assayed by root growth measurements (Thomine et al., 2000). Disruption of AtNramp3 in Arabidopsis (T-DNA tagged line) led to a moderate increase in Cd resistance (increased root growth compared with the control, in medium with increased Cd concentration), but there was no signiﬁcant difference in Fe, Mn, and Zn content or Cd accumulation levels in the plants (Thomine et al., 2000). The Saccharomyces cerevisiae SMF1 and SMF2 genes encode membranetransport proteins that are able to transport Mn, as well as other metals, including Cd (Liu et al., 1997). In yeast, Cd uptake by the SMF1 protein is downregulated by the BSD2 protein, preventing the accumulation of Cd in the cell. It was shown that inactivating the BSD2 gene resulted in bsd2 mutant cells that accumulated high levels of Cd, because SMF1 is stabilized, instead of entering the vacuole where it is degraded (Liu et al., 1997; Liu and Cizewski Culotta, 1999). Metals also play an important role in the post-translational regulation of the SMF1 protein. (d) P-type ATPase. P-type ATPases have been identiﬁed in a wide range of organisms as diverse as bacteria, yeasts, and humans. This large superfamily uses ATP to energize the transport of a variety of ions across biological membranes. These P-type ATPases are distinguished in their formation of a phosphorylated intermediate (hence, called P-type) during the reaction cycle (Williams et al., 2000; Axelsen and Palmgren, 2001). Several bacterial ATPases are involved in Cd transport, like Escherichia coli ZntA catalyzing Cd, Zn, and Pb efﬂux (e.g., Silver, 1996; Lee et al., 2001). In Arabidopsis, the P1B ATPase subfamily contains eight metal transporting members, four of which (the proteins are named HMA1-4) are thought to be involved in Zn/Co/Cd/Pb transport across the plasmalemma (Mills et al., 2003). They are also called CPx-ATPase because they share the common feature of a conserved intramembrane cysteine-proline and either a cysteine (Cys), histidine (His), or serine motif (CPx motif), which is thought to function in metal transport. The type of metal-binding motif, as well as the type of residues close to the motifs, may be involved in the metal selectivity or afﬁnity for a particular metal at the binding site (Williams et al., 2000). Yeasts expressing AtHMA4 are more resistant to Cd. In Arabidopsis, AtHMA4 is expressed at the highest levels in roots and is downregulated in roots after exposure to 1 mM CdSO4 for 30 h (Mills et al., 2003). Some P1B P-type ATPases may have potential as Cd transporters, but their exact role in plants remains to be elucidated. (e) CDF family. Members of the cation diffusion facilitator (CDF) protein group of metal transporters that are involved in the transport of Zn, Co, and Cd have been identiﬁed in bacteria, fungi, plants, and animals. Eukaryotic members

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of the CDF family (or cation efﬂux family; Ma¨ser et al., 2001) share a His-rich region, which may be involved in metal binding. Certain members of the CDF family are thought to function in metal efﬂux or vacuolar uptake; some are found in plasma membranes, while others are in intracellular membranes (reviews in Williams et al., 2000; Ma¨ser et al., 2001). For example, the yeast ZRCl is involved in Cd tolerance, probably by transporting Cd into the vacuole (Li and Kaplan, 1998; Ma¨ser et al., 2001). In plants, the Zn transporter of A. thaliana (AtZAT1) was the ﬁrst CDF member to be characterized (Van der Zaal et al., 1999). When overexpressed in transgenic Arabidopsis, a slight, but signiﬁcant increase in Zn tolerance is observed at high Zn exposure, and Zn accumulates in the roots, but not in the shoots (Van der Zaal et al., 1999). However, Northern blot analysis indicates that ZAT1 is expressed throughout the plant (Williams et al., 2000). It might be involved in vacuolar sequestration of Zn (Van der Zaal et al., 1999; Guerinot, 2000). Antisense plants were viable, and had a wild-type level of Zn resistance and accumulation (Van der Zaal et al., 1999). By reconstituting the ZAT1 protein into proteoliposomes, Bloss et al. (2002) showed that 109Cd transport rate by ZAT1 represents only 1% that of Zn. Moreover, when incubated with Cd, the bacterium Ralstonia metallidurans expressing AtZAT1p did not accumulate different amounts of Cd, compared with controls (Bloss et al., 2002). Other ZAT-related proteins (or metal tolerance protein, MTP) are found in Arabidopsis and other plants. MTPs from Thlaspi goesingense may play a role in the vacuolar sequestration of Cd (Ma¨ser et al., 2001; Persans et al., 2001). More studies are needed on CDF proteins to determine their functions, expression patterns, and possible role in Cd transport. (f) Cation/proton antiporters. Antiporter proteins can exchange protons (Hþ) for metal ions in the vacuole sap, causing the accumulation of the metals in the vacuole. Such a transport was shown for Cd across the vacuole membrane of oat roots (Salt and Wagner, 1993; Gries and Wagner, 1998; Gonzalez et al., 1999). It has also been shown for tobacco roots vesicles (Koren’kov et al., 2002). However, it is not clear whether this mechanism is a lower afﬁnity metal transport, analogous to that which is associated with the Ca2þ/Hþ antiporter (Williams et al., 2000). Further investigation is needed to determine whether distinct proteins transport Ca and Cd (Koren’kov et al., 2002). It has been suggested that the CAX1 and CAX2 (calcium exchanger 1 and 2) genes, which encode putative divalent metal/Hþ antiporters from Arabidopsis (Hirschi et al., 1996), may also transport Cd (see below and Fox and Guerinot, 1998). Hirschi et al. (1996) suggested that CAX2, which is located in the vacuolar membrane in Arabidopsis, may be involved in metal (e.g., Cd) transport by plants. Yeast vacuolar Ca transport was increased when nine amino acids of the CAX1 Ca domain were inserted into CAX2 (CAX2 –9; Shigaki and Hirschi, 2000; Shigaki et al., 2001). Tobacco (cv. KY160) expressing CAX2 under control of the 35S promoter accumulated three times more Cd in the roots and 15% more Cd in stems than controls. It also accumulated more Ca and Mn, suggesting that CAX2

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has broad substrate selectivity. Modiﬁcation of this transporter may be an important component of future strategies to improve plant divalent ion tolerance (Hirschi et al., 2000). CAX4, cloned from an Arabidopsis cDNA library, is expressed in all tissues analyzed, although seemingly at levels lower than CAX2 (Cheng et al., 2002). CAX4 RNA levels increased slightly, in response to Mn, Ni, and Na. CAX4 suppressed, although weakly, the Cd sensitivity of a yeast IRT1-expressing strain (IRT1 transports Cd into the cytosol and thereby is assumed to make the wild-type yeast strains more Cd sensitive) (Cheng et al., 2002). This suggests that CAX4 may transport Cd. In tobacco, CAX4 is located in the vacuolar membrane (Cheng et al., 2002). Alterations at the N-terminus of the CAX transporter genes may modulate their ion transport properties. (g) LCT1. The wheat low afﬁnity cation transporter (LCT1) cDNA (Schachtman et al., 1997) was found to increase the uptake of Ca and Cd when expressed in yeast cells (Clemens et al., 1998). A mild pH dependence in Cd uptake in transgenic yeast was noticed, with uptake rates highest at pH 6. Calcium interfered strongly with the uptake of Cd. It has been proposed that in plants, LCT1 functions as a Ca transport system and might contribute to Cd transport, except in soils with high Ca levels (Clemens et al., 1998). Recently, Amtmann et al. (2001) expressed wheat LCT1 in a salt-sensitive yeast mutant lacking a Na export pump. The transformed yeast showed enhanced Na accumulation and loss of intracellular K (were NaCl sensitive). However, high K and Ca concentrations in the growth medium inhibited Na uptake through LCT1 and hence, rescued the growth of the LCT1-transformed yeast mutant on saline medium. LCT1 cellular localization in plants is unknown (plasma or internal membrane). (h) MATE family. The multidrug and toxic compound extrusion (MATE) family has been recently deﬁned (Brown et al., 1999). Li et al. (2002) isolated a MATE efﬂux protein from Arabidopsis, At DTX1 (for detoxiﬁcation 1), which is probably located in the plasma membrane. KAM3 mutant bacterial cells are Cd sensitive. They did not grow on the medium containing 10 mM Cd or higher, but when transformed with AtDTX1, they grew in the presence of up to 100 mM Cd. This suggests that AtDTX1 may function as an efﬂux carrier to extrude toxic compounds in plant cells. Such systems are well established in bacteria. Based on their results, Li et al. (2002) speculated that AtDTX1 could be required to export the ions into the xylem for long-distance transport. This study shows that plant cells possess at least one efﬂux mechanism, in addition to internal sequestration mechanisms, for metal detoxiﬁcation. But, it remains to be seen if efﬂux systems are restricted to long-distance transport tissues. (i) Cation channels. While relatively little is known about Cd passage through the channels of plant membranes, available published evidence suggests that plasmalemma Ca channels may play a prominent role in the uptake of Cd into the cytosol. Cadmium has been considered a channel blocker in animal systems, but a number of studies have demonstrated its permeability through Ca channels (see Perfus-Barbeoch et al., 2002). Calcium is established as a signal transduction

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messenger and ampliﬁer in stomatal function (White, 2000). Recently, Perfus-Barbeoch et al. (2002) concluded that Cd affects guard cell regulation in an ABA-independent manner after its entry into the cytosol through Ca channels, but not K channels, perhaps by perturbing Ca-calmodulin or Ca-ionmediated signaling processes. The effects of low Cd treatment (10 pM) are perhaps particularly interesting because they suggest that if root cell Ca channels are similarly sensitive to Cd, channel-mediated permeation may be a signiﬁcant route for entry of Cd into roots under low-level Cd exposure, as occurs in the typical ﬁeld situation. It is apparently not known if tonoplast channels (e.g., SV channels) may participate in Cd efﬂux from root cells during Cd translocation to the shoot. These channels are thought to be permeable to various monovalent and divalent cations (White, 2000).

3.

Phytochelatins, Metallothioneins, and Glutathione

Phytochelatins (PCs), metallothioneins (MTs), and glutathione (GSH) are cysteine-rich, low-molecular-weight polypeptides that can bind various metals. The role of plant PCs and MTs in metal tolerance and detoxiﬁcation have been the subject of various recent reviews (e.g., Cobbett and Goldsbrough, 2002). (a) Metallothioneins in plants. In mammalian systems, MTs can bind to metals such as Cd, to protect the cell from toxicity. Animal and yeast MTs are small (approximately 60 amino acids, of which 20 are Cys) gene-encoded proteins consisting of two domains and capable of binding a total of seven divalent metal ions. In their reduced state they provide thiols for metal chelation through mercaptide bonds. The arrangement of Cys residues in different MTs may affect the metal-binding speciﬁcity. Metallothionein proteins are difﬁcult to isolate because of their high sensitivity to oxygen of the thiol groups. The ﬁrst evidence of MTs in plants (wheat) was provided by Lefebvre et al. (1987), and the ﬁrst MT-like gene isolated and sequenced in monocotyledon plants was apparently rgMT-1, a rice stress-inducible MT-like gene (Hseih and Huang, 1998). A number of MT-like proteins have been cloned from several plant species and appear to be expressed at relatively high levels (Zhou and Goldsbrough Peter, 1994; Choi et al., 1996; Liu et al., 2002). However, there is no evidence that they function in metal scavenging in plants (Rauser, 1995; Zenk, 1996), and the level of knowledge about MTs is poor, compared with the insight gained about PCs. Nonetheless, the list of MT-like genes found in plants has grown to 58 from a range of plants (including tomato) and tissues (reviewed in Rauser, 1999). Recently, it was found that banana MT3 gene expression was greatly enhanced in response to CdSO4, copper sulfate (CuSO4), and zinc sulfate (ZnSO4) (Liu et al., 2002). In a brief study, Watanabe et al. (2001) expressed yeast CUP1 gene in sunﬂower at the callus stage and found increased

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resistance to Cd. Hong et al. (2000) improved the stability of human MT (hMT) protein in transgenic E. coli by designing synthetic genes for dimeric and tetrameric hMTs. The oligomeric MT bound twice the amount of Cd as did monomeric hMT. (b) Phytochelatins. The term phytochelatin (PC) was proposed by Grill (1987) to designate a class of peptides induced by metal exposure in plants. Phytochelatin biosynthesis occurs within minutes of Cd exposure. The general structure of PCs is (g-Glu-Cys)n-Gly ðn ¼ 2 – 11Þ: Phytochelatin variants can have residues other than glycine (Gly) at the C-terminus (Zenk, 1996). According to Zenk (1996), PC binding afﬁnity in plant suspension cells is Cd . Pb . Zn . Sb . Ag . Hg . As . Cu . Sn . Au . Bi. Phytochelatins are not gene encoded, but are instead the product of a biosynthetic pathway. They are synthesized in plants from GSH or its analogues by the phytochelatin synthase (PCS or PC synthase), an enzyme activated by Cd and other metal ions (Grill, 1987; Grill et al., 1989). Genes encoding PCS have been recently identiﬁed in yeasts and plants (wheat, Arabidopsis) and homologues were also found in the nematode Caenorhabditis elegans (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999). The Arabidopsis PCS1 (AtPCS1 ¼ CAD1) gene suppresses the Cd-sensitive phenotype in yeast (Ha et al., 1999; Vatamaniuk et al., 1999). The level of AtPCS1 mRNA increased 2.1-fold in wild-type 5-day-old Arabidopsis seedlings subjected to 50 mM Cd, compared with non-treated plants (Lee and Korban, 2002). AtPCS1 is transcriptionally regulated by Cd at the early stage of development, but this regulation disappears as the plants grow older (Lee and Korban, 2002). In 10- and 21-day-old plants, Ha et al. (1999) and Vatamaniuk et al. (1999), respectively, did not detect transcriptional regulation of AtPCS1. A second Arabidopsis gene, AtPCS2, when expressed in yeast, confers Cd resistance at exposure levels up to 100 mM CdCl2, when compared to control yeasts (Cazale´ and Clemens, 2001). In Arabidopsis, AtPCS2 is not upregulated by exposure to 10 mM Cd and is expressed at a relatively low level, compared with AtPCS1, in most plant tissue. However, it has been argued that it may be the predominant PC synthase in some tissues or environmental conditions, because this enzyme has been preserved as a functional PCS through evolution (Cobbet, 2001). Transgenic S. cerevisiae, expressing the wheat PCS gene, TaPCS1, showed an increase in Cd accumulation (Clemens et al., 1999). The ﬁnding that an S. cerevisiae strain that lacked visible vacuoles showed TaPCS1-mediated Cd tolerance suggests that PCs can act as a cytosolic buffer for Cd and other metal ions (Clemens et al., 1999). Transcriptional regulation of TaPCS1 was found in 4-day-old wheat seedlings. In Arabidopsis, Cd stress induces the production of various enzymes involved in Cys synthesis, an important component of GSH and hence, PCs (Harada et al., 2002). Glutathione is not only necessary for PC synthesis, but also for Cd·GSH

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transport via YCF1-type ABC transporters. Therefore, overexpression of g-glutamylcysteine synthetase (g-ECS), a key enzyme in the biosynthetic pathway of GSH, to increase the biosynthesis of GSH may enhance Cd tolerance. For example, Indian mustard (Brassica juncea) seedlings overexpressing the E. coli gene GSH1 (encoding g-ECS) in their chloroplasts had higher levels of GSH, g-Glu-Cys, and PCs (Zhu et al., 1999b). Transgenics showed increased tolerance to Cd, and shoot Cd concentrations were 40– 90% higher than that in the wild type (Zhu et al., 1999b). Xiang et al. (2001) expressed the cDNA encoding g-ECS (the GSH1 gene, May and Leaver, 1995) under the control of the CaMV 35S promoter in both the sense and antisense orientations in Arabidopsis. The resulting plants had GSH levels from 3 to approximately 200% of the level in wild-type plants. However, Arabidopsis plants with elevated levels of both g-ECS and GSH did not show increased Cd resistance when exposed to Cd. Overexpression of glutathione synthetase (GS), another enzyme of the GSH synthetic pathway, also resulted in higher GSH levels. Zhu et al. (1999a) overexpressed the GS from the E. coli GSH2 gene in Indian mustard and obtained transgenic plants accumulating three times more Cd than the wild type when exposed to Cd. Interestingly, yeast strain Dgsh2 (deﬁcient in GS) had the same capacity to remove Cd from the medium after 24 h as did control cells, while strain Dgsh1 (deﬁcient in g-ECS) accumulated about twice as much Cd as controls after the same time period (Gomes et al., 2002). Perhaps, g-ECS can bind Cd and be a substrate for YCF1 transporter. Pilon-Smits et al. (2000) expressed a bacterial GSH reductase in B. juncea targeted to the plastids. Interestingly, Cd levels in shoots were half as high as those in the shoots of control plants, while root Cd levels were roughly the same. Transgenic plants had higher GSH levels than controls both in the presence or absence of Cd. No difference was observed in plant growth. (c) Metallothioneins and phytochelatins in tobacco. In early work, Wagner and Trotter (1982) found that Cd induces a ligand protein in tobacco that binds Cd in mercaptide bonds. Reese and Wagner (1987) demonstrated the formation of Cd-induced small peptides in tobacco leaves (cv. KY14) and cells (cv. Wisconsin 38). The peptides displayed properties that differed substantially from those of animal MTs. Hirt et al. (1990) provided evidence that Cd-binding peptides of N. tabacum (cv. Xanthi) suspension cells appear to be PCs. Vo¨geli-Lange and Wagner (1990) showed that PC – Cd complexes were sequestered in the vacuole of mesophyll protoplasts of tobacco plants exposed to Cd. In a later work on N. rustica, Vo¨geli-Lange and Wagner (1996) found that when more than 5 mM Cd was present in the growth medium, the number of g-ECS repeat units positively correlated with the Cd concentration in the plant. Wagner (1993) argued that, at low levels of Cd, as is found in most soils (i.e., up to 0.3 mM), Cd would mostly be complexed by vacuolar citrate, and it would only be at high levels of Cd exposure that PCs might play a role. Many argued that GSH might be sufﬁcient to sequester Cd at low exposure levels. However, this hypothesis is

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challenged by the observation that PC-mutants of Arabidopsis are highly sensitive to Cd levels as low as 0.6 mM. Schneider and Bergmann (1995) studied the regulation of GSH synthesis and the effect of Cd exposure on g-ECS and GS in suspension cultures of tobacco (cv. Samsun). The activities of the two synthetases showed slight increases of 1.6- and 1.8-fold, respectively, after 3 days of Cd exposure at 100 mM. Maserti et al. (1998) observed the formation of 10 kDa peptides in suspension cell cultures of a hybrid of N. langsdorfﬁi £ N. glauca exposed to 50 and 200 mM Cd. They hypothesized that these peptides were PCs. Fenyk et al. (1998) investigated the interrelationship between PCs, organic acids, and Cd tolerance in cell cultures of N. plumbaginifolia and concluded that the Cd tolerance was not always associated with intracellular accumulation of PCs and organic acids. By co-treatments of cadmium with As or arsenate (AsO4), the tobacco (cv. BY-2) cell content of PC3 and PC4 was lower than in the Cd-treated cells, whereas PC2 content was higher, and Cd content was higher, as well (see also Nakazawa et al., 2001, 2002). To our knowledge, none of the genes encoding for enzymes of the PC biosynthetic pathway or for MT/MT-like proteins have, to date, been isolated from N. tabacum. However, several PC and MT or MT-like genes from other species have been transferred into N. tabacum in an effort to learn more about the function of these proteins in plants (see Section V.C.1). In the late 1980s, Maiti et al. (1988, 1989, 1991) introduced and stably expressed a mouse MT gene in tobacco (cv. KY14 and Petit Havana). Six-week-old plants (cv. KY14) were exposed to ﬁeld-like Cd concentrations (0.02 mM) for 15 days. Transgenic tobacco leaves accumulated about 24% less Cd than untransformed controls, while roots had about 5% more Cd than the controls (Maiti et al., 1989). Yeargan et al. (1992) expressed the mouse MT-I under control of the 35S promoter in Burley tobacco (cv. KY14) and did tests in the ﬁeld (tobacco ﬁeld soil with no additional Cd). This led to a Cd concentration in the shoots of mature tobacco that was about 14% lower than that of control plants. The transformed ﬁeld plants had 12% fewer leaves and were 9% shorter than controls. Copper concentration was approximately 10% higher in the bottom nine leaves; no difference in the level of Zn was observed. The same gene was introduced in tobacco cv. Petit Havana. When grown in solution culture, the transformed plants did not accumulate less Cd in the leaves (rather, about 2% more), but had increased Cd content in the roots (48% higher) when compared with controls. When grown in the ﬁeld, transformed tobacco cv. Petit Havana had a similar Cd content in the shoots as the controls. Root Cd content could not be assessed because the smallest roots could not be recovered from the ﬁeld (Yeargan et al., 1992). It is unknown whether the differences observed between the two varieties were due to variety-speciﬁc differences in Cd translocation from root to shoot. Yan et al. (1995) expressed chimeric genes encoding an E. coli periplasmic glutamine-binding protein (GBP) and a chicken MT in tobacco. The GBP –MT construct (containing a signal peptide) provided secretion of the fusion protein to the cell wall which resulted in

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“somewhat less leaf Cd accumulation” (with a reported range of 15– 22% less Cd in the leaves) when compared with cytoplasmic-targeted lines (no signal peptide) (see also Yan et al., 1996). This result suggests no advantage to apoplastic targeting of MT. However, the oxidative state of a compartment can greatly impact Cd binding to MT. The same laboratory expressed a mouse MT in the vascular tissue of KY14 tobacco, but no signiﬁcant impact on Cd tissue partitioning was observed (Wang and Wagner, 1995). Misra and Gedamu (1989, 1990) introduced a chimeric human MT-II gene (CaMV35S:hMTIIpg:NOS30 ) in N. tabacum (cv. W38). The growth of transgenic tobacco seedlings was unaffected by up to 0.1 mM Cd and the heavy-metal tolerance trait showed Mendelian inheritance. Cuttings of transformed tobacco (stem with three leaves) exposed to 1 mM CdCl2 showed tolerance for up to 7 days (Misra and Gedamu, 1989). This work has apparently not been extended to test root expression of MT. Brandle et al. (1993) examined Cd levels in four lines of ﬁeldgrown ﬂue-cured transgenic tobacco (cv. Delgold) expressing a Chinese hamster MT gene driven by the 35S promoter. No signiﬁcant differences were found in leaf- and root-Cd levels of the transformants, as compared with controls. Only the lower leaves of all transformed lines showed slightly increased Cd levels (4.5 –4.9 ppm) compared with controls (4.3 ppm). Transgenic lines were equal to, or performed more poorly than the control plants with regard to yield, day to ﬂower, and leaf number. The expression of the Chinese hamster MT-II under control of the 35S promoter in transgenic tobacco (cv. Delgold) resulted in a signiﬁcant reduction in Cd accumulation to about 70% (71 ^ 30%) of that in control plants after 2 weeks of exposure to Cd (Hattori et al., 1994). Pautot et al. (1989) introduced artiﬁcial gene constructions based on a mouse MT-I gene into tobacco, under control of the mMT-I and nopaline synthase promoter, but no successful transcription or translation was observed. Heterozygous and homozygous tobacco lines expressing a MT gene were found to contain less Cd in their leaves (49 and 36% less in heterozygous and homozygous lines, respectively; Dorlhac De Borne, 1996). The expression of the mammalian MT-II under the control of the double 35S promoter (Kay et al., 1987) in tobacco seedlings led to a substantial reduction (60 –70% lower than controls) of Cd levels in tobacco (cv. PB D6) leaves (Elmayan and Tepfer, 1993, 1994). Transformed tobacco translocated only about 20% of the Cd absorbed, while controls (35S2-GUS) had a transfer of about 50% (Elmayan and Tepfer, 1994). The MT expression was about 20 times higher than that observed by Brandle et al. (1993). Since tobacco leaf lamina and midribs are often used differently in manufacturing tobacco products, they were analyzed separately. In ﬁeld trials, the Cd levels in leaf lamina were considerably reduced (73% lower than controls). The Cd concentration in control leaf lamina (4.44 ppm) was reduced to 1.18 and 1.75 ppm in transgenic tobacco hemizygous and homozygous for the MT gene, respectively. However, the reduction in leaf-lamina Cd correlated with an increase in Cd in the midribs, stems and root (Dorlhac De Borne et al., 1998).

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Morphologically, the plants were normal, except that they ﬂowered earlier than the controls (see also Dorlhac De Borne, 1996). Pan et al. (1993) introduced a chimeric gene containing two cloned tandemly repeated copies of the alpha-domain (Cd-binding) of the human MT-IA gene into tobacco cells. Growth of transgenic tobacco plants was unaffected by up to 200 mM Cd while the growth of control plants was strongly affected by 10 mM Cd (Pan et al., 1994a,b). Mo et al. (1996) also prepared a 35S mMT-I genetically modiﬁed tobacco, which was tolerant to 100 mM Cd in the growth medium. Zhao (1998) expressed the MT-I in tobacco plants, leading to higher accumulation of Cd, Zn, and Cu in roots and stems (especially for Cd). The quantity of Cd and Zn absorbed was higher in acidic soil than it was in basic soil, whereas the opposite was true for Cu. The quantity of combined metals accumulation in the transgenic plants increased as the MT content increased. More recently, Zhang et al. (2001) introduced the chimeric gene containing a cloned mouse MT processed gene or a cloned mouse MT domain mutant alpha gene into tobacco (cv. NC89). Both types of transgenic plants had signiﬁcantly longer roots and signiﬁcantly higher fresh weight than control plants when grown in Murashige and Skoog (MS) medium with up to 300 mM CdCl2. The herbicide phosphinothricin (PPT) tolerance trait co-segregated with Cd tolerance. The transgenes were stably integrated and inherited. Suh et al. (1998) expressed a wound- and pathogen-inducible MT gene of N. glutinosa in transgenic N. tabacum (cv. Samsun NN) and found tolerance to high levels of Cd. Besides MT genes, genes in the biosynthetic pathway of PC have also been introduced into tobacco. Creissen et al. (1999) overexpressed the E. coli gene GSH1 (encoding g-ECS) in N. tabacum (cv. Samsun NN) chloroplasts. This increased the GSH levels in leaves threefold but, surprisingly, also caused light intensity-dependent necrosis. Harada et al. (2001) found that in Cd-stressed tobacco (cv. Xanthi), the activity of cysteine synthase (CS) was increased to nearly three times that of untreated plants within 7 days. Overexpression of cytosolic CS confers Cd tolerance in tobacco. However, in Arabidopsis, no increase in activity for up to 2 days was found, regardless of the Cd exposure (Harada et al., 2002). CS is certainly regulated at the post-translational level (e.g., protein modiﬁcation, dimerization). (d) A role of phytochelatins in cadmium tolerance. Despite the ability of PCs to form complexes with transition metal ions and its apparent, prominent role in Cd2þ detoxiﬁcation within plant cells, there is no evidence that an elevated production of PCs is responsible for the resistance to toxic metals in all plants. The PC response to Cd may depend on the metal concentration. Wagner (1993) suggested that PCs would play a role only when Cd levels in the soil are high, whereas when Cd levels are low, the metal would be complexed with GSH and vacuolar organic acids. It should be noted that speciation modeling of plant cell vacuoles using computer simulation predicted a signiﬁcant complexation of Cd by citrate (Wang et al., 1991). Ebbs and colleagues investigated the role of PCs

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in metal tolerance in the hyperaccumulator, T. caerulescens, and the related non-accumulator, T. arvense. Although both species produced PCs in response to Cd, they concluded that PCs did not seem to be involved in metal tolerance in T. caerulescens (Ebbs et al., 2002). Indeed, while T. caerulescens had a higher Cd concentration, its total PCs levels were generally lower than the non-accumulator. Results that point to the lack of a signiﬁcant role for PCs have been obtained for other metal-tolerant plant species, such as Silene vulgaris and potato cell lines (see Ebbs et al., 2002). The formation of complexes between metals and PC can be a transient process and, according to Leopold et al. (1999), it is not necessarily important for metal tolerance of plants, which are able to grow on metal polluted soils under natural conditions. In fact, not all metal-tolerant plants exhibit increased production of PCs (Mehra and Tripathi, 1999). For instance, no metal – PC complexes were found in metal-tolerant plants of S. vulgaris and two other species grown on a medieval Cu mining dump (Leopold et al., 1999). This implies that PCs do not necessarily play a general role in metal tolerance, even if they are produced in response to Cd. Therefore, other mechanisms should be responsible for Cd resistance in this case. To what extent these ﬁndings and suggestions apply to tobacco is unknown.

4.

Other Molecular and Biochemical Approaches

Other transporters or compounds may be linked to Cd transport, sequestration, or detoxiﬁcation. A transcriptome analysis of 500 mM Cd-induced genes in Arabidopsis showed that Cd stress activated transcription factors, genes involved in signal transduction pathways, protein denaturation, oxidative stress, and sulfur metabolism (Suzuki et al., 2001). An unidentiﬁed Cd-induced (CdI) gene in Arabidopsis, CdI19, appears to be a metal-binding protein (Suzuki et al., 2001, 2002). When transformed in S. cerevisiae, the strain carrying CdI19 had increased resistance when grown in a medium containing 50 mM Cd (fast growth rates). This protein is localized at the plasma membrane and is able to selectively bind metal by interaction with a CXXC motif (Suzuki et al., 2002). The authors concluded that this protein is able to trap and ﬁx Cd, and ameliorate the toxic effect of Cd in the cell (Suzuki et al., 2002). Cheong and Kwon (1999) demonstrated the importance of the impact of metal ions on the sulfur reductive assimilation pathway. Sulfate in soils is chemically inert and must be reduced to sulﬁde (divalent Cd ions possess high afﬁnity towards sulfhydryl groups) to be incorporated into sulfur-containing compounds, such as Cys. The Arabidopsis AHL gene encodes a 30 (20 ),50 -biphosphate nucleotidase, an enzyme that contributes to the rapid sulfur ﬂux through the assimilatory pathway hydrolyzing 30 -phosphoadenosine 50 -phosphate (PAP) in the PAPS-utilizing reactions (PAPS is reduced to sulﬁte by PAPS reductase). The

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AHL-encoded enzyme is sensitive to metal ions, in particular Cd, by which it is completely inhibited. As a result, the supply of Cys might be limited and affect the synthesis of Cys-containing peptides such as GSH, PCs, and MTs. The authors hypothesize that the overexpression of the AHL gene in plants may lead to increased tolerance to metals.

5.

Summary

Mechanisms for sequestering Cd in plants exposed to low level Cd, which occurs in typical ﬁeld conditions, are still poorly understood. At high Cd exposure PCs appear to be involved in Cd detoxiﬁcation, but there appear to be different mechanisms of Cd tolerance. Attempts to introduce Cd sequestering proteins, particularly animal and plant MTs, into plants to alter Cd accumulation and tissue distribution, have yielded variable results. Efforts to genetically modify various plasma membrane and tonoplast metal transporters (Section V.A.2) to alter Cd accumulation and tissue distribution have only just begun, but the preliminary results of these efforts are promising.

B. BREEDING STRATEGIEStTO REDUCE CADMIUM In crop plants, quantitative variation is a feature of many important traits, such as yield, quality, or disease resistance. The means of analyzing quantitative variation and, especially, uncovering its potential genetic basis are therefore of prime importance for breeding purposes. Cadmium accumulation in plants is undoubtedly controlled by multiple genes. These genes contribute quantitatively, in a developmental stage-speciﬁc, tissue-speciﬁc, and environment-speciﬁc manner to Cd transport, accumulation, and sequestration in the plant. Strong variability of Cd tolerance and accumulation has been observed between varieties of the same species or between species. This variability can be exploited using traditional breeding and represents a valuable source of genetic variation for the development of plant types that accumulate less Cd in the shoot or that hyperaccumulate Cd (see Section V.C.1 for a discussion of phytoextraction and, for example, Srikanth and Anees, 1999). This source of genetic variability remains, for the most part, untapped. In recent years DNA molecular marker technology has been developed and successfully used to assist breeding. DNA markers near genes of interest have been used as tools for selection in conventional breeding programs. Thus, marker-assisted selection may provide a powerful means for selecting low-Cdaccumulating genotypes, avoiding the costly process of Cd-level determination. An example is provided by Penner et al. (1995), who identiﬁed two randomly

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ampliﬁed polymorphic DNA (RAPD) markers associated with low- or high-Cd content in durum wheat. The marker was further used to determine the genetic basis of Cd resistance in 20 introduced durum wheat lines. This group was able to accurately predict the Cd content in 18 of 20 (90%) exotic lines. In addition to the use in marker-assisted breeding, DNA marker studies of Cd accumulation traits could provide the means for understanding the complexity of the genetic control of Cd accumulation and possibly lead to the identiﬁcation of major genes involved in Cd transport or sequestration. Other breeding options and selection techniques exist to generate plants that accumulate either lower or higher Cd levels, e.g., by somaclonal variation or by screening and testing mutagenized (using chemicals or irradiation) populations of plants for high level of Cd tolerance. For example, Navarro et al. (1999) isolated two Cd-tolerant mutants, as initially assessed by root growth, from ethyl methyl sulfonate (EMS) mutagenized Arabidopsis seeds. One mutant, named cdht1, showed an LD50 of 200 mM Cd versus an LD50 of 110 mM Cd for the wild type. The mutants, cdht1 and cdht4, accumulated 2.3 times less Cd than wild-type plants when exposed to 150 mM CdCl2, suggesting a Cd-exclusion mechanism.

C. SOIL CADMIUM REMEDIATION Other potential strategies for reducing the Cd content in plant shoots would be to extract Cd from soil, using plants, for example (see Section V.C.1), or to reduce soil Cd phytoavailability through the use of soil amendments or biological organisms (see Sections V.C.2 and V.C.3). Some of these strategies are brieﬂy discussed, as follows. 1.

Phytoremediation

Phytoremediation is the use of plants to restore contaminated land and water, either by extracting, volatilizing, stabilizing, or inactivating soil organic or inorganic pollutants (Chaney et al., 1997; Salt et al., 1998; Meagher, 2000; Lasat, 2002). This relatively low-cost alternative has received considerable attention in recent years. For Cd, a common phytoremediation strategy involves removing the metal from the soil and accumulating it in above-ground parts of plants that can be easily harvested (phytoextraction; Salt, 2000). Such plants must be able to tolerate and concentrate high levels of Cd (hyperaccumulators) in their harvestable parts if they are to be used on polluted soils (Chaney et al., 1997). In addition to this trait, an ideal plant for Cd phytoremediation should be able to rapidly uptake Cd from the soil, and should have a high rate of Cd translocation to the shoot, with rapid growth and high biomass, so as to decontaminate the polluted site in a reasonable number of

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harvests. And, it should not be an attractive food source for herbivores (it should contain herbivore-deterrent substances). Another difﬁculty arises from Cd distribution in the soil, which may vary from site to site. Hence, root characteristics (e.g., depth and density of the root system) must be taken into account (Keller et al., 2003). Although at present no natural plant has all of the desirable characteristics for Cd phytoremediation, some successful cases of phytoremediation of contaminated sites have been reported. For example, the Cd hyperaccumulator, T. caerulescens (Ganges ecotype), extracted approximately 8.3 mg/kg (43%) Cd present in a contaminated loamy soil (19 mg/kg Cd) in three successive croppings within 391 days (Lombi et al., 2001). Because tobacco can produce a fairly high biomass and accumulates substantial Cd in the shoot, its use in phytoextraction has also been investigated (Guadagnini et al., 1999; Guadagnini, 2000; Kayser et al., 2000; Wenger, 2000; Ruso et al., 2001; Wenger et al., 2002; Keller et al., 2003). Because the use of conventional plants to decontaminate a Cd-polluted soil may take decades, several, mutually non-exclusive means can be envisaged to ameliorate Cd phytoextraction efﬁciency. Agronomic practices may be optimized to enhance biomass, and hence Cd uptake. Conventional breeding can also be used to improve plants for metal phytoremediation (Chaney et al., 2000), although difﬁculties may arise from potential sexual incompatibilities. Moreover, this approach may take time. Other breeding options exist. For example, using in vitro breeding (somaclonal variation) and selection techniques on tobacco callus cultures, Guadagnini (2000) isolated tobacco (cv. Badischer Geudertheimer and cv. Forchheim Pereg) variants containing up to 1.7 times the shoot Cd concentration of parent plants when grown in metal-contaminated soils (pot experiment). Various types of soil amendments (synthetic, natural, or biological) (see Section V.C.3 for a discussion of biological soil amendments) may be added to enhance Cd phytoavailability. For example, as Cd is solubilized at low pH, an artiﬁcial soil acidiﬁcation can be performed. In a greenhouse pot experiment, Wenger et al. (2002) used sulfur amendments to increase Cd uptake by tobacco (cv. Bad. Geudertheimer) grown on two soils contaminated with metals, a Haplic Luvisol (acidic) and a Calcaric Regosol (calcareous) soil. The treatment decreased soil pH and increased the soluble, NaNO3-extractable Cd fraction in both soils. In the acidic soil, an approximate fourfold increase in Cd shoot concentration was observed. However, because plant biomass decreased by 36– 45%, Cd removal under this treatment did not exceed 2.5 times that of controls. In the calcareous soil, Cd uptake by tobacco was only slightly increased, but plant biomass did not decrease. In this soil, Cd removal did not exceed approximately 1.3 times that of controls. Plant uptake of Cd can also be enhanced through the addition of chelators (chelate-assisted phytoremediation, see Salt et al., 1998). Ethylene glycol tetraacetic acid (EGTA) can increase the bioavailability of Cd (Blaylock et al., 1997). The addition of EDTA has been used for Pb phytoextraction (e.g., Vassil et al., 1998; Salt, 2000), although its use

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may not be advisable due to leaching of Pb– EDTA complexes. Moreover, its effect on Cd uptake by plants, which may vary according to soil characteristics, can be the reverse (i.e., decrease in Cd in the shoot) (Kirkham, 2000). Recently, nitrilotriacetate (NTA), a chelator that is more biodegradable than EDTA, was tested in ﬁeld- and pot-studies for phytoextraction of Cd by tobacco (cv. Badischer Geudertheimer) (Kayser et al., 2000; Wenger et al., 2002). In a ﬁeld experiment using polluted soils, NTA treatments resulted in the enhanced uptake of Cd by tobacco, although to a lesser extent than in preliminary greenhouse tests (Kayser et al., 2000). Wenger et al. (2002) found that NTA treatments did not result in a signiﬁcant increase in the Cd concentrations in the tobacco shoot, while shoot biomass decreased signiﬁcantly. At present, there are seemingly no amendments leading to a strong increase in Cd accumulation in aerial (harvestable) parts in different soil types, without negative side effects, such as the accumulation of the amendments in the soil, leaching, toxicity to the plant, and decreased biomass. Another means to increase the efﬁcacy of Cd phytoextraction is through the genetic engineering of plants (Pilon-Smits and Pilon, 2002), speciﬁcally, plants that have been genetically engineered to accumulate higher levels of metal(loid)s (e.g., Bizily et al., 2000; Grichko et al., 2000; Dhankher et al., 2002). Transgenic Indian mustards grown on a metal-contaminated soil accumulated signiﬁcantly more Cd in their leaves than controls (Bennett et al., 2003). Recently, tobacco was engineered to accumulate higher levels of Cd in the leaves. The S. cerevisiae CUP1 MT gene combined with a gene encoding a peptide with six His, under the control of the CaMV 35S promoter, was introduced into tobacco (cv. Wisconsin 38) (Macek et al., 2002). When grown in a sand media with 0.2 mg Cd/l, transgenics showed increased Cd accumulation in the shoot, compared to controls. After 6 weeks of treatment, one line accumulated 90% more Cd in the shoots than controls (31.3 ^ 2.7 and 15.8 ^ 2.9 ppm, respectively), while roots had two times less Cd (10.6 ^ 2.8 and 19.9 ^ 2.1 ppm, respectively) than controls. In contrast, Cd accumulation by transformants containing only the yeast MT gene did not differ from that of controls, as was also found by Thomas et al. (2003). However, these results need to be veriﬁed under conditions that better mimic a typical ﬁeld situation. Genetic engineering may provide the means for developing phytoremediation into a viable industry. As various proteins and compounds can transport or bind Cd in plants (see Section V.A.2), a multi-gene approach, with constitutive or tissue-speciﬁc expression, has potential for preparing a transgenic plant that is useful for Cd phytoremediation. An example is provided by Dhankher et al. (2002), who combined two genes in Arabidopsis, the E. coli arsC gene, which encodes arsenate reductase under the control of a light-inducible promoter (hence, strongly expressed in leaves), and a constitutively expressed g-ECS gene to increase extraction of, and tolerance to, As. The resulting transgenics extracted more As than plants expressing either gene alone. Once the mechanisms

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governing Cd uptake (including rhizospheric interactions and soil Cd speciation), tolerance, and accumulation are better understood, the production of efﬁcient transgenic plants for Cd phytoremediation should be possible. However, while this strategy for soil clean-up may prove valuable to decontaminate nonagricultural Cd-polluted soils, it may not be the most relevant strategy to follow in order to reduce the Cd content in agricultural products.

2.

Non-biological Amendments and Cadmium Stabilization in Soil

As discussed in the above section on phytoremediation, various synthetic and natural, non-biological amendments have been tested as a means to remediate metal-polluted sites either by enhancing Cd mobilization for subsequent phytoremediation (see Section V.C.1) or by decreasing it to avoid metal uptake by plants. For example, the addition of beringite (an alkaline aluminosilicate) and zeolites to a soil can decrease Cd uptake by plants (Mench et al., 1994a; Oste et al., 2001, 2002). A few studies investigated the impact of soil amendments on the Cd uptake by tobacco, but the soils used were heavily contaminated, thus not indicative of the typical agricultural ﬁeld situation. Mench et al. (1994b) tried to immobilize Cd in a soil from a sewage-sludge application ﬁeld trial (soil A) and a soil from an agricultural area near a non-ferrous metal smelter (soil B). They added different chemicals to reduce the Cd content of tobacco shoots (cv. PB D6). Control tobacco contained 120 and 40 ppm when grown in soil A and soil B, respectively. The addition of hydrous Mn-oxide (HMO) signiﬁcantly reduced the Cd levels in tobacco grown in both soils, with reductions of 41 and 63% in soil A and soil B, respectively. The addition of Thomas phosphate basic slags yielded different responses according to soil type: In soil A, the Cd content of tobacco leaves increased (153 ppm), while in soil B, it decreased to 30 ppm. When hydrous Fe oxide was added, a signiﬁcant increase was found in soil A (177 ppm in leaves), but no change was found in soil B (38 ppm in leaves). Sappin-Didier et al. (1993) evaluated the feasibility of adding chemical compounds to immobilize Cd, Zn, Ni, Pb, and Cu in two soil types (an arenic udiﬂuvent and a silty lime soil). The addition of HMO and steel shots at 1% (w/w) to a silty lime soil reduced Cd in the shoots of N. tabacum (cv. PBD6) by 60 and 41%, respectively, compared to untreated soil (Sappin-Didier et al., 1993). In a subsequent publication, Sappin-Didier and Gomez (1994) studied the Cd concentrations in tobacco (cv. PBD6) grown on two soils amended with steel shots. Soil 1 was heavily polluted with sewage-sludge amendments (contained 108 ppm Cd, 248 ppm Ni) and soil 2 originated from an industrial site (contained 18 ppm Cd, 1112 ppm Pb, 1434 ppm Zn). Interestingly, control tobacco grown in soil 1 showed symptoms of ferric chlorosis and necrosis on leaves (probably due to the high Ni content of the

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sewage sludge), which were reduced by the use of steel shot. Nickel concentrations were also decreased (from 162.7 ppm in controls to 33.2 ppm in the steel-shot treatment). In soil 2, steel shot signiﬁcantly reduced the Cd concentration in tobacco leaves by 40%, but slightly increased that of Pb (mean: 36.56 ppm in control tobacco and 50.96 ppm in tobacco grown with steel shot). The use of steel shot signiﬁcantly reduced both water-extractable Cd by 75 and 87% in soil 1 and soil 2, respectively, and Ca(NO3)2-extractable Cd by 57 and 69%, respectively (Sappin-Didier and Gomez, 1994). In soil, steel shot oxidizes into Fe oxide (maghemite and lepidocrite), and these negatively charged compounds can adsorb cations, such as Cd, although other mechanism may occur, as well (Sappin-Didier and Gomez, 1994). The Cd contents of tobacco (control) grown in soils 1 and 2 without steel shot were 120.4 ^ 23.64 and 40.56 ^ 9.02 ppm, respectively. Tobacco grown with steel shot contained less Cd than the controls grown in both soils, but only the values for soil 2 were signiﬁcantly different (100.73 ^ 7.7 and 24.54 ^ 1.3 ppm in soil 1 and soil 2, respectively). Although soil Cd immobilization does not clean the soil, this strategy may, nevertheless, represent a valuable temporary solution to avoid Cd uptake by tobacco and other plants. Current published data indicate that the use of amendments may reduce the Cd concentration in tobacco leaves. However, the long-term efﬁciency, the fate and behavior of the amendments in real, long-term ﬁeld situations, is unknown. More knowledge has to be gained on the resistance of the amendments to chemical weathering, on amendment-microbial and rhizospheric interactions, as well as on the fate and effects of the amendments in soils with different characteristics, and the effects of amendments with different plant species/cultivars. Particular attention should be paid to possible negative side effects, such as enhancement of uptake of other undesirable metals. Cadmium solubility in soils can be reduced by the addition of hydroxyapatite, but this treatment can increase As and Cr solubility (Seaman et al., 2001). Amendments may also increase leaching of Cd and other metals, by increasing the concentration of dissolved organic matter (DOM), for example, which may result in higher leaching of metal – DOM complexes (Oste et al., 2002).

3.

Microorganisms

Fungi as well as soil and root-colonizing bacteria can play an important role in the availability of Cd for plant uptake. For example, some fungi may be used as natural biosorbents of Cd (Jarosz et al., 2002; Simonovicova et al., 2002). Various ectomycorrhizae, predominantly associated with trees, can ameliorate metal tolerance and affect the Cd content of forest trees (Jentschke and Godbold, 2000; Turnau et al., 2002). (a) Arbuscular mycorrhizae. Arbuscular mycorrhizal (AM) fungi (Zygomycetes, Order Glomales) are microscopic fungi able to form associations with the

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roots of many angiosperms, including tobacco. Cadmium uptake by plants may be decreased (e.g., for food-chain safety) or increased (e.g., for phytoextraction) by the presence of mycorrhizae (e.g., El-Kherbawy et al., 1989; Heggo et al., 1990; Guo et al., 1996; Leyval et al., 1997, 2002; Oudeh et al., 2002; Liao et al., 2003). For example, under Cd-stress conditions (100 ppm), the presence of Glomus intraradices signiﬁcantly increased the concentration of Cd in shoots of pea (cv. Frisson and cv. VIR4788) and decreased root concentration in cv. Frisson (Rivera-Becerril et al., 2002). Genetic variability in Cd accumulation existed among three pea cultivars studied (Rivera-Becerril et al., 2002). In addition, the presence of this AM fungus had positive effects on the plants, as shoot, root and pod biomass were decreased by Cd in non-mycorrhizal plants. However, the response at low Cd exposure, not investigated in this study, may differ. Indeed, results by El-Kherbawy et al. (1989) and Heggo et al. (1990) suggest that the original soil Cd concentration plays a role in the plant responses to mycorrhizae (isolated from alfalfa grown in a soil uncontaminated with metals; Fig. 6). For example, mycorrhizae reduced the Cd concentration in alfalfa shoots grown in a high Cdcontaminated silt loam soil at pH 6.0 and 6.7, but at pH 7.2, Cd uptake was increased, compared with controls (El-Kherbawy et al., 1989). The increase in pH led to a decrease in DTPA-extractable soil Cd. Heggo et al. (1990) found a similar

Figure 6 Cd uptake by plants, according to soil Cd concentration (DTPA extractable), after the addition of only bacteria or bacteria and mycorrhizae to various soils. Different soils were used in the soybean study (A), whereas the same silty loam (SL) soil was used in the alfalfa study (B). In the latter, the pH was experimentally changed, leading to a change in the Cd–DTPA concentration of the soil. Data from El-Kherbawy et al. (1989) and Heggo et al. (1990). The letters used to identify the soils in the soybean study follow Heggo et al. (1990).

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trend in soybeans. Cadmium uptake by mycorrhizal soybean decreased in the high Cd-contaminated soils, while the opposite pattern was more likely to be found in less Cd-contaminated soils (Heggo et al., 1990). However, other factors also have to be taken into account, such as soil characteristics, presence of competing ions, extent of extraradical hyphae and root colonization by mycorrhizae. Although data are few, the results from available published studies indicate that AM fungi may be able to sorb Cd and affect Cd uptake by some plants. However, different fungal species have different Cd sorption abilities and different effects on Cd transfer to the plant. Soil characteristics (e.g., Cd concentration, competing ions), as well as the plant species or cultivar, may inﬂuence the response of the mycorrhizae. More research is needed in these areas; in particular, studies are needed to investigate the molecular response of mycorrhizae and the host plant to Cd stress upon AM fungi colonization because very little is known in this area (Taylor and Harrier, 2003). Recently, using functional complementation, Lanfranco et al. (2002) characterized a full-length cDNA encoding an MT in the AM fungus Gigaspora margarita (GmarMT1). When transformed with this cDNA, the Cd-sensitive Dyap-1 yeast mutant, as well as the heavy-metal-sensitive (particularly Cu) Dcup1 yeast mutant, had increased Cd-tolerance, although this gene was not upregulated upon Cd exposure. A more thorough understanding of the molecular basis of mycorrhizal Cd uptake, sequestration, and transfer to the plant will provide new opportunities for engineering fungi to enhance the efﬁciency of soil immobilization of Cd or phytoextraction. (b) Bacteria. Bacteria may affect Cd uptake by plants (see, for example, Fig. 6), and several strains may be used as biosorbents for the bioremediation of Cdpolluted sites or wastewaters (e.g., Devinny and Chang, 2000; Carlot et al., 2002). However, their effect on plant Cd uptake in the ﬁeld is poorly understood. In a ﬁeld experiment after amendment of 5 ppm of CdCl2, inoculation with the commercially available, Cd-tolerant Klebsiella mobilis, CIAM 880, resulted in a twofold decrease in barley-grain Cd concentration, compared with controls (no bacteria), and grain yield was increased (Pishchik et al., 2002). Bacteria can be genetically engineered to be used in the remediation of heavymetal-polluted sites (Valls and de Lorenzo, 2002). Using genetically modiﬁed E. coli, Bang et al. (2000a,b) were able to enhance the removal of Cd from soil, probably by precipitating it as Cd-sulﬁde. Bacteria have also been engineered to overexpress MT genes or PC-like synthetic genes (Bae et al., 2000; Mejare and Bulow, 2001; Valls and de Lorenzo, 2002). For example, E. coli expressing the Neurospora crassa MT gene (Pazirandeh et al., 1995) and Mesorhizobium huakuii expressing a tetrameric human MT gene (Sriprang et al., 2002) accumulated more Cd than controls. The bacterium, R. metallidurans (previously Alcalignes eutropha) CH34, overexpressing a mouse MT on the cell surface, immobilized approximately 70% of the Cd accessible to Nicotiana benthamiana in soil. Moreover, the plant biomass and the chlorophyll content were increased fourfold (Valls et al., 2000). Peptides (or proteins) can also be

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designed to enhance metal speciﬁcity, like engineered bacteria producing a protein containing a heavy-metal-binding motif (Pazirandeh et al., 1998).

VI. CONCLUSION During the past several decades, a considerable amount of knowledge has been obtained on the effects of Cd on plant physiology and how this metal is taken up and compartmentalized in plants (Section III). Transport, accumulation, and compartmentalization of Cd appear to be controlled by a number of mechanisms that may provide multiple options for reducing Cd accumulation by tobacco and other plants (Section V.A). In the absence of well-deﬁned targets for engineering endogenous metal-accumulation- and sequestration-mechanisms, the wellcharacterized animal MT genes have been the focus of initial attempts to genetically modify Cd accumulation in tobacco. The results of these studies can be considered promising, but not yet satisfactory, in view of the goal of reducing and eventually eliminating the transfer of Cd from tobacco and other crops to humans. A complementary approach is to act on the PC-mediated detoxiﬁcation system. Because it is a multi-step process, interference with any of these steps may decrease the capacity of the plant for Cd detoxiﬁcation. But, ﬁrst, it must be shown that PCs are substantial contributors to Cd accumulation and tissue partitioning under low-level Cd (ﬁeld-like) exposure. And, with regard to the phytoremediation objective, altering the expression of genes in the PC biosynthetic pathway may not have a signiﬁcant impact on Cd detoxiﬁcation. Genes for enzymes of GSH synthesis may hold more promise. A potentially successful approach to reducing Cd accumulation is the modiﬁcation of genes that encode proteins that are able to transport Cd, although several challenges need to be overcome (e.g., control at the translational and at post-translational levels; amount and cellular location of the transporters; differences in function, and regulation of transporter activities). Besides studies on plant physiology and metabolism, additional information is needed to increase our understanding of interactive effects between Cd and various extrinsic factors (e.g., other metals, soil parameters, agronomic practices, as discussed in Section IV). A better understanding is needed to optimize limitation of Cd uptake by plants (e.g., in situ remediation) and also to maximize uptake and accumulation toward a phytoextraction objective (Section V.C). Characteristics of the soil (e.g., soil type and pH, Cd concentration, concentration of competing ions, agronomic practices) in which the plant is grown have a substantial impact on Cd bioavailability and plant uptake. But rhizospheric processes (e.g., root exudation, root-colonizing microorganisms) probably also play an important role; additional studies of these processes are needed. Certain agricultural practices may introduce Cd into tobacco ﬁelds (e.g., Cd-contaminated P-fertilizers) and atmospheric

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deposition can be a signiﬁcant source of Cd in soils, particularly in industrialized areas. The relative importance of these factors can vary both at micro- and macrogeographical levels and further study is needed to determine the relative contributions of these factors to the Cd load in different tobacco-growing regions. There is much to be learned about the complex interactions between plants and Cd. Moreover, as was frequently repeated throughout this review, often-used experimental conditions (e.g., hydroponics, the use of very high to unrealistic Cd concentrations, soils in which Cd was added as a salt, the use of seedlings or young plants instead of mature plants) do not reﬂect realistic ﬁeld conditions. Inferences made from hydroponic or pot studies to the ﬁeld can be questionable. However, understanding the interaction effects of various metals on Cd uptake is clearly more difﬁcult to achieve in ﬁeld situations than in pot or solution culture experiments. Furthermore, many studies used high Cd concentrations in the growth media, combined with fairly short exposure times, which may not mimic the plant’s response in real, ﬁeld-like conditions. Therefore, conclusions drawn from such studies may not hold true for conventional ﬁelds. Ultimately, well-controlled ﬁeld tests must be performed (under conditions used in production of the crop) if we are to understand the relevance of possible mechanisms or determine if modiﬁcation of a mechanism is relevant to crop production. Given the complexity of the processes underlying Cd accumulation in tobacco and other plants, options for effectively reducing the metal content in tobacco leaves can only be realized using a system-based, integrated approach.

ACKNOWLEDGMENTS We thank Alec Hayes, Anthony Tricker, and Roger Walk for helpful comments on the manuscript, and Eileen Y. Ivasauskas for help in editing the manuscript.

REFERENCES Adamu, C. A., Mulchi, C., and Bell, P. F. (1989). Relationships between pH, clay, organic matter and CEC and heavy metal concentrations in soils and tobacco. Tob. Sci. 33, 96– 100. Alloway, B. (1995). “Heavy Metals in Soils”. Blackie Academic Professional, London. Alloway, B. J., and Steinnes, E. (1999). Anthropogenic additions of cadmium to soils. In “Cadmium in Soils and Plants” (M. J. McLaughlin and B. R. Singh, Eds.), pp. 97 –123. Kluwer Academic Publishers, Dordrecht. Amtmann, A., Fischer, M., Marsh, E. L., Stefanovic, A., Sanders, D., and Schachtman, D. P. (2001). The wheat cDNA LCT1 generates hypersensitivity to sodium in a salt-sensitive yeast strain. Plant Physiol. 126, 1061–1071. Andersson, A., and Pettersson, O. (1981). Cadmium in Swedish winter wheat: regional differences and their origin. Swed. J. Agric. Res. 11, 49 –55.

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THE IMPACT OF GRAZING ANIMALS ON N2 FIXATION IN LEGUME-BASED PASTURES AND MANAGEMENT OPTIONS FOR IMPROVEMENT John C. Menneer,1 Stewart Ledgard,2 Chris McLay3 and Warwick Silvester1 1

University of Waikato, Private Bag, Hamilton, New Zealand AgResearch Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand 3 Environment Waikato, P.O. Box 4010, Hamilton, New Zealand

2

I. Introduction II. Animal Treading A. Plant Damage and Burial by Hoof Action B. Soil Compaction: Mechanical Impedance Effects on Legumes C. Soil Compaction: Aeration and/or Waterlogging Effects on Legumes D. Signiﬁcance of Plant and Soil Factors, and Limits of Pasture Tolerance III. Animal Grazing A. Diet Selection and Defoliation Effects B. Direct Effects of Defoliation on N2 Fixation IV. Animal Excretion A. Increased Soil N and Grazing Avoidance of Excretaaffected Areas B. Direct Effects of Excreta N on N2 Fixation V. Strategies to Minimise the Impacts of Grazing Animals A. Pasture Management to Aid Legume Production B. Choice of White Clover Cultivar and Companion Grasses C. Tactical Use of N Fertiliser VI. Farm-scale Management Practices A. Soil Management: Preventing Treading and Compaction B. Restricted Grazing and Supplementary Feeding in Winter/Spring C. Technical Based Decision Making for Improved Management VII. Summary and Conclusions Acknowledgments References

181 Advances in Agronomy, Volume 83 Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved. DOI 10.1016/S0065-2113(04)83004-9

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Recent moves toward greater intensiﬁcation of legume-based pasture systems have raised concerns regarding the impact of grazing animals on legume production and symbiotic N2 ﬁxation. This review uses recent research to further our understanding of grazing animal impacts (via treading, defoliation, and excretion) on the N2 ﬁxing performance of legume-based pastures. Options for improving farm management to minimise adverse animal impacts and improve legume performance and N2 ﬁxation are also covered with emphasis on white clover (Trifolium repens). In general, effects on N2 ﬁxation involve both soil and plant processes and are mediated by large-scale changes in legume morphology and physiology and/or by inﬂuencing the legume – grass competitive interaction. For example, defoliation of legumes by grazing animals causes a marked decrease in nitrogenase activity within several hours and recovery takes anywhere from 5 to 21 days depending on the severity of defoliation. Similarly, new research has shown that animal excreta can have prolonged effects on decreasing N2 ﬁxation (e.g., urine decreases N2 ﬁxation by up to 70% with effects lasting for up to 286 days). The magnitude of animal impacts from treading, defoliation, and excretion, individually or as a whole varies greatly and are closely tied to farm management practices and the edaphic features of the entire farm system. Key farm/pasture management strategies identiﬁed to optimise N2 ﬁxation in legume-based pastures include: selecting suitable legume and grass cultivars, restricting grazing intervals, altering seasonal grazing intensity, use of mixed animal types, strategic conservation cuts, and management to reduce soil physical damage. Future research should include the use of validated dynamic models to integrate treading, defoliation, and excretion and predict effects on legume productivity and N2 ﬁxation. Such an approach provides the best opportunity to determine the overall response of the legume system and deﬁne key requirements for management strategies. q 2004 Elsevier Inc.

I. INTRODUCTION Grazing animals have profound effects on individual plants and plant communities in several interrelated ways (Balph and Malechek, 1985; Vallentine, 2001) including: (1) physical impacts on soil and plant material through treading, (2) plant defoliation, and (3) nutrient removal by grazing and redistribution through excreta. These effects are common to all grazed pastoral systems, but in legume-based pastures they play a major role in regulating the efﬁciency of N2 ﬁxation by pasture legumes. The effects that are relevant to legume-based pastures are depicted schematically in Fig. 1. At the individual

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Figure 1 Schematic representation of how grazing animals may affect N2 ﬁxation in legumebased pasture systems.

plant level, grazing animal effects are manifest by changes in legume morphology and physiology, and at the community level they act through modifying the balance of competition between plants in the legume –grass association (Schwinning and Parsons, 1996a). When either of these plant or community related processes cause the legume to be disadvantaged through the inﬂuence of the grazing animal then legume performance can be adversely affected (e.g., Brock et al., 1988; Cluzeau et al., 1992; Menneer et al., 2001, 2003). In grazing systems this is reﬂected by decreasing legume production, persistence and/or a diminishing legume content in the sward, especially if management ignores the legume component. Competition between the grass and legume component in legume – grass swards can also be inﬂuenced by their differing susceptibilities to various other factors such as nutrient deﬁciencies, pests and diseases, and climatic stresses, and these aspects have been discussed in various reviews (e.g., Ledgard and Steele, 1992; Woodﬁeld and Caradus, 1996).

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The magnitude of legume response to treading, defoliation, and excretion is largely determined by the intensity of animal grazing. As grazing intensity increases, it ampliﬁes the negative impacts of the grazing activities (e.g., Curll and Wilkins, 1983; Greenwood and McKenzie, 2001; Menneer et al., 2001, 2003). Other farm system attributes that have been reported to contribute to the adverse effects of grazing on the legume component and N2 ﬁxation include: animal type, pasture management, grazing regime (e.g., continuous versus rotational), and soil properties (e.g., Hay and Baxter, 1984; Murphy et al., 1995a,b; Fothergill et al., 2000; Nolan et al., 2001). Future farming systems are likely to become increasingly intensive due to the limited availability of prime agricultural land and a need to meet greater world food demands. Under this scenario a greater reliance on efﬁcient N2 ﬁxation to meet the N requirements of high-yielding pastures is desirable to reduce the economic and environmental costs of N fertiliser use (Mosier, 2002). In addition, current farming trends of increased dependence on grazing-based systems and a move away from the housing of animals to reduce costs and labour requirements have also led to a greater reliance on legume-based pastures for meeting plant N requirements (Leep et al., 2002). If future legumebased pasture systems are to derive a consistent and signiﬁcant contribution from N2 ﬁxation, and operate at a high level of efﬁciency, then an understanding of the full effects of grazing animals and the underlying processes involved is necessary. For example, it has been reported that intensive grazing of pasture in winter by cattle is often associated with very high stocking rates to ration feed at a time of low pasture growth, and this greatly increases the possible negative impacts on N2 ﬁxation from treading damage and high rates of excreta return (Ledgard et al., 1996a). This review reports the effects of grazing animals in legume – grass pasture systems. Most emphasis is on white clover (T. repens)–grass associations, and reference to other legume-based agricultural systems is for comparative purposes only. The impacts of treading, defoliation, and excreta (Sections II, III, and IV, respectively) on legume growth and efﬁciency of N2 ﬁxation and the underlying processes involved are described. The role of key management practices to reduce grazing animal impacts and optimise legume production and N2 ﬁxation will be discussed in Sections V and VI.

II. ANIMAL TREADING Animal treading of pasture can affect plants directly by plant injury, death and/or burial, and indirectly through soil compaction and puddling resulting from hoof penetration (e.g., pugging) in wet soil and treading on dry soil (Greenwood and McKenzie, 2001). Evidence from limited research with

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pasture legumes and more extensively with crop legumes in compacted soils (Voorhees et al., 1976; Asady and Smucker, 1989; Henderson, 1991; Cook et al., 1996; Grath and Arvidsson, 1997; Mapfumo et al., 1998) indicates that increased soil bulk density is likely to have two component effects on legume growth, productivity, and N2 ﬁxation in grazing systems. Firstly, it can cause an increase in mechanical impedance to root penetration, and secondly, a reduction in aeration and/or an increase in waterlogging of soil. Although numerous studies in grazing systems have shown large negative effects of treading on grass and legume production (Table I), most have failed to adequately separate plant damage effects from the soil physical effects and few have described the underlying processes involved or related them to effects on N2 ﬁxation (Fig. 1).

Table I Summary of Treading Effects on Grass, Clover and Total Pasture Production in Mixed Grass/White Clover Pastures (not all Components Measured in some Studies) Species DM as % difference from non-trodden control Animal Stocking rate type (animals ha21 day21) Ryegrass White clover Grass/clover mix Cattle

Sheep

2.5 20 2.7 67 ha21 7 h 133 ha21 7 h 15 29 29 118 10 49 15 29 10 78 25 100

nd nd nd nd ns ns þ 13a 256a 217 264 257 268 242 250c nd nd

nd 281 nd nd þ 7a,b 274a,b 216a 290a 226 295 28 230 25 259c nd nd

nd, not determined; ns, not signiﬁcant. Varied with season. b Varied with soil moisture. c Varied with species of associated grass species in sward. a

þ1 224 270 27 240 nd þ 12a 269a 218 266 218b 240b nd nd 27 229

Reference Edmond (1970) Edmond (1970) Cluzeau et al. (1992) Nie et al. (2001) Nie et al. (2001) Brown (1968b) Brown (1968b) Brown (1968a) Brown (1968a) Edmond (1958a) Edmond (1958a) Edmond (1962) Edmond (1962) Edmond (1964) Edmond (1964) Edmond (1970) Edmond (1970)

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A. PLANT DAMAGE

AND

BURIAL

BY

HOOF ACTION

Studies in mixed legume –grass pastures under treading have shown large effects of treading on grass and white clover production with little difference between sheep and cattle (Table I). In the majority of these studies it is difﬁcult to determine the relative contribution of soil compaction versus direct damage to plants, but it is likely that plant damage is the key factor responsible for reduced pasture yield in these shorter-term (less than 1 year) investigations. For example, in the numerous studies of animal treading by Edmond (1958a,b, 1962, 1963, 1964) that ranged in duration from 3 to 10 months, plant yield reductions following treading were mainly caused by direct damage to plants by hoof action rather than changes to soil physical properties. In one of these studies using white and red clover (Trifolium pratense) grown in mixture with grasses, Edmond (1962) measured white clover yield reductions of 22% on dry soil, 23% on moist soil, and 30% on saturated soil when treading of 24 sheep equivalents per hectare was compared to no treading over 3 months. White clover had a greater tolerance to treading than red clover with the latter showing a two-fold greater reduction in yield. Compared to white clover, red clover plants (and lucerne, Medicago sativa) grow from a central crown containing basal buds that are more sensitive to treading damage (Lodge, 1991; Frame et al., 1998). In general, the sensitivity of individual pastures species to treading depends on the intensity of grazing and the plant species (e.g., Table II). Notwithstanding, white clover is more susceptible to treading compared to several of its

Table II Pasture Species Tolerance to Treading as Measured by Percent Reduction of Pasture Yield from Sheep Treading at Two Stocking Ratesa Yield reduction (%)b Species Perennial ryegrass (L. perenne) Kentucky bluegrass (P. pratensis) Roughstalk bluegrass (Poa trivialis) Short-rotation ryegrass (L. perenne) White clover (T. repens) Browntop (Agrostis tenius) Timothy (Phleum pratense) Cocksfoot (D. glomerata) Red clover (Trifolium pratense) Yorkshire fog (H. lanatus) a

20 sheep ha21 day21

80 sheep ha21 day21

5 6 0 9 10 24 22 26 37 57

23 31 50 56 60 60 62 80 87 91

Relative to nil treading. Data from Edmond (1964). Data is based on mean DM yield over 11 months and nine separate treading events.

b

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common-companion species (e.g., ryegrass or poa). In recent studies (e.g., Cluzeau et al., 1992; Menneer et al., 2001), white clover further proved its greater susceptibility to treading damage compared to ryegrass by producing much lower yields than its sward associate after treading for up to 4 months. Treading has also been shown to affect clover content with early research on temperate pastures in Europe and the United Kingdom (Bates, 1935; Davies, 1938) ranking Lolium perenne, Poa annua, Poa pratensis, and T. repens as the most resistant to treading damage. Recently, Menneer (2003) measured white clover content in a mixed clover – grass sward in the medium term after a single treading event, and recorded a clover content of 10% under severe treading compared to 40% in the nil-treading control soil. In intensively grazed clover –grass systems, differences in grass and clover tolerance to treading could infer a general growth advantage to associated grasses over clover. Other research has shown that competitive interactions between grass and clover are important in governing clover performance and content in mixed pastures, e.g., self-regulation by clover – grass swards of soil inorganic N concentration (Chapman et al., 1996; Schwinning and Parsons, 1996a,b). Consideration, therefore, should be given to the choice of companion species with clover in mixed swards and their potential to interact through treading. Compared to the upright tufting growth habit of ryegrass and poa, white clover with its prostrate growth form appears to be more prone to burial and stolon fragmentation. In two recent studies using white clover the aerial biomass of stolon decreased by 50– 60% (Cluzeau et al., 1992; Menneer et al., 2001), in the short term (ﬁrst 48 days) after severe treading. Burial of stolon tissue in mixed clover – grass grazing systems is not unusual though, with workers in New Zealand (Hay and Chapman, 1984; Hay et al., 1987; Harris, 1994) and Scotland (Marriott and Smith, 1992; Gooding and Frame, 1997) demonstrating a seasonal cycle of stolon burial in winter (up to 80 –90%), stolon fragmentation and re-emergence of growing points in spring, followed by surface stolon development over summer –autumn (up to 40% stolon burial). Along with worm castings, animal treading is an important factor controlling stolon burial (Hay et al., 1987; Marriott and Smith, 1992). In high rainfall areas or on poorly drained soils where treading causes excessive burial of stolon material the balance between stolon growth and decomposition may be such that the amount of white clover in the sward decreases (Marriott and Smith, 1992). In addition, the smaller plant units resulting from spring fragmentation of parent plants may be more vulnerable to direct treading effects thereby reducing white clover content and yield. Decreased white clover yield due to treading damage or burial is not the only contributor to losses of ﬁxed N in pasture as direct effects on N2 ﬁxation can also occur. For example, Menneer (2003) measured a small proportion of clover N derived from atmospheric N2 (%Ndfa) in mixed clover – grass pasture during the

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Table III Pasture Production and N2 Fixation over 48 days After Pugging. Yield Data are the Sum of Two Harvests. Treatments Followed by a Different Letter for each Plant Measurement are Signiﬁcantly ðP < 0:01Þ Differenta Pugging severity Nil Total pasture yield (kg DM ha21) Clover yield (kg DM ha21) Clover % of total pasture yield %N derived from N2 ﬁxation Total N ﬁxed (kg ha21 48 days21)

2219a 244a 12a 88a 10.8a

Moderate

Severe

SED

1319b 94b 6b 79a 3.8b

527c 12b 2b 47b 0.4b

240 50 2.9 8.2 2.3

a

Data from Menneer et al. (2000).

ﬁrst 48 days after a severe pugging event by dairy cows (Table III). This short-term decrease in %Ndfa in combination with a measured decrease in annual white clover yield culminated in a signiﬁcant reduction in total N ﬁxed, under severe pugging. This study, highlighted the potential for direct negative effects of treading on N2 ﬁxation, as well as losses of white clover yield via plant damage, and indicates that other indirect processes due to treading (e.g., mechanical impedance and/or reduced aeration) may also operate where severe treading occurs.

B. SOIL COMPACTION: MECHANICAL IMPEDANCE EFFECTS ON LEGUMES In grazing systems, there are no reported effects of increased mechanical impedance due to compaction by animal treading on legume growth and N2 ﬁxation. This is in spite of many studies reporting increased soil bulk density in grazed pastures due to treading (Table IV). Therefore, in this section, pot experiments and ﬁeld studies using wheeled agricultural machinery are reviewed with respect to soil compaction and the associated effects of increased mechanical impedance on legume growth and N2 ﬁxation (e.g., Frame, 1985; Cook et al., 1996; Grath and Arvidsson, 1997). In general, the effects of increased mechanical impedance on plant growth and function are largely caused by restricted root growth and reduced soil water availability, and their inﬂuence on decreasing water and nutrient uptake (Bennie, 1991; Henderson, 1991; Cook et al., 1996). Some studies (e.g., Passioura, 1991; Cook et al., 1996) suggest hormonal signalling from the impeded roots may be involved in slowing the growth of shoots.

N2 FIXATION IN LEGUME-BASED PASTURES

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Table IV Effect of Treading by Livestock on Bulk Density of Soils in Grazed Pasture Systems.a Studies Included are Both Short-Term (Months) and Long-Term (Years)

Animal type Stocking rate/treatment Sheep

Cattle

0 –50 ha21 day21 25, 50 ha21 day21 25, 50 ha21 day21 0 –22 ha21 day21 2.5 –37 ha21 day21 7.4 –22 ha21 day21 Light versus intensive 3 £ above control 0 –20 ha21 day21 0 –40 ha21 day21 0 –50 ha21 day21 0 –1.06 ha21 year21 350 ha21 for 8 h 0 –0.9 AUM ha21 0 –4.4 AUM ha21 0 –4.8 AUM ha21 400 ha21 for 12 h Sheep versus cattle 80 ha21 for 24 h

Depth (mm)

Bulk density (g cm23)

Reference

60 0–50 0–50 0–60 0–38 0–50 0–50 0–60 0–80 0–51 0–50 0–80 20–84 0–75 0–75 0–75 0–50 0–50 50–100

1.08 ! 1.28 1.12 ! 1.42b 1.10 ! 1.26c 0.89 ! 1.05 1.15 ! 1.43 1.27 ! 1.57 0.83 ! 1.06 1.16 ! 1.28 1.17 ! 1.26 1.34 ! 1.61d 1.04 ! 1.30e 1.00 ! 1.29 1.42 ! 1.50 1.02 ! 1.07 0.89 ! 1.07 0.75 ! 0.90 0.52 ! 0.76 1.12 ! 1.37 0.96 ! 1.06

Edmond (1958a,b) Curll and Wilkins (1983) Curll and Wilkins (1983) Willatt and Pullar (1983) Langlands and Bennett (1973) Carter (1977) Greenwood and McNamara (1992) Russell (1960) Greenwood et al. (1997) Stephenson and Veigel (1987) Daniel et al. (2002) Taboada and Lavado (1988) Kelly (1985) Naeth et al. (1990) Naeth et al. (1990) Naeth et al. (1990) Singleton and Addison (1999) Murphy et al. (1995a) Drewry and Paton (2000)

AU, animal unit, a measure of grazing pressure equivalent to a dry cow weighing 450 kg; AUM, animal unit per hectare for 1 month. a Modiﬁed from Greenwood and McKenzie (2001). b Without excretal return. c With excretal return. d Mean of ﬁve sampling dates. e After 10 years of treatment.

1.

Legume Shoot and Root Growth

Research with pasture legumes has shown reduced root elongation and diameter, and a decrease in shoot dry weight as bulk density increases (Table V). For example, the adverse effects of mechanical impedance (under controlled conditions without the effects on soil aeration) have been measured for white clover, with Cook et al. (1996) reporting marked reductions in root length, and root and shoot dry weight, when plants potted in sand were subjected to an increase in bulk density at 20 mm depth from 1.50 to 1.80 g cm23 (Table IV). In the same study, comparisons of white clover with several grass species (L. perenne and Agrostis capillaris) revealed that the effect of increased bulk density on root length appeared to be less with white clover. This may be due to the greater ability of dicotyledonous species (which have a thick seminal taproot)

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Table V Summary of Effects of Increasing Soil Bulk Density Under Controlled Conditions on Shoot and Root Growth of Selected Pasture Legumes Bulk density (g cm23)

Soil type

Base level

Treatment level

White clovera (T. repens L.) Subterraneum cloverb (T. subterraneum L.)

Sand culture

1.50

Silt loam

1.10

Lucernec,d (M. sativa L.)

Clay loam

1.15

Sandy loam

1.20

1.70 1.80 1.2 1.4 1.6 1.27 1.38 1.50 1.38 1.56 1.74

Species

Percent decrease in Shoot Biomass

Root Biomass

Root length

Root diameter

38 37 Nil 38 52 18 40 78 38 53 84

45 35 – – – Nil Nil 31 Nil Nil 57

37 44 ns 55 82 – – – – – –

– – ns 41 62 – – – – – –

ns, not signiﬁcant. Data from Cook et al. (1996). b Data from Nadian et al. (1996). c Data from Mapfumo et al. (1998). d May have also been limited by reduced aeration. a

to penetrate compacted soil than monocotyledonous plants (with thinner roots) (Materechera et al., 1991), and could modify the competitive interaction of clover in legume –grass based pastures. Other research (Nadian et al., 1996) with subterranean clover (Trifolium subterraneum) examined root length and diameter across an increasing continuum of bulk densities (1.10 – 1.60 g cm23). Plant growth parameters were not affected until bulk density exceeded 1.20 g cm23 and then shoot growth was reduced by up to 52%. Similarly, in lucerne (M. sativa) grown in pot culture, increased bulk density reduced shoot and root growth (by up to 78 and 57%, respectively; Mapfumo et al., 1998). The pot experiments reported above were carried out using seedling plants, and extending this work to pastures that are dependent on the vegetative propagation of legumes could be problematical. For example, in established clover – grass pastures (. 2 years), white clover seedling recruitment is usually rare and plants typically spread by vegetative growth (Brock et al., 2000; Gustine and Sanderson, 2001). Compared to clover seedlings which have a seminal taproot, stolon fragments have shallow, weakly-taprooted nodal roots and could differ in their response to soil compaction. This could be important in spring when clover – grass pastures rely on successful root initiation and the establishment of small

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191

fragmented stolon units to maintain the sward clover population. In one report that used white clover stolon cuttings in cores of compacted ﬁeld soil, shoot and root growth were reduced, but differentiation of root resistance effects from reduced aeration was not possible (Blaikie and Mason, 1993). Currently, insufﬁcient information exists to rank grass and legume species according to their tolerance of compaction, and potential use in compacted pasture soils. Recent work with lucerne (Mapfumo et al., 1998) has shown that plant response to compaction is greatly inﬂuenced by soil texture and the component of plant response measured (e.g., leaf, roots, or branches). Thus, future research and comparisons between pasture species grown in compacted soils will need to relate observed changes in growth to the response factor measured and the soil characteristics. In addition, pot experiments and ﬁeld studies are required to establish compaction effects across a broader lower-endrange of bulk density values (e.g., 0.75 –1.0 g cm23) to include coarser textured soils that comprise some temperate pasture systems (e.g., New Zealand). Field studies in pasture soils compacted by wheeled agricultural machinery (e.g., silage making operations) generally conﬁrm the work of pot experiments, and show that soil compaction can reduce legume productivity (e.g., red clover and lucerne; Davies and Hughes, 1980; Frame, 1985; Rechel et al., 1987; Henderson, 1991). For example, in mixed swards of grass/red clover, compaction by wheel trafﬁc reduced red clover yield by 17– 25% and was mainly linked to an increase in bulk density (from 1.24 to 1.40 g cm23) (Frame, 1985). Similarly, ﬁeldwork in Australia by Henderson (1991) with subterranean clover and medic (M. littoralis) showed that restricted root growth and function led to reduced shoot growth of 30% when bulk density increased from 1.32 to 1.50 g cm23 in the top 0 –50 mm soil depth after wheel trafﬁc. In these ﬁeld studies, plant growth limitations were probably not only due to mechanical impedance, but also to the effects of reduced soil aeration (discussed in Section II.C). Nonetheless, the evidence reviewed here strongly suggests that the magnitude of bulk density increases seen in grazed pasture due to animal treading (Table IV) could potentially have a negative effect on legume root and shoot yield through increased mechanical impedance to roots. In legume-based pastures, ﬁxed N in roots can potentially be 30 –60% of the ﬁxed N in leaves (e.g., white clover, Jorgensen and Ledgard, 1997; subterranean clover, McNeill et al., 1997). Thus, any reduction in root biomass due to increased root resistance will have serious implications for total N2 ﬁxation and the cycling of N in pasture systems, as well as the causative effect of reduced above ground biomass. 2.

N2 Fixation

There appears to be no published studies on the effects of soil compaction per se resulting from animal treading on nodulation and N2 ﬁxation in pasture

192

J. C. MENNEER ET AL. Table VI Summary of Effects of Increasing Bulk Density on Nodulation and N2 Fixation by Crop Legumes Soil bulk density (g cm23)

Species Soybean

Speciﬁc Base Nodule Nodule nitrogenase level Compacted number weight activity na 1.16 1.20

Common bean 1.20 Field pea

Percent decrease in

na

na 1.28 1.40 1.60 1.40 1.60 na

30 19 23 46 15 30 60

36 26 17 24 11 30 –

– ns 10 46 26 48 –

Reference Voorhees et al. (1976) Lindemann (1982) Tu and Buttery (1988) Tu and Buttery (1988) Tu and Buttery (1988) Tu and Buttery (1988) Grath and Hakansson (1992)

na, not available; ns, not signiﬁcant. NB: Data signiﬁcant unless otherwise stated.

legumes. Hence, the discussion here relies on studies using crop legumes grown in arable soils compacted by wheeled agricultural machinery. Generally, these studies have not determined the relative importance of mechanical impedance and soil aeration status speciﬁcally. Studies with crop legumes have related increases in soil compaction to decreases in nodulation and N2 ﬁxation (Table VI). Under areas of compaction in ﬁeld grown soybean (Glycine max), Voorhees et al. (1976) found decreases of about 20 – 30% in nodule numbers and a 36% smaller total nodule mass. Similarly, soybean and common bean (Phaseolus vulgaris) grown in pots at bulk densities from 1.20 to 1.60 g cm23 showed a 30 –50% reduction in nodule number and a 25 –30% reduction in nodule fresh weight per plant (Tu and Buttery, 1988). Nitrogenase speciﬁc activity (using the acetylene reduction assay) was reduced by about 50% (Tu and Buttery, 1988). Such a reduction in nitrogenase speciﬁc activity suggests that while impedance may impact dramatically on root biomass and nodulation, nodule functioning may also be affected by other factors, in particular, poor soil aeration.

C. SOIL COMPACTION: AERATION AND/OR WATERLOGGING EFFECTS ON LEGUMES The adverse effect of poor aeration and/or waterlogging on growth and N2 ﬁxation of pasture legumes has been reported by many workers and is caused by a lack of O2 for root metabolism (affecting nutrient and water uptake) and N2

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ﬁxation (Finn et al., 1961; Hoveland and Mikkelsen, 1967; Arrese-Igor et al., 1993) (Table VII). In addition, increased accumulation of toxic products (e.g., Mn, Fe, and ethylene) under poor aeration/waterlogging has also been shown to negatively affect the growth of both legumes and non-legumes (e.g., Hoveland and Mikkelsen, 1967; Ponnamperuma, 1984; Pezeshki, 1994). These negative effects on legume growth and N2 ﬁxation are quite variable and can differ with the extent of exposure and species tolerance to conditions of poor aeration and/or waterlogging.

1.

Legume Shoot and Root Growth

In general, reduced aeration or waterlogging has a large negative effect on legume shoot and root growth (Table VII). For example, Finn et al. (1961) measured root and shoot yield decreases for lucerne, white clover, and lotus across a range of different soil aeration (0, 14, and 20% air-ﬁlled porosity), with similar relative decreases in root yield. Lucerne was most sensitive to reduced aeration. In subsequent work by Hoveland and Mikkelsen (1967) the tolerance to waterlogging in three different species of clover was reported to be in the order of Persian (Trifolium resupinatum) $ strawberry (Trifolium fragiferum) . Ladino white (T. repens L. regal) . intermediate white clover (T. repens L. S1), when plants were either intermittently ﬂooded (3 and 6 days out of every 10 for 4 months) or continuously ﬂooded. The white clover cultivars were most sensitive to ﬂooding and experienced a decrease in shoot dry matter yield of up to 62% under the most extreme conditions of ﬂooding treatment. Differences in response between white clover cultivars were also evident with the larger-leafed ladino cultivar appearing to be far more tolerant of ﬂooding than the smallerleafed intermediate cultivar (Table VII). The reduced clover yields in this study were due to a combination of root decay and reduced root growth, decreased N uptake, and possibly Mn toxicity. In contrast, there have been reports of increased legume growth in waterlogged soil or under low O2 conditions. For example, Pugh et al. (1995) reported that white clover subjected to a period of prolonged waterlogging (9 weeks) resulted in an 81% increase in shoot yield and no change in root yield compared to a normally watered regime. Other workers too (e.g., lucerne, Arrese-Igor et al., 1993; lotus, James and Crawford, 1998) have measured similar legume growth enhancement or maintenance when plants have been exposed to waterlogging and/or reduced aeration for extended periods, especially when other anaerobic stresses are avoided (e.g., toxic levels of Mn, Fe, and ethylene). In these cases and others with crop legumes (cowpeas, Dakora and Atkins, 1990a, 1991; soybean, Parsons and Day, 1990) important structural adaptations of enhanced production of lenticels and/or aerenchyma have been cited as key

194

Table VII Summary of Shoot and Root Dry Matter After Waterlogging, or Reduced Aeration (if Stated) of the Major Pasture Legume Species Under Controlled Conditions

Species White clover Ladino type Ladino type Intermediate type Ladino type White clover White clover Miscellaneous clovers Strawberry clover (T. fragiferum) Persian clover (T. resupinatum) Red clover (T. pratense) Strawberry clover (T. fragiferum) Alsike clover (T. hybridum) Subterranean clover cv. subterraneum cv. yanninicum cv. brachycalcycinum

Days of waterlogging or reduced O2

Shoot DM

230, 216 229, þ 11 243, 262 223, 230

Root DM

Reference

211, 217, 225 233, 230, 29 240, ns, ns 229, ns, ns 280 ns

Finn et al. (1961) Finn et al. (1961) Hoveland and Mikkelsen (1967) Hoveland and Mikkelsen (1967) Heinrichs (1970) Pugh et al. (1995)

10a 30a 3, 6, 10b 3, 6, 10b 20 60

223, 236, 221, 220, 270 þ70

3, 6, 10b 3, 6, 10b 20 20 20

25, 224, 218 0, 218, 215 260 250 260

ns ns 250 240 260

Hoveland Hoveland Heinrichs Heinrichs Heinrichs

21 21 21

226 ns 244

245 226 263

Francis and Devitt (1969) Francis and Devitt (1969) Francis and Devitt (1969)

and Mikkelsen (1967) and Mikkelsen (1967) (1970) (1970) (1970)

J. C. MENNEER ET AL.

Percent difference from control in

10a 30a 60 at 1% pO2 14

281, 245, 230 290, 269, 258 ns 272c

290, 242, 229 287, 279, 254 250 –

Finn et al. (1961) Finn et al. (1961) Arrese-Igor et al. (1993) Shiferaw et al. (1992)

10a 30a 14 14 60c

þ 11, 26, þ 11 224, 216, þ 11 250c 245c þ 40

227, 210, 226 234, 229, 22 – – ns

Finn et al. (1961) Finn et al. (1961) Shiferaw et al. (1992) Shiferaw et al. (1992) James and Crawford (1998)

ns, not signiﬁcant. NB: data signiﬁcant unless otherwise stated. a Plants subjected to 0, 25, and 40 cm moisture tensions, representing air-ﬁlled porosities of 0, 14, and 20%, respectively; waterlogged for 10 or 30 days out of 60 days. b 3, 6, and 10 days waterlogging out of 10 days for 4 months. c Total plant weight reported only (roots þ shoots).

N2 FIXATION IN LEGUME-BASED PASTURES

Lucerne M. sativa M. sativa M. sativa M. sativa Lotus Lotus corniculatus L. corniculatus L. corniculatus L. pendunculatus L. uliginosus

195

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J. C. MENNEER ET AL.

features that enable the increased supply of O2 through tissue air spaces from shoot to root in low O2 environments.

2.

N2 Fixation

Studies investigating the effects of soil aeration and waterlogging on the N2 ﬁxing performance of various crop and pasture legumes have shown nodulation and N2 ﬁxation can decrease for up to 30 days, but in many longer-term examples (c. 60 days) effects can actually be positive (Table VIII). In the medium term, decreased nitrogenase activity in legumes under reduced aeration and/or waterlogging primarily occurs as a result of reduced oxygen supply to the nodule (Arrese-Igor et al., 1993; Pugh et al., 1995). Early studies (Minchin and Pate, 1975; Minchin and Summerﬁeld, 1976) using seedling pea species (cowpea and ﬁeld pea) with well-formed nodules showed nodule tissue production decreased by up to 60% and nitrogenase activity per plant by 70% after 16 days of waterlogging. Later, Pugh et al. (1995) observed similar effects when normally watered white clover plants were subjected to waterlogging for 7 days causing a dramatic reduction in N2 ﬁxation (96%). Over the long term, legumes can apparently adjust to waterlogging and experience enhanced N2 ﬁxation. For example, white clover when subjected to constant waterlogging for 9 weeks increased N2 ﬁxation substantially, compared to normal watering (Pugh et al., 1995). Supporting these ﬁndings using a controlled O2 environment to avoid anaerobic stresses, Arrese-Igor et al. (1993) measured no difference in nitrogenase speciﬁc activity in lucerne at ambient and sub-ambient pO2. This suggests that lucerne is capable of adapting to low aeration and developing a more efﬁcient N2 ﬁxation system. As in other studies with crop and non-crop legumes (e.g., Dakora and Atkins, 1990a,b; Parsons and Day, 1990; James et al., 1992) this work has highlighted the importance of various structural adaptations in low oxygen environments, and the inﬂuence this has on nodulation and nitrogenase activity. In addition to the previously mentioned production of lenticels and/or aerenchyma, are modiﬁcations of cells within the nodule cortex and the infected zone to increase gas diffusion to bacteriods carrying out N2 ﬁxation (Dakora and Atkins, 1990a,b, 1991; Minchin, 1997). These cellular modiﬁcations include increases in the ratio of uninfected cells to microbial-infected cells, and increases in the size of intercellular spaces in both the cortex and the infected region (Atkins et al., 1993). Pugh et al. (1995) also noted that vacuole enlargement within infected cells could help increase O2 availability to bacteriods. Adaptive cellular mechanisms such as these are thought to operate in conjunction with the “variable diffusion barrier” that surrounds the site of N2 ﬁxation and regulates O2 diffusion into the nodule (Dakora and Atkins, 1990a,b, 1991; Minchin, 1997).

Table VIII Summary of Nodulation and N2 Fixation Under Conditions of Waterlogging or Reduced Aeration (if Stated) in Selected Pasture and Cropping Legumes

Species Pasture legumes White clover White clover Kenya white clover (Trifolium semipilosum) Lucerne (M. sativa) Lucerne Lotus (L. corniculatus) Lotus (L. pendunculatus) Lotus (L. uliginosus) Crop legumes Cowpea (Vigna unguiculata) Cowpea (V. unguiculata) Fababean (Vicia faba) Garden pea (Pisum sativum)

Days of waterlogging or reduced O2

Nodule weight

Speciﬁc nitrogenase activity

Reference

60 7 14

nd nd ns

þ 34 296 nd

Pugh et al. (1995) Pugh et al. (1995) Shiferaw et al. (1992)

14 60 at 1% pO2 14 14 60a

2100 250 280 268 þ35

nd ns nd nd ns

Shiferaw et al. (1992) Arrese-Igor et al. (1993) Shiferaw et al. (1992) Shiferaw et al. (1992) James and Crawford (1998)

69 at 1% pO2 4–32 21–50 16

263 227 to 259 nd 256

270b 218 to 246c þ 10 to þ 60c 262

Dakora and Atkins (1990a) Minchin and Summerﬁeld (1976) Gallacher and Sprent (1978) Minchin and Pate (1975)

197

nd, not determined; ns, not signiﬁcant. NB: Data signiﬁcant unless otherwise stated. a Measured on 55th day of 69 day study. b Depended on nodule/plant adaptations. c Comparison of two waterlogging treatments with and without aeration of O2 (i.e., pO2 0.241 versus 0.094).

N2 FIXATION IN LEGUME-BASED PASTURES

Percent difference from control in

198

J. C. MENNEER ET AL.

The beneﬁts of these adaptations in low O2 environments (e.g., “puddled” pasture soils) is not known, but it is likely that the duration of waterlogging, and the degree of plant response to other factors associated with waterlogging (e.g., Mn, Fe, and ethylene accumulating in the root zone) will also have important effects. In reality, waterlogging in pasture soils is usually shortterm/intermittent (e.g., puddling after pugging) so longer-term adaptive mechanisms for enhancing O2 supply may not counteract the negative plant response to anaerobosis. On the other hand, if compaction via treading is causing a gradual but cumulative decline in soil aeration, or in soils with poor drainage, the development of legume varieties which form adaptations for O2 transport may be advantageous.

D. SIGNIFICANCE OF PLANT AND SOIL FACTORS, AND LIMITS OF PASTURE TOLERANCE The relative signiﬁcance of plant and soil effects resulting from treading processes on legume growth and N2 ﬁxation in legume-based pastures is difﬁcult to determine. This is mainly because of the large inﬂuence that farm management has on the magnitude of treading processes, and also from differences in soil properties between farm systems. Although damage to plants appears to be the main effect under treading (especially at high stocking rates), systematic studies are required to determine the relative contribution of direct effects of plant damage versus indirect effects of soil compaction. In particular, future work with intensive farming systems should recognise that compaction of pasture soils is likely to be a long-term and cumulative process of soil degradation involving changes in soil strength and aeration (e.g., Daniel et al., 2002; Tables IV and V). To this end, studies may need to have a much longer term focus (years rather than months) to establish if increasing compaction is causing a slow but important winding down of legume productivity and N2 ﬁxation. To achieve this, further insight is required regarding the application of suitable soil physical indices along with modelling of legume growth response and N2 ﬁxation under ﬁeld conditions to determine impacts at the farm scale. Establishing critical limits of bulk density that affect plant growth in compacted soils is difﬁcult and large variations exist in reported values due to the inﬂuences of soil water content (Eavis, 1972), soil texture (Jones, 1983), and plant species tolerance (Materechera et al., 1991). Additionally, some studies have revealed that even slight increases in bulk density can have adverse affects on the growth of some key pasture species (e.g., ryegrass, Houlbrooke et al., 1997). Therefore, it may be of more use to establish guideline values of bulk density for individual soil types (or properties) and plant species that represent a continuum of incremental reductions in key growth factors rather than deﬁning critical upper limits. This approach was adopted by Mapfumo et al. (1998) using

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199

lucerne and a brome grass in two different soil types (a clay loam and sandy loam), which were compacted to a range of bulk densities and growth changes recorded, after which linear regression equations were used to estimate the threshold bulk densities that reduced yields by 25, 50, and 75% of the control (Table IX). Threshold values for decreased shoot dry weight were the same for both species but different for the two soils, and threshold values for root dry weight reductions were different for both soil type and species. This work clearly shows the necessity to consider differences in soil texture, species tolerance, and the response factor measured when establishing “critical” levels of bulk density causing mechanical impedance problems in pasture soils. Air-ﬁlled porosity is a frequently used indicator for assessing soil aeration status in grazed pasture (e.g., Greenwood and McNamara, 1992; Singleton and Addison, 1999). As would be expected, increases in bulk density of compacted pasture soils are paralleled with a decrease in air-ﬁlled porosity. However, plant function and growth is not normally affected unless the air-ﬁlled porosity falls below about 10% (Grable, 1971). It is generally assumed that values of air-ﬁlled porosity between 10 and 25% provide adequate aeration but with some limitations to O2 diffusion under certain conditions, and above 25% airﬁlled porosity provides good aeration (Stepniewski et al., 1994). As with bulk density, critical values of air-ﬁlled porosity vary with plant type and soil texture, because of differences in air-ﬁlled pore geometry and stability, and subsequent effects on O2 diffusion. For example, Bakken et al. (1987) found that O2 diffusion was reduced to zero when air-ﬁlled porosity was 10% for a

Table IX Estimated Threshold Bulk Densities to Reduce Relative Yield (%) of Shoot and Root Dry Matter for Lucerne and Brome Grass Grown in Clay Loam and Sandy Clay Loam Soils with Subsurface Compactiona Threshold bulk density (g cm23) Clay loamb Parameter Shoot biomass

Root biomass

a

Relative yield (%)

Brome

Lucerne

Brome

Lucerne

75 50 25 75 50 25

1.28 1.40 1.52 1.31 1.45 1.58

1.28 1.40 1.52 1.55 1.79 2.02

1.36 1.55 1.74 1.38 1.59 1.81

1.35 1.55 1.74 1.45 1.69 1.93

Data from Mapfumo et al. (1998). Base level soil bulk density was 1.15 g cm23. c Base level soil bulk density was 1.20 g cm23. b

Sandy loamc

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J. C. MENNEER ET AL.

Figure 2 Relationship between relative pasture yield for mid-summer and macroporosity at 0– 50 mm soil depth ðP ¼ 0:001; r 2 ¼ 0:49Þ: (After Drewry et al., 2001.)

clay soil, but in a sandy soil zero O2 diffusion did not occur until the air-ﬁlled porosity was 2%. Despite many authors reporting low air-ﬁlled porosity in grazed pasture, only recently has work began to investigate the relationship between yield response and air-ﬁlled porosity with ryegrass (Drewry et al., 2001). No similar work has yet been done for white clover. Under simulated treading to reduce plant damage effects, Drewry et al. (2001) produced a pasture response curve which indicated that the optimum air-ﬁlled porosity for ryegrass yield was about 16– 17%, and a critical air-ﬁlled porosity (for . 10% yield reduction) was approximately 10– 11% (Fig. 2). Differences in soil texture and seasonal weather patterns (wet or dry periods) meant that this curve could undergo a lateral shift in either direction and requires additional calibration. Further work is also needed to see if a similar relationship exists for pasture legumes, especially in legume – grass mixtures where any differences in effect may inﬂuence the legume – grass competitive interaction and thus the persistence of legumes in the sward.

III. ANIMAL GRAZING Defoliation by grazing animals can affect the size of individual legume plants or plant parts (e.g., leaf size), plant density, and from a physiological

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201

standpoint N2 ﬁxation and photosynthesis (Fig. 1). Key components of defoliation that can have either a positive or negative effect on legume productivity and N2 ﬁxation in pasture relate directly to the foraging behaviour and management of the grazing animal: viz., selection by the animal (of certain plants or plant parts) and, frequency and intensity at which plants are defoliated.

A. DIET SELECTION 1.

AND

DEFOLIATION EFFECTS

Diet Selection

Pasture species selection by the grazing animal can have a marked inﬂuence on the legume content of mixed pastures (e.g., Ledgard and Steele, 1992). Selection for legume varies with the degree of selectivity and the feeding style attributes of the animal type (Table X). In turn these attributes can lead to differences in legume content of pasture. For example, white clover content of swards grazed by different animals is generally in the order: cattle . goats . red deer $ sheep (Hunt and Hay, 1989; Sheath and Hodgson, 1989; Wright et al., 1992; Semiadi et al., 1995). Because of white clover’s lower position in the sward the height at which different animals graze also has a considerable bearing on the amount of white clover ingested (Milne et al., 1982). These differences in grazing selection behaviour between animals can be used advantageously by mixed grazing of different animal types to modify sward composition in favour of white clover (or grass if need be) (Curll et al., 1985a,b; Collins, 1989; Murphy et al., 1995b; Nolan et al., 1999, 2001). For example, Nolan et al. (2001) highlighted the modifying inﬂuence

Table X Feeding Style Attributes of Different Livestock Speciesa

Species Cattle Sheep Goat Deer

LUb

Method

Selectivity

Type

Typical minimum sward height grazed (cm)

0.73–1.0 0.13–0.2 0.1–0.2 0.1–0.4

Tear with tongue Biting/shearing Biting/shearing Biting/shearing

Low High High High

Grazer Grazer/browser Browser/grazer Browser/grazer

6 3 6 ,3

Data from Hearn (1995), Milne et al. (1998), Crofts and Jefferson (1999), Mayle (1999), and Cosgrove and Hodgson (2002). a Modiﬁed from Bullock and Armstrong (2000). b LU, livestock unit (1 dry cow, 600 kg).

202

J. C. MENNEER ET AL.

of mixed grazing with sheep and cattle on white clover content in mixed clover –grass swards. After a 3-year period of rotational grazing with mixed cattle and sheep, versus sheep only, white clover contents were signiﬁcantly different at 13.5 versus 6.1%, respectively (Nolan et al., 2001). The mixing of goats with cattle or sheep has also been shown to improve white clover content in the sward and can signiﬁcantly increase cattle and sheep live weight gains (De Pozo et al., 1997; Osoro et al., 2000). The improved white clover content occurs because of goats’ top-down grazing style which results in more grass being ingested than the lower lying white clover foliage (De Pozo et al., 1996).

2.

Defoliation Frequency and Intensity

While the selective preferences of the grazing animal are of considerable importance for manipulating pasture composition, the frequency, intensity, and timing of defoliation have an overriding effect on white clover content in legume-based pastures (Curll and Wilkins, 1982; Brock et al., 1988; Parsons et al., 1991a,b; Brock and Hay, 1996; Frame et al., 1998). Defoliation frequency depends solely on stocking rate, whereas the intensity of defoliation depends on stocking rate and the duration of grazing, both of which are features of the chosen grazing management system (e.g., continuous versus rotational) (Lemaire and Chapman, 1996). Through grazing management, defoliation frequency and intensity interact to determine the competitive balance between white clover and grass for light and space and can have a profound effect on white clover productivity. Early work in mixed swards indicated that white clover is favoured by frequent and intense defoliation with short rest periods (Brougham, 1959; Graham et al., 1961; Bland, 1967; Ward et al., 1996). Brougham (1959), for example, found that white clover content and yields were highest where grazing was most frequent and closest, but with a rest between grazings (i.e., short rotation), and was attributed to the reduced effects of shading from ryegrass under such conditions. In general, these early studies highlighted the importance of rest periods between defoliation events that were not prolonged but still gave adequate time for white clover regrowth without excess shading. Undoubtedly, this early work along with subsequent research (e.g., Widdup and Turner, 1983; Clark et al., 1984; Frame and Newbould, 1984, 1986; Newton et al., 1984; Brock, 1988; Brock et al., 1988; Hay et al., 1988; Orr et al., 1990; Laws and Newton, 1992; Elgersma et al., 1998; Nolan et al., 2001) has consolidated the commonly held belief that rotational grazing management with its inherent rest periods tends to enhance white clover content as opposed to continuous grazing management which is

N2 FIXATION IN LEGUME-BASED PASTURES

203

Figure 3 White clover content (bars) and dry matter yield (lines) under rotational grazing (shaded and A) and continuous grazing (open and W) at a stocking rate of 14 ewes ha21. (Derived from Newton et al., 1984.)

often reported to reduce white clover content (but see Section V.A.2) and yield by overly frequent and intense defoliation and/or continual selective pressures (Fig. 3). Under rotational grazing, the severe removal of leaf and stolon tissue during defoliation events is usually offset by the generally longer interval between defoliations which allow plants to rebuild photosynthetic area compared to continuous grazing. This allows more leaves per stolon to accumulate, and a high photosynthetic potential and greater plant size. Additionally, competition with grass reduces since grass tiller density and leaf appearance rate decline (Grant and Barthram, 1991), thereby allowing more space for white clover invasion (Brock et al., 1988). Larger, more complex structured white clover plants are conducive to white clover survival in the sward, whereas smaller, simple structured plants are more vulnerable to edaphic and animal stresses (Brock et al., 1988; Hay et al., 1989). The latter have been blamed for white clover decline or “crashes” that may occur on a 3 – 4 year cycle in swards continuously grazed by sheep (Fothergill et al., 1996). These cycles of declining white clover content under continuous grazing are most evident with sheep because of their high selectivity for clover, though cattle under continuous grazing can also reduce sward white clover contents, albeit gradually (Gibb and Baker, 1989; Laidlaw et al., 1995). Herbage rejection by cattle near dung pats offers patches of white clover a rest from defoliation, and has been suggested to help maintain white clover content compared to sheep grazed swards (Frame et al., 1998).

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Although white clover is the most extensively used legume in mixed legume – grass pasture systems, red clover, and to a lesser extent lucerne, which are generally better suited to conservation, are sometimes used in multispecies mixtures for grazing. With lucerne and red clover, the effects of defoliation frequency are clearer than with white clover. Both species do not persist under frequent and severe defoliation (Lodge, 1991; Frame et al., 1998) and so are better suited to rotational, lenient grazing with sufﬁcient time between grazings to allow for plant recovery. Lucerne regrowth is from crown buds using stored reserves, and the plant needs 4– 6 weeks for reserve replenishment (Lodge, 1991). In contrast, red clover relies on terminal meristems for regrowth and needs to maintain adequate residual leaf area for subsequent growth. Persistence of both lucerne and red clover in grazed mixed swards is unstable and appears to be limited to 2 or 3 years for red clover and 4 years for lucerne (Sprent et al., 1996; Frame et al., 1998).

B. DIRECT EFFECTS

OF

DEFOLIATION

ON

N2 FIXATION

Nitrogen ﬁxation in pasture legumes is greatly reduced or ceases almost immediately after defoliation (Vance et al., 1979; Cralle and Heichel, 1981; Davidson et al., 1990; Ta et al., 1990). In early literature, this decrease in nitrogenase activity resulting from defoliation was attributed to reduced photosynthesis and an associated fall-off in photosynthate supply to the nodule for nodule function and growth (Boller and Heichel, 1983; MacDowall, 1983; Ryle et al., 1986; Ta et al., 1990). An alternate model (Hartwig and Nosberger, 1994; Hartwig and Trommler, 2001) is that defoliation leads to a reduction in shoot demands for nitrogen (reduced N sink strength) and that continuing N2 ﬁxation leads to an accumulation of N compounds in the nodules. These N compounds trigger an increase in resistance to O2 diffusion (by an unknown mechanism) resulting in an inhibition of respiration, and thereby decreased nitrogenase activity. Alternatively, N2 ﬁxation may become a low-priority sink for carbon, which is redirected to regrowth and away from the export of N from nodules. In white clover, nitrogenase activity decreases within several hours after defoliation (Moustafa et al., 1969; Chu and Robertson, 1974; Ryle et al., 1985) and recovery takes anywhere from 5 to 21 days depending on the severity of defoliation. Interestingly, Ryle et al. (1985) observed nodule dry weight decreases by about 30% (as did Chu and Robertson, 1974) in white clover after defoliation, with recovery to original nodule weights after about 6 –9 days depending on the photosynthetic ability of the remaining tissue. This indicates that the adverse effect of defoliation on nitrogenase activity is not entirely due to loss of photosynthetic area. Similarly, results have been obtained using other pasture legumes with nitrogenase activity decreasing by 60 – 95%

N2 FIXATION IN LEGUME-BASED PASTURES

205

immediately after defoliation, and recovery to control levels taking between 5 and 24 days, depending on the species, severity of defoliation, and growing conditions (Table XI). In intensively grazed mixed legume – grass swards the immediate effect of defoliation on nitrogenase activity suggests that N2 ﬁxation may be operating at sub-optimal levels, particularly if insufﬁcient time is allowed between grazing events for return of N2 ﬁxation to maximum rates. In a rotationally grazed mixed clover –grass sward, Murphy et al. (1986) showed a rapid decline in N2 ﬁxation occurred at each grazing event, but regrowth was accompanied by a recovery in N2 ﬁxation shortly (about 20 days) after grazing ceased (Fig. 4). Thus, the length of grazing interval needs to be carefully balanced; ﬁrstly, to ensure recovery of N2 ﬁxation, and secondly, to minimise the risk of shading by companion grasses and the potential for further reductions of N2 ﬁxation (Chu and Robertson, 1974; Halliday and Pate, 1976). In the study by Murphy et al. (1986), increased soil N from animal excreta may have also contributed to the lower levels of N2 ﬁxation that were measured after grazing (e.g., Hoglund and Brock, 1978) and is further discussed in Section IV. Other more complicated factors also need consideration when pasture is intensely grazed, with recent work (e.g., Menneer et al., 2003) showing defoliation intensity can also affect N2 ﬁxation (%Ndfa) by regulating soil N availability through manipulation of the botanical composition of the sward. Menneer et al. (2003) measured increased %Ndfa in a more severe cutting height treatment, but only during the summer months. This was attributed to an increase in drought-tolerant grasses and weeds that had a greater ability to grow and absorb soil N than ryegrass, which led to reduced soil N availability and enhanced the potential for N2 ﬁxing activity.

IV. ANIMAL EXCRETION In intensive high-producing pasture systems, dung and urine patches can cover more than a third of grazed pasture in any 1 year (Haynes and Williams, 1993; Whitehead, 1995). Urine N is in highly mineralisable forms (mainly as 70 – 90% urea-N) compared to dung N, and within 3– 5 days is rapidly converted to plantavailable N in soil (e.g., Menneer et al., 2003). This can result in inorganic soil N under urine patches up to 10 times greater than under a dung pat, and more than 30 times greater than areas unaffected by excreta (Afzal and Adams, 1992). Where cattle graze, animal rejection of herbage on or near dung pats is also a problem, and depending on the climate and stocking rate can represent 35 – 40% of the total sward area and last up to 1 year (Marsh and Campling, 1970).

206

Table XI Summary of Defoliation Effects on Nitrogenase Activity of Selected Pasture Legumes

White clover (T. repens)

Lucerne (M. sativa)

Lotus (L. corniculatus)

% Decrease in N2 ﬁxation or nitrogenase activity within 48 h of defoliation

Number of days for recovery of N2 ﬁxation (speciﬁc activity)

Reference

50 100 44b and 83c 90 na 90 60 100 60 85

70 85 Nil and 70 62 95 91 80 78 88 70

5–9a 10 Nil and 5 21 24 12 14 12 18 11

Ryle et al. (1985) Chu and Robertson (1974) Davidson et al. (1990) Moustafa et al. (1969) Kim et al. (1993) MacDowall (1983) Ta et al. (1990) Cralle and Heichel (1981) Vance et al. (1979) Cralle and Heichel (1981)

na, not available. NB: Data signiﬁcant unless otherwise stated. a Depended on age of plants; N2 ﬁxation determined from N content. b Successive defoliations every 10 days for 40 days. c Single defoliation after 40 days.

J. C. MENNEER ET AL.

Species

Defoliation severity (% shoots removed)

N2 FIXATION IN LEGUME-BASED PASTURES

207

Figure 4 N2 ﬁxation rate (nitrogenase activity) in a grass– clover sward as inﬂuenced by a series of grazings: arrows indicate times of grazing events. (Redrawn from Murphy et al., 1986.)

In mixed clover –grass pasture, high soil N from urine and animal rejection of herbage on or near dung pats adversely affects legume growth and N2 ﬁxation mainly by altering the legume –grass competitive interaction and/or depressing N2 ﬁxation (Fig. 1). While smothering/burial or scorching of plant tissue by excreta can also affect legume performance, it is far less important than changes caused by the primary processes described earlier.

A. INCREASED SOIL N AND GRAZING AVOIDANCE OF EXCRETA- A FFECTED AREAS Typically, increased levels of soil N due to excreta return, particularly urine, have a large negative effect on white clover content and in many cases also white clover yield (Table XII). This commonly measured decrease in white clover content under urine is a dilution effect caused by what is often an impressive increase in grass yield (e.g., up to 166%; Menneer et al., 2003) without an accompanying increase in white clover yield. Similarly, the rejection of herbage by grazing animals on or near dung pats can cause low white clover contents and yield (Vinther, 1998). Death of white clover nodes also occurs directly beneath dung pats shortly after deposition (about 25 days) (MacDiarmid and Watkin, 1972). To some degree this effect is offset by old dung-pat sites providing fresh bare patches for colonisation by white clover (Weeda, 1967), and can result in white clover dominance for up to 18 months on such sites.

208

Table XII Effect of Animal Excreta on Various Components of White Clover Performance and N2 Fixation, Measured Under Cutting Unless Otherwise Stated

Urine Ball et al. (1979) Curll and Wilkins (1983) Grazed at 25 sheep ha21 Grazed at 50 sheep ha21 Ledgard et al. (1982) Marriott et al. (1987) Menneer et al. (2003) Vinther (1998) Dung Vinther (1998)f a

Decrease in total N ﬁxed (%)

Duration of study (days)

Minimum N2 ﬁxation activitya

Days for recovery of N2 ﬁxation activity to baseline

–

48 ! 12

–

53

5b

.53

13 Nil 27 72e Nil Nil

58 ! 45 58 ! 18 43 ! 22 28 ! 5 44 ! 33 60 ! 30

– – 72d – 43 45

150 150 120 81 365 120

– – 30b 10b 25c 28c

– – .90 .80 289 .120

Nil

40 ! 30

20

120

75c

120

Units are either: speciﬁc nitrogenase activity or c %Ndfa. d Estimate calculated using N2 ﬁxation value derived by acetylene reduction assay method. e Some severe scorching of herbage by urine also involved. f Measurements within 0–10 cm of dung pat. b

J. C. MENNEER ET AL.

Excreta type

Change in white clover content (%)

Decrease in white clover yield (%)

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209

In mixed clover – grass swards the negative effects of excreta on clover performance are largely due to strong competition from its grass companion for light and space. White clover, because of its relatively low position in the canopy is easily overcome by shading when sward heights are excessive (e.g., when soil N is high or herbage is rejected). High soil N and herbage refusal also increases grass tiller density so reducing the space available for white clover invasion (Davies, 2001). In clover – grass swards, some workers (e.g., Schwinning and Parsons, 1996b) propose that the spatial heterogeneity of soil inorganic N generated by the patchiness of excreta return may be important for desynchronising legume – grass interactions and reducing the risk of large population ﬂuctuations at the ﬁeld scale. This is the key reason for the typical spatial variability of legume content and N2 ﬁxation that occurs in intensively grazed pastures. The extent of competition for light and space between white clover and grass is dependent on defoliation intensity (Curll and Wilkins, 1983; Evans et al., 1992; Menneer et al., 2003). For example, Menneer et al. (2003) measured the effect of a single application of cow urine (746 kg N ha21) under differing cutting severity, through to complete recovery of N2 ﬁxation and white clover production. As in some other studies (e.g., Ledgard et al., 1996b; Vinther, 1998) in mixed clover – grass pastures with applied urine or N fertiliser, Menneer et al. (2003) observed that the major effect of urine on N2 ﬁxation was by the direct effect of increased soil N availability on reducing the N2 ﬁxing capability of the plant. Appropriate grazing management is therefore important for countering the negative effect of animal excreta on white clover performance, especially in intensively grazed pastures where excretion often covers a large proportion of the grazed area.

B. DIRECT EFFECTS

OF

EXCRETA N ON N2 FIXATION

Where conditions of high inorganic soil N prevail, N2 ﬁxing activity in legumes is reduced because of the plant’s preference for uptake of N from soil over the more energy dependent process of N2 ﬁxation (Phillips and DeJong, 1982; Davidson and Robson, 1985). In mixed clover –grass pasture this is reﬂected in a rapid (within 2 days) decline in N2 ﬁxation of up to 90% upon deposition of animal urine (Table XII). Reported decreases in N2 ﬁxation rates near dung affected areas are much less than near urine affected areas, because of the lower availability of dung N, with values falling by only 2– 10% within 10 cm of the pat edge (Jorgensen and Jensen, 1997; Vinther, 1998). Under urine patches, the rate of recovery of N2 ﬁxation is variable and depends on the time taken for soil inorganic N to return to back-ground levels (e.g., 30– 162 days; Ball et al., 1979; Marriott et al., 1987; Vinther, 1998; Menneer et al., 2003). Recovery of N2 ﬁxation per unit of legume growth, therefore, may

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be as quick as 40 – 50 days (Ball et al., 1979; Marriott et al., 1987) or as long as 120 – 289 days under urine patches (Vinther, 1998; Menneer et al., 2003). Only one of these studies (Menneer et al., 2003) measured the full recovery in clover growth and N2 ﬁxation from urine deposition. In that study, a marked reduction in N2 ﬁxation due to urine began within 3 days and low N2 ﬁxation values persisted for up to 289 days even though inorganic N had fallen to back-ground levels in the 0– 150 mm soil depth by 160 days (Fig. 5). This delayed recovery of N2 ﬁxation in the absence of high soil N (also measured in the study of Vinther, 1998), may be due to the remobilisation of stored N from roots to shoots, uptake of inorganic N from below the soil sampling depth, and/or the delayed reestablishment of active nodules (Munns, 1977; Marriott et al., 1987). Overall, the prolonged effect of decreased N2 ﬁxation after urine application resulted in a 38% decrease in total N ﬁxed from 232 to 145 kg N ha year21 (Menneer et al., 2003). At the farm scale not all pasture is inﬂuenced by urine, with the proportion of affected pasture depending on stocking rates, length of the grazing period, frequency of excretion, and area covered by individual urine patches (Haynes and Williams, 1993). On New Zealand dairy farms, the average stocking rate is 2.8 cows ha21 year21 (LIC, 2003). Haynes and Williams (1993) reported a typical urination frequency of 10 urinations per day, and the average area covered of 0.30 m2. Using these reported values and a Poisson distribution to allow for the overlapping of urine patches (Peterson et al., 1956), we calculate that urine would be deposited on 25% of the grazed area each year. However, the area affected by urine usually extends well beyond the wetted urine patch (Whitehead, 1995).

Figure 5 Effect of a single application of urine (740 kg N ha21) on percent N2 ﬁxation using the N-labelling technique (X), compared to a non-urine control (W), and on soil inorganic NHþ 4 -N (O) and NO2 3 -N (K), during a 12-month ﬁeld study. (Redrawn from Menneer et al., 2003.). 15

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Affected areas of 0.5 – 0.7 m2 have been reported for dairy cows (Richards and Wolton, 1976; Latinga et al., 1987), and could potentially result in up to 46% of grazed pasture being affected by urine deposition each year. Using a typical estimate of total N2 ﬁxation with no input of urine of 250 kg N ha21 year21 (e.g., Ledgard and Steele, 1992; Menneer et al., 2003) we calculate N2 ﬁxation would decrease by 10% (25 kg N ha21 year21) on an annual basis, using the area covered by urine of 25% and the 38% percent decrease of total N ﬁxed reported by Menneer et al. (2003). This reduction due to urine could be as high as 40% by accounting for the total area affected by urine compared with pasture receiving no urine.

V.

STRATEGIES TO MINIMISE THE IMPACTS OF GRAZING ANIMALS

Optimising white clover performance and N2 ﬁxation by minimising grazing animal effects is only possible through judicious pasture and soil management and the implementation of effective farm management practices. This may require the adoption of special grazing management strategies (e.g., mixed grazing) and the integration of novel farm management practices (e.g., animal stand-off pads) to achieve the desired goal. However, a proactive approach such as this is highly dependent on the management skill and decision making ability of the grazier, and only by blending on-farm experience with off-farm support can informed decisions be made (Fig. 6).

A. PASTURE MANAGEMENT

TO

AID LEGUME PRODUCTION

One of the key aspects of pasture productivity in mixed legume – grass swards is the need to adequately manage defoliation by grazing animals so legume performance and N2 ﬁxation are optimised. In farm systems the frequency and intensity at which livestock graze, their dietary preference/grazing attributes, and the impact these processes have on legume performance can be regulated through strategic grazing or cutting management practices that overlie the principal grazing method employed (e.g., continuous versus rotational). Seasonal differences in the growth and development of legumes and grasses, and the subsequent effect this has on the legume –grass competitive interaction, require that grazing management strategies be ﬂexible and suit the changing seasonal pattern of plant growth (Table XIII). For example, recent work indicates that the sward condition of white clover (leaf area and plant size) during autumn is a key determinant of white clover content in the following spring (Fothergill et al., 1997, 2000; Luscher et al., 2001; Wachendorf et al., 2001a,b).

212 J. C. MENNEER ET AL. Figure 6 Farm management strategies and decision-based considerations for optimising legume performance and N2 ﬁxation in mixed grass–legume based pastures to reduce the detrimental impacts of grazing animals.

Grazing management Continuous-sheep

Continuous-cattle Rotational-cattle Rotational-sheep

Strategic management technique

Seasonal timing

Effect on clover content

Reference

Resting and cutting Resting and cutting Resting and cutting

Early summer Early-mid summer Late summer

UUU U UUU

Mixed grazing Hard grazing

All year Late spring to early summer All season

UUU UU

Barthram and Grant (1994, 1995) Gooding et al. (1996) Curll and Wilkins (1985) and Fothergill et al. (2000) Nolan et al. (2001) Gibb and Baker (1989)

UU

Murphy et al. (1995a,b)

Spring Late summer

UUU UUU

Hay and Baxter (1984) Sheldrick et al. (1993)

Mixed grazing (sheep following cows) Switch to continuous grazing Resting and cutting

N2 FIXATION IN LEGUME-BASED PASTURES

Table XIII Summary of Grazing Management Strategies that Provides Either Marginal (U), Good (U U), or Signiﬁcant (U U U) Gains in White Clover Performance

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1.

Late-Summer and Autumn Pasture Management Strategies

At about mid-late summer, pasture management strategies can take advantage of declining grass tillering and maximum stolon development to promote white clover presence in the sward (Laidlaw and Vertes, 1993). Relevant management strategies include a rest from grazing and a conservation cut in mid- or latesummer (Curll and Wilkins, 1985; Barthram and Grant, 1995; Gooding et al., 1996; Fothergill et al., 2000; Table XIV), lenient grazing (Curll, 1982; Curll and Wilkins, 1982), and introducing mixed or alternate grazing by cattle rather than sheep only (Garwood et al., 1982; Gibb et al., 1989; Evans et al., 1992). All of these strategies can improve autumn white clover content, as well as reducing the chance of stolon exposure and “burn-off” in drier climates (Brock and Hay, 1996). Implementing a late-summer silage cut also reduces the summer build-up of soil N in the root zone, thereby assisting with the beneﬁt of increased white clover content in autumn. In addition, using a mid-late summer cutting strategy has the added gain of decreasing the potential for subsequent leaching losses of soil N during winter. Although cutting of mixed clover – grass pasture removes a large proportion of white clover leaf tissue; growing points and newly developing leaves are much less affected than grass (Fothergill et al., 2000). This gives white clover a competitive advantage compared to the grass component which is slower to recover (depending on the season), and probably compensates for shading effects that occur during the initial resting phase of the cutting strategy (Fothergill et al., 2000). Establishing a high-quality white clover sward condition in autumn may help offset the winter period of stolon fragmentation and the reduced overwintering capability of smaller plants into spring (Bouchart et al., 1998;

Table XIV Effect of Seasonal Timing of Resting (Conservation) on Subsequent White Clover Abundance in Mixed Grass/Legume Pasture Seasonal timing of conservation References

White clover densitya Gooding et al. (1996) White clover contentb Curll and Wilkins (1985) Barthram and Grant (1995) Laidlaw et al. (1992)

Unrested control

Early spring

Mid-late spring

Late spring to early summer

Mid to late summer

48

–

33

48

67

– 15 15

– – 22

31 8 –

37 ns –

40 40 –

Number of 80 mm £ 80 mm squares in which clover was present out of 100. White clover content (% DM basis).

a b

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Fothergill et al., 2000; Goulas et al., 2001). This may be critical if pastures are to be intensely grazed on a regular basis during the winter (e.g., Australia, New Zealand) when treading-induced fragmentation can heighten the risk of plant loss.

2.

Mid-Spring to Mid-Summer Pasture Management Strategies

From mid/late spring to mid-summer, grass growth is rapid and white clover is less able to compete for the upper layers of the mixed sward canopy (Woledge et al., 1990; Frame and Laidlaw, 1998). This is probably the reason for the reported deleterious effect that resting for silage has on white clover content in late-spring or early-summer (see Barthram and Grant, 1995; Gooding et al., 1996). Because of increased grass growth at this time of year, grazing management becomes a critical factor in determining subsequent white clover content. Several New Zealand studies (e.g., Hay and Baxter, 1984) have found that continuous grazing (i.e., increased frequency of grazing) with sheep during this period, followed by rotational grazing for the remainder of the year has longterm beneﬁts for white clover performance (Table XV). Similar advantages to white clover growth have been observed in Europe when grazing intensity has been increased to maintain low sward heights during spring and early-summer in mixed clover – grass pasture continuously grazed with cattle (Gibb et al., 1989; Teuber and Laidlaw, 1995). Intense grazing at this time of year was found to increase annual N2 ﬁxation by 33% in pastures in Argentina (Reﬁ et al., 1989) and 10% in pastures in New Zealand (Brock et al., 1983). Increasing grazing intensity also increases competition between animals, lessens avoidance of grazing dung-affected areas and in doing so reduces shading of white clover by grasses. Furthermore, during late-spring and early-summer, soil has low

Table XV Yield of White Clover Grown with Ryegrass After Different Spring Grazing Managements in a Sheep System which is Usually Rotationally Grazeda White clover yield (kg DM ha21) Spring management Continuous Grazing every 2 weeks Grazing every 3 weeks Grazing every 4 weeks a

Data from Hay and Baxter (1984).

Summer

Annual total

1865 1500 1165 875

2750 2450 2020 1820

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J. C. MENNEER ET AL.

susceptibility to pugging damage so high stocking rates are less likely to injure and/or bury stolon tissue.

B. CHOICE

OF WHITE CLOVER CULTIVAR AND COMPANION GRASSES

1. White Clover Morphology Even though white clover can adapt to defoliation through changes in plant size and density (as discussed in Section III.A.2), the degree of compensation is not always sufﬁcient to maintain white clover productivity and persistence (Brock and Hay, 1996). This has led to a large research effort to produce cultivars that cover a range of morphology and has given farmers a variety of white clover cultivars from which to choose with improved suitability for different farm systems. Schematically presented in Fig. 7 are guidelines for choice of suitable white clover cultivar in relation to the farm system. In general, the choice of cultivar is governed by their adaptability to particular grazing and cutting managements (e.g., Evans et al., 1992). Under grazing, reduced white clover yield is usually due to removal of stolons which affect foliage growth, and so differences in branching ability of cultivars are important, particularly under frequent grazing. With the frequent defoliation of continuous grazing, smaller-leaved cultivars with their higher branching and growing point capabilities are more persistent and produce better than larger-leaved cultivars which have fewer stolons and a lower stolon density (Table XVI). In contrast, the infrequent defoliation of rotational grazing favours production and persistence of larger-leaved cultivars, which can compete with grasses for light as the sward height increases during the resting interval. However, the superiority of larger-leaved cultivars under rotational grazing cannot be taken for granted, at least with sheep (Brock and Hay, 1996). In this case, selective grazing of large leafed cultivars which have fewer stolons reduces the capacity of plants to regenerate and persist. Current work in selective breeding programs is starting to improve branching density while maintaining a particular leaf size and this is showing signs of increasing white clover productivity and persistency under rotational sheep grazing (e.g., T. repens “Kopu II” and “Crusader,” Woodﬁeld et al., 2001). A difﬁculty with small-leaved cultivars is the lengthy time required for establishment in the sward and low initial white clover contents, but this can be overcome by blending small- and medium-leaved cultivars, thus increasing white clover yield in the establishment year (Evans et al., 1992).

N2 FIXATION IN LEGUME-BASED PASTURES

Figure 7 Guidelines for choice of white clover cultivar for farm systems. (Updated from Evans et al., 1992.)

217

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Table XVI Effect of Leaf Size in Different White Clover Cultivars on Clover Content of Swards Under Continuous or Rotational Grazing, both at 22.5 ewes ha21, a Individual leaf area (mm2)

Clover content of pasture (% DM)

Cultivar

Leaf size description

Rotational grazing

Continuous grazing

Rotational grazing

Continuous grazing

Grassland Tahora Grasslands Huia Grasslands Pitau Grasslands Kopu LSD0.05b

Small Intermediate Large–intermediate Large

209 275 408 558

130 115 130 166

13.3 11.0 15.1 19.5

20.8 13.1 7.0 7.3

35

2.8

a

Data from Brock (1988). Least signiﬁcant difference of the mean at a P , 0:05:

b

2.

Grass Species and Cultivar Growth Attributes

The choice of grass species and variety is also an important consideration given that differences in growth characteristics between grass types may inﬂuence the competitive interaction of the grass– white clover association. In particular, with ryegrass the lower tiller density of tetraploid versus diploid cultivars, and differences in the seasonal growth pattern between some types are worthy of consideration. Although studies are limited and restricted to sheep grazing, results have shown improved white clover growth when grown with early maturing ryegrass cultivars rather than late maturing cultivars, and with tetraploids rather than diploids (Swift et al., 1993; Gooding et al., 1996; Sanderson and Elwinger, 1999). These advantages stem from the timing of white clover’s growth peak (in late season) with the less vigorous phase of ryegrass growth, and the greater openness of the sward for providing more space and light when using tetraploid ryegrass cultivars. Sowing ryegrass in new pasture at or below the recommended rate (c. 12 kg ha21) can also aid establishment and early growth of white clover. Chestnutt and Lowe (1970) comprehensively reviewed the relative contribution made by clover when grown in association with a range of different grasses. The least compatible companion grass for clover was Dactylis glomerata, while L. perenne and Festuca pratensis were the most compatible. Subsequent work has shown that the various fescue species and cultivars (e.g., F. pratensis, F. rubra, and F. arundinacea) as well as Phleum pratense and Cynosurus cristatus are often more compatible with clover than ryegrass (e.g., Pederson and Brink, 1988; Frame, 1990; Gooding and Frame, 1997). However, compared to ryegrass, fescue species typically lack persistence in pasture and P. pratense is less

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productive. Notwithstanding, these species are often grown in mixed legume – grass pastures where regional climate conditions limit the performance of ryegrass. For example, F. arundinacea is deeper rooting than ryegrass and is therefore more productive in dry summer conditions (Stevens and Hickey, 2000), whereas, P. pratense is a winter-active grass species more suited to moist soils in cool-temperate regions (Caradus, 1978, 1988; Maunsell and Scott, 1996). When either of these species has been grown in association with clover, the clover component is still able to make a signiﬁcant contribution to both pasture production and animal performance (e.g., Ayres et al., 2000; Hyslop et al., 2000). Compared to tillering grass types, stoloniferous and rhizomatous grasses have generally been shown to be counterproductive to clover performance when grown as its sward companion (e.g., Bakken et al., 1987; Frame, 1990). Both Agrostis stolonifera and Holcus lanatus form a dense vegetative cover close to the ground and compete vigorously for light and space against clover (Brougham et al., 1978; Turkington et al., 1979; Frame, 1990; Barthram, 1997). However, this effect is undeniably inﬂuenced by pasture management. Research (Stringer, 1997) with the stoloniferous grass species Cynodon dactylon showed that clover content was markedly increased as sward heights were reduced. This highlights the potential that grazing management can have on manipulating the legume –grass competitive interaction with grass species that are often considered incompatible with clover. In general, the utility of combining different ryegrass or grass types and white clover cultivars needs further evaluation under new farm system managements and the implications for white clover performance more fully assessed. Furthermore, as new grass cultivars with differing vigour and persistence become available further testing of their compatibility with clover will be necessary (e.g., Pederson et al., 1999).

C. TACTICAL USE

OF

N FERTILISER

An overwhelming amount of evidence exists showing the effects of N fertiliser on pasture production per se, as well as white clover performance and N2 ﬁxation (e.g., Curll et al., 1985b; Evans et al., 1992; Ledgard et al., 1996b; Elgersma et al., 2000). Typically, where N fertiliser is used, total pasture production is increased because of increased grass yield while that of the white clover component declines and N2 ﬁxation decreases markedly (Table XVII). In some studies (e.g., Ledgard et al., 1996b; Table XVII), the direct effect of N fertiliser on reducing N2 ﬁxing activity is largely responsible for causing the loss in annual ﬁxed N (e.g., up to 60%) with reductions in white clover yield contributing to a far lesser extent. However, this is not always the case and in other studies (e.g., Frame and Boyd, 1987a; Nesheim et al., 1990; Elgersma et al., 2000), depending on the level of N application, white clover yield losses of between 50 and 80% have been observed within a year of frequent N fertiliser use, and can therefore be

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Table XVII Effect of N Fertilisation on Annual White Clover Production, Content, and N2 Fixation in a Mixed Legume/Grass Pasture Rotationally Grazed by Sheepa N fertilizer White clover White clover Proportion of treatment production content clover N ﬁxedb (kg DM ha21 year21) (kg DM ha21 year21) (%) (%) 0 390 SED

3602 2974 196

28 19 NR

58.4 33.4 2.6

Total N ﬁxedc (kg N ha21 year21)

111 47 9

NR, not reported. Data from Ledgard et al. (1996b). b Estimated using the 15N isotope method. c Fixed N in herbage. a

a major contributing factor in reducing the amount of N ﬁxed in these systems. The reason for declining white clover growth under N fertiliser is due to a combination of factors brought about principally by competition for light and nutrient resources from its more vigorous and upright-growing grass associate. These factors include reduced photosynthesis, a reduction in growing point densities because of diminished assimilate allocation and stolon branching, and possibly increased competition for soil nutrients (Laidlaw and Withers, 1989; Hoglind and Frankow-Lindberg, 1998). While the repetitive use of N fertiliser is often reported to be detrimental to white clover performance, some studies have shown that even under high regular N fertiliser applications white clover content can be maintained, but only if sward management prevents shading from the grass component (Frame and Boyd, 1987a). For example, in pasture grazed by dairy cows, Barr (1996) and Harris and Clark (1996) reported that under high N fertiliser use (up to 400 kg N ha21 year21) white clover can persist and contribute usefully to production, provided the additional grass grown is fully utilised. In view of the signiﬁcant direct effect of N fertiliser on N2 ﬁxation, maintaining a sward with high clover content but of greatly diminished N2 ﬁxing capability is of limited advantage from an N2 ﬁxation efﬁciency standpoint. To overcome this drawback the tactical use of N fertiliser is often advocated, particularly at times when rates of legume growth and N2 ﬁxation are low. If the tactical use of N is paired with speciﬁc grazing management strategies, it could provide the best compromise in maintaining optimal pasture production without substituting N fertiliser for N2 ﬁxation or jeopardising the long-term performance of white clover. It is common for farmers to apply N fertiliser in early-spring to boost early-season grass production. Unfortunately, this tactic can have a negative effect on white clover performance (Frame and Boyd, 1987b), unless grazing

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Table XVIII Effect of Spring and/or Autumn N Fertilisation on White Clover Content (% DM Basis) in a White Clover/Grass Sward Under Mowing (Mean of 3 years)a Autumn N application (kg ha21) Spring N application (kg ha21)

0

25

50

75

0 25 50 75

48 43 38 34

48 36 33 27

41 40 33 29

45 36 31 27

a

Data from Frame and Boyd (1987b).

management strategies are used to control subsequent grass growth and minimise shading effects on white clover. More recently, studies in the northeast USA (Stout and Weaver, 2001; Stout et al., 2001) predicted that a single application of N fertiliser (45 kg N ha21) in spring gave the largest gains in pasture production without compromising clover growth or N2 ﬁxation. This was provided if a target sward harvest height of 15 cm was not exceeded, since above this height clover content declined rapidly. Alternatively, applying N fertiliser in autumn seems to have less effect on white clover content than in spring (Table XVIII). Thus, to realise the full potential of N2 ﬁxation in mixed ryegrass – white clover pastures, N fertiliser use should be minimised or strict grazing/cutting management strategies used to control total herbage height, particularly after spring-applied N.

VI. FARM-SCALE MANAGEMENT PRACTICES Within the farm system a number of opportunities exist for improving farm management practices and increasing legume performance and N2 ﬁxation in mixed legume – grass pastures. Generally, changes at the farm system level involve integrating new management practices with an increased awareness of the limitations and optimal utilisation of plant and soil resources. This is best carried out by using “quantiﬁable assessment tools” to evaluate pasture and soil conditions, and by engaging the assistance of other farm colleagues and off-farm expertise. Because animals are an intrinsic part of any grazing system, realistic management options are required which do not compromise the business goals of the farming enterprise.

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A. SOIL MANAGEMENT : PREVENTING TREADING AND COMPACTION In farming systems, good soil management should aim to create optimal physical conditions for legume growth through preventative management strategies. To minimise treading damage to soils, a sound knowledge is required of the different landscape units and soil types that comprise the farm and their susceptibility to structural damage in wet conditions. Information such as this should be incorporated into far-sighted grazing management plans to ensure restricted or zero grazing of vulnerable areas is easily implemented when conditions necessitate, without placing undue strain on other farm activities. Additionally, in pasture that suffers from waterlogging in winter, improving the drainage of the soil, for example by mole/tile draining, also reduces the likelihood of treading damage to soil (e.g., Davies and Armstrong, 1986). Farm systems that include areas for forage cropping (e.g., maize silage) could provide an opportunity for renovating compacted pasture soils through breakingdown compacted layers, increasing soil aeration, and by providing an extended rest from animal grazing impacts. For this strategy to be effective the forage crop should be rotated and tillage only performed when soil moisture conditions are ideal, so to avoid any additional compaction by agricultural machinery and to optimise soil rejuvenation. A further beneﬁt of legume growth and N2 ﬁxation would be incurred after pasture resowing as a result of soil N depletion during the cropping phase, and reduced insect pests (e.g., clover nematodes, Yeates, 1977; Yeates and Hughes, 1990). Recently, indicators of soil physical condition have been developed (e.g., macroporosity, penetration resistance, microbial biomass, worm counts, e.g., Ditzler and Tugel, 2002; Drewry et al., 2002) and can be used on-farm alongside soil fertility measures to indicate limiting conditions for plant growth. For example in New Zealand some current research has resulted in a “macroporosity test” for on-farm use to determine limiting soil physical conditions for pasture yield (Drewry et al., 2001, 2002). This work is still in its infancy and requires further calibration across a range of soil types and farm management practices before it can be widely used.

B. RESTRICTED GRAZING AND SUPPLEMENTARY FEEDING IN WINTER /S PRING An effective method of reducing treading damage to pasture in wet conditions is the practice of restricted grazing and supplementary feeding. This concept requires farmers to identify the risk of damage to pasture at key times during winter/spring and remove livestock from paddocks and onto an area(s) designated for animal stand-off and supplementary feeding. Alternately, animals

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may graze for 4 – 6 h and then be put on a stand-off area for the remainder of the day. In dairy systems, stand-off areas may be highly sophisticated feed-pad systems such as “herd houses” that are entirely covered, have concrete pads and collect and contain efﬂuent in sub-surface storage tanks for later disposal. In practice, simpler uncovered feed pads with either a concrete pad or a bark/saw dust pad on a road metal or lime base with drainage of efﬂuent to storage ponds are more common in Australia (e.g., Moran and Wamungai, 1992) and New Zealand. Even less complicated is the practice of standing-off cows in races/lanes for part of a day during wet soil conditions. In sheep and beef farm systems a more rudimentary approach is sometimes taken in wet periods with animals stocked on a “sacriﬁce paddock” and fed hay. This approach contains treading damage to one area of the farm rather than causing indiscriminate damage to larger areas. This strategy has largely been replaced in current times by the more deliberate method of destocking for winter and keeping livestock off paddocks that are susceptible to treading damage. In New Zealand, the use of stand-off pads (cf. feed pads) on dairy farms is becoming increasingly common, and gives the farmer the option of “on/off” grazing during winter and early-spring when ground conditions may be overly wet. The extent of feed-pad use varies with climate. In milder regions intermittent use in winter or early-spring is often all that is required to prevent treading damage to pasture. In wetter-cooler regions, feed pads are sometimes occupied for extended periods in winter (e.g., 75 days in Southland, New Zealand) because of limited pasture growth and the greater potential for damage by treading. In Europe with the current move towards extended grazing in some milder regions similar tactics to those above could be applied, i.e., using an extended but restricted or partial grazing regime through the winter in conjunction with cow housing and supplementary feeding. This strategy would minimise winter damage to pasture and has the added beneﬁt of keeping sward heights in check to favour white clover growth and productivity for spring grazing. Despite the increasing popularity of feed-pad use in Australasia, there is little research on the beneﬁts of these systems on pasture productivity let alone white clover productivity. One Australian study (Moran and Wamungai, 1992) using partial restrictions to grazing during winter (6 h day21) and supplementary feeding with maize silage on a feed pad gave improvements in pasture quality (increased subterranean clover content) while still maintaining a high winter stocking rate and milk production. Other work by De Klein (2001) estimated that zero grazing for an extended period in winter (123 days) while supplying cut pasture to cows on a feed pad could increase annual pasture production by 2 – 8%. However, this estimate only considered the effect of better utilisation of applied efﬂuent collected from the feed pad; the beneﬁcial effects from lowering the impact of other grazing animal factors (e.g., treading and defoliation, Sections II and III, respectively) were not considered and could arguably further boost this value. Furthermore, effects on white clover productivity were not considered in

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the De Klein (2001) study, but presumably the frequent cutting of pasture for feed-pad supply would be advantageous for white clover, giving the potential for higher white clover content in spring swards and increased N2 ﬁxation (Roberts et al., 1989; Bax and Thomas, 1992). Schwinning and Parsons (1996b) estimated that white clover can take advantage of sward patchiness and areas of low soil N in grazed pasture for growth and survival. This behaviour of white clover allows it to escape light restrictions caused by shading of N stimulated grass. Substituting grazing with cutting and mechanically applying animal efﬂuent to pasture both create a more even spatial distribution of soil N and therefore improved sward height uniformity, which eliminates opportunities for white clover to escape light limiting situations. To compensate for this, keeping pasture cover low during periods of nil grazing by using a frequent/hard cutting management regime would improve light penetration to the lower sward canopy and probably assist in white clover growth and persistence. While the few studies reviewed here allude to the beneﬁts of partial or zero grazing in winter, the increasing use of feed-pad systems necessitates further research with a broader investigative approach to consider the additional beneﬁts for grass and legume that might occur through reduced treading and defoliation impacts, as well as by the improved utilisation of animal excreta. This future work should take into account the effect of variations in feed-pad use (intermittent, partial, or extended) brought about by differences in regional climatic conditions together with a fuller assessment of their economic viability.

C. TECHNICAL BASED DECISION MAKING IMPROVED MANAGEMENT

FOR

In farm systems, management skill and decision making have a great bearing on white clover performance, N2 ﬁxation, and overall pasture productivity. To assist the farmer in making accurate knowledgeable decisions regarding the legume productivity component of farm management a number of useful “tools” and off-farm interactions (e.g., farmer study groups) offer options for optimising clover performance and N2 ﬁxation (Fig. 6). Participatory farmer study groups are a relatively recent concept (last 5– 10 years), but have been successful in addressing complex farm system changes and environmental problems where expert-only driven approaches have failed (e.g., Engel, 1991; Okali et al., 1994). Typically, these study groups involve linking farmers, scientists, and policy agents to provide a broad base of knowledge and experience to tackle on-farm issues and to facilitate technology transfer. In New Zealand, for example, a study group in a predominantly dairying region was formed in 1995 to identify relevant on-farm issues and determine ways of dealing with these (Tarbotton et al., 1997). The study group, comprising eight dairy

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farmers, a farm systems analyst, a soil scientist, and two policy agents, soon identiﬁed pugging by dairy cows as causing considerable damage to pasture during winter and spring (up to 10% area affected on one farm). Using the pooled knowledge and expertise of all the group participants, a number of management strategies and options were identiﬁed to prevent winter and spring pugging (Table XIX). These were successfully implemented and have achieved the desired outcome of greatly reducing pugging and damage to pasture (Tarbotton et al., 1997). The study group is now in its 7th year and has continued to use the participatory process approach to solving on-farm problems, one of which is seeking to ﬁnd ways of improving white clover performance in white cloverbased pastures (Bateup et al., 2001). Although multidisciplinary on-farm study groups are not widespread, the application of the group discussed here highlights what can be achieved by bringing together individuals having a breadth of experience and expertise. In Europe, some farmers are recognising the value of such an approach and have called for a greater participation of agricultural scientists and farm advisors in farm discussion groups, especially in the light of some recent changes to farm system operations (e.g., a move to grazing-based dairy systems and extended grazing) (John et al., 2000). The challenge for these study groups will be in dealing with the multiple facets of farm optimisation under a new farm system, for example, livestock management in winter, pasture utilisation throughout the year, and white clover performance and N2 ﬁxation. Some simple and inexpensive tools which aid decision making are available for farm use. These include: soil tests (e.g., nutrient status, macroporosity, and penetration resistance), pasture yield meters (e.g., rising plate meters), recording on-farm meteorological information, and weather forecasting (short and longterm). In conjunction with other farm decision support mechanisms (e.g., practical computer models, advisors, study groups, and tacit knowledge) these tools, if used effectively, will further enhance the potential for optimising legume content as well as farm productivity. For example, adopting a standardised method of pasture herbage assessment (e.g., rising plate meter) provides for greater consistency of yield information and gives conﬁdence in making reliable grazing management decisions for better utilisation of pasture (Lile et al., 2001). Inclusive to this decision making process should be all the key pasture production elements, including that of optimising legume performance and N2 ﬁxation. In achieving this, careful thought needs to be applied in deciding on which type of pasture management strategies are best utilised and their appropriate timing of implementation (e.g., seasonal timing of certain grazing regimes, duration of rest interval, and timing of silage cuts). Using an approach like this is especially useful where certain farm objectives (e.g., ﬁnishing lambs in autumn or increasing milk production in summer) are known to be beneﬁted by swards containing high clover content (Askin et al., 1987; Harris and Clark, 1996;

Observations (1) Soil condition prior to grazing in the wetter months of June, July, August, September, October (2) Soil moisture content How soft is the ground? Is the area waterlogged?

(3) Know the topography of the paddock. The above three give the basic paddock information and should be used in conjunction with 4 and 5 (4) Actual and forecasted weather, particularly previous rainfall. Also, wind speed and direction in combination with actual rainfall

(1) Recognise the potential for damage (2) Grade paddocks according to their susceptibility to damage Followed by one or more of the following options: (a) Implement on/off grazing using: races, cow sheds, dry peat land or a sacriﬁce paddock (b) Grazing off farm, or carrying feed to cows on a stand-off pad (c) Decrease stocking pressure by using quicker rotation, short term only. Increase the area; cows are grazing, e.g., no break feeding or use larger breaks

(5) Grass colour, i.e., dirt on grass within the area grazed beyond acceptable levels

Management options

Overcoming implications

Outcomes

(1) On/off grazing: This will minimise damage and is convenient, beneﬁcial, cheap, and allows easy feed rationing

Put in longer hours and be prepared to change priority on your time

Time well spent. Short term, there was less cost. Long term no negative effects. The problem was perceived to be greater than that which eventuated. No effect on cow condition

Implications: Cows get sore feet, damage to races, possible loss of cow condition, damage to gateways and fences, labour intensive (2) Grazing either off farm or use of a stand-off pad: no pugging Implications: Going off farm is expensive, and depends on land availability and results in loss of control of cow condition and stock management On farm pad has a high labour input of feeding, requires buying or making large quantities of feed, efﬂuent has to be spread (3) Speeding up rotation: short term for 1–2 days. Early in winter it is a quick short-term solution Implications: Uses too much feed. Eating into reserve feed is the key point. Have reduced grass cover

NB: Example of process-ﬂow indicated by arrows and shading. a Data from Tarbotton et al. (1997).

Short term, take care and shift cows slowly, long term do more repairs and maintenance Keep yard clean and free of debris Use a footbath and put copper sulphate on the races or on mats. Separate and treat cows with severely sore feet Restrict the movement on races to a small area

Research the cost of grazing off farm and supplements on farm Forward planning to control sward heights at home

Success—no permanent damage, sore feet healed quickly without long-term negative effects Increase the area: Keep it short term, a few days at a time, and consider nitrogen as an option

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Is water ponded on the ground surface?

Management strategies

226

Table XIX The Results of a Workshop at a New Zealand Dairy Farm Study Group Documented the Understanding, Management and Outcomes Developed to Prevent Winter Pugginga

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Papadopoulos et al., 2001; Woodward et al., 2001; Wu et al., 2001). Thus, speciﬁcally targeting high legume contents on some parts of the farm to meet these objectives are likely to be most advantageous for farm productivity. Indeed, new research is examining the potential of growing grass and clover separately within the same paddock, to achieve strategic goals of high clover content for increased clover intake and animal production (e.g., Marotti et al., 2002; Cosgrove et al., 2003).

VII. SUMMARY AND CONCLUSIONS In this review, the impacts of treading, defoliation, and excretion by grazing animals on the efﬁciency of N2 ﬁxation have been considered along with preventative management options to improve legume performance in mixed legume – grass pasture systems. Grazing animal activities have a signiﬁcant impact on the efﬁciency of N2 ﬁxation, which is mediated by large-scale changes to legume morphology and physiology both at the individual plant level and/or by inﬂuencing the legume – grass competitive interaction. The magnitude of animal impacts, both individually and as a whole, varies greatly and are closely tied to farm management practices and the edaphic features of the entire farm system. Future research focus should include increasing the competitiveness of white clover for light and space resources, and further evaluation of companion grasses with attributes such as lower tillering and earlier maturity so as to desynchronise their growth peaks and provide gap opportunities for clover exploitation. Excretion by grazing animals has a major effect on the efﬁciency of N2 ﬁxation by altering soil N status. This in turn directly affects N2 ﬁxing activity for extended periods, or operates through reducing clover productivity from shading by grasses if grazing management allows. Exploring the potential for use of clover cultivars that can continue high N2 ﬁxation under elevated soil N status is tempting, but ﬂow-on effects to N cycling would ﬁrst need to be considered, in particular, the likelihood of increased N losses to the environment. With regard to treading and soil compaction, more detailed research is needed to determine the broader and longer-term implications of impacts on N2 ﬁxation and clover productivity in the ﬁeld. Indications are that if soil aeration is limited by compaction over extended periods then white clover may be able to compensate by utilising cellular and structural adaptations to adjust to low O2 levels. Damage and burial of plants by treading is also of considerable importance and appears to be a key factor in determining successful overwintering and subsequent spring white clover content in clover –grass associations. In farm systems where animal intensity is high but defoliation pressure is relatively low (due to supplementary feeding) treading damage may have a greater relative effect on white clover performance than defoliation and

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excretion processes. The use of models may provide the best means to unravel the complexity of treading processes, their interaction with defoliation and excretion, and the overall response of the legume system. By far the greatest means of practical improvement in N2 ﬁxation efﬁciency in legume-based pasture systems is by evaluating the management decisions of the farm system and identifying alternate pasture management strategies or farm management practices to achieve optimal legume presence and N2 ﬁxation in the sward. These management strategies may include: varying seasonal grazing intensity, mixed animal type grazing, strategic silage cuts, and removal of animals from pasture in wet conditions. Future research will need to develop guidelines that facilitate the adoption/integration of these strategies and other novel approaches into different farm systems. In addition, research should more clearly deﬁne the potential economic and environmental advantages from greater reliance on N2 ﬁxation technology. Farmer involvement in collaborative study groups which blend together the expertise of farmer, scientist, farm advisor, and other industry specialists in a participatory team process will provide the best opportunity to realise these goals and achieve improved legume performance and N2 ﬁxation.

ACKNOWLEDGMENTS The ﬁrst author is grateful to the New Zealand Agricultural Marketing and Research Development Trust for ﬁnancial assistance; John Brock, Mike Hay, and Jim Crush for constructive comments on the manuscript.

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Roberts, D. J., Frame, J., and Leaver, J. D. (1989). A comparison of a grass/white clover sward with a grass sward plus nitrogen under a three-cut regime. Res. Dev. Agric. 6, 147–150. Russell, J. S. (1960). Soil fertility changes in the long-term experimental plots at Kybybolite, South Australia. I. Changes in pH, total nitrogen, organic carbon, and bulk density. Aust. J. Agric. Res. 11, 902–926. Ryle, G. J. A., Powell, C. E., and Gordon, A. J. (1985). Defoliation in white clover: regrowth, photosynthesis and N2 ﬁxation. Ann. Bot. 56, 9 –18. Ryle, G. J. A., Powell, C. E., and Gordon, A. J. (1986). Defoliation in white clover: nodule metabolism, nodule growth and maintenance, and nitrogenase functioning during growth and regrowth. Ann. Bot. 57, 263– 271. Sanderson, M. A., and Elwinger, G. F. (1999). Grass species and cultivar effects on establishment of grass–clover mixtures. Agron. J. 91, 889–897. Schwinning, S., and Parsons, A. J. (1996a). Analysis of the coexistence mechanism for grasses and legumes in grazing systems. J. Ecol. 84, 799 –813. Schwinning, S., and Parsons, A. J. (1996b). A spatially explicit population model of stoloniferous N-ﬁxing legumes in mixed pasture with grass. J. Ecol. 84, 815–826. Semiadi, G., Barry, T. N., Muir, P. D., and Hodgson, J. (1995). Dietary preferences of sambar (Cervus unicolor) and red deer (Cervus elaphus) offered browse, forage legume and grass species. J. Agric. Sci. 125, 99 –107. Sheath, G. W., and Hodgson, J. (1989). Plant –animal factors inﬂuencing legume persistence. In “Persistence of Forage Legumes” (G. C. Marten, A. G. Matches, R. F. Barnes, R. W. Brougham, R. J. Clements, and G. W. Sheath, Eds.), pp. 361–374. ASA/CSSA/SSSA, Madison, WI. Sheldrick, R. D., Lavender, R. H., and Martyn, T. M. (1993). Management options for increasing white clover content of swards without re-sowing. Grass Forage Sci. 48, 223 –230. Shiferaw, W., Shelton, H. M., and So, H. B. (1992). Tolerance of some subtropical pasture legumes to waterlogging. Trop. Grassl. 26, 187 –195. Singleton, P. L., and Addison, B. (1999). Effects of cattle treading on physical properties of three soils used for dairy farming in the Waikato, North Island, New Zealand. Aust. J. Soil Res. 37, 891–902. Sprent, J. I., and ’t Mannetje, L. (1996). The role of legumes in sustainable farming systems: past, present and future. In “Legumes in Sustainable Farming Systems”. “Occasional Symposium No. 30” (D. Younie, Ed.), pp. 2– 14. British Grassland Society, Aberdeen, UK. Stephenson, G. R., and Veigel, A. (1987). Recovery of compacted soil on pastures used for winter cattle feeding. J. Range Manag. 40, 46–48. Stepniewski, W., Glinski, J., and Ball, B. C. (1994). Effects of soil compaction on soil aeration properties. In “Soil Compaction in Crop Production” (B. D. Soane and C. van Ouwerkerk, Eds.), pp. 167–189. Elsevier, Amsterdam. Stevens, D. R., and Hickey, M. J. (2000). Effects of binary seed mixtures of ryegrass, cocksfoot and tall fescue on pasture production. Proc. NZ Grassl. Assoc. 62, 141 –146. Stout, W. L., and Weaver, S. R. (2001). Effects of early season nitrogen on nitrogen ﬁxation and fertilizer-use efﬁciency in grass–clover pastures. Commun. Soil Sci. Plant Anal. 32, 2425–2437. Stout, W. L., Weaver, S. R., and Elwinger, G. F. (2001). Effects of early season nitrogen on grass– clover swards in the Northeastern USA. Agron. J. 93, 1000–1005. Stringer, T. L. (1997). Effect of Bermudagrass height on clover establishment. Crop Sci. 37, 1663–1665. Swift, G., Vipond, J. E., McClelland, T. H., Cleland, A. T., Milne, J. A., and Hunter, E. A. (1993). A comparison of diploid and tetraploid perennial ryegrass and tetraploid ryegrass/white clover swards under continuous sheep stocking at controlled sward heights. 1. Sward characteristics. Grass Forage Sci. 48, 279–289. Ta, T. C., MacDowall, F. D. H., and Frais, M. A. (1990). Utilization of carbon and nitrogen reserves of alfalfa roots in supporting N2-ﬁxation and shoot regrowth. Plant Soil 127, 231 –236.

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Taboada, M. A., and Lavado, R. S. (1988). Grazing effects on the bulk density in a Natraquoll of Argentina. J. Range Manag. 41, 500 –503. Tarbotton, I. S., Paine, M. S., Wedderburn, M. E., and Carlyon, M. (1997). A method for improving farm practice: a case study of pugging. Proc. NZ Grassl. Assoc. 59, 79 –85. Teuber, N., and Laidlaw, A. S. (1995). Effect of herbage rejection by steers on white clover (Trifolium repens) branching and development in continuously stocked grass–clover swards. J. Agric. Sci., Cambridge 124, 205 –212. Tu, J., and Buttery, B. (1988). Soil compaction reduces nodulation, nodule efﬁciency, and growth of soybean and white bean. Hortic. Sci. 23, 722 –724. Turkington, R., Cahn, M. A., Vardy, A., and Harper, J. L. (1979). The growth distribution and neighbour relationships of Trifolium repens in a permanent pasture. III. The establishment and growth of Trifolium repens in natural and perturbed sites. J. Ecol. 67, 231–243. Vallentine, J. F. (2001). “Grazing Management”, 2nd edn. Academic Press, USA. Vance, C. P., Heichel, G. H., Barnes, D. K., Bryan, J. W., and Johnson, L. E. (1979). Nitrogen ﬁxation, nodule development, and vegetative regrowth of alfalfa (Medicago sativa L.) following harvest. Plant Physiol. 64, 1–8. Vinther, F. P. (1998). Biological nitrogen ﬁxation in grass–clover affected by animal excreta. Plant Soil 203, 207–215. Voorhees, W. B., Carlson, V. A., and Senst, C. G. (1976). Soybean nodulation as affected by wheel trafﬁc. Agron. J. 68, 976–985. Wachendorf, M., Collins, R. P., Connolly, J., Elgersma, A., Fothergill, M., Frankow-Lindberg, B. E., Ghesquiere, A., Guckert, A., Guinchard, M. P., Helgadottir, A., Luscher, A., Nolan, T., NykanenKurki, P., Nosberger, J., Parente, G., Puzio, S., Rhodes, I., Robin, C., Ryan, A., Staheli, B., Stoffel, S., and Taube, F. (2001). Overwintering of Trifolium repens L. and succeeding spring growth: results from a common protocol carried out at twelve European sites. Ann. Bot. 88, 669–682. Wachendorf, M., Collins, R. P., Elgersma, A., Fothergill, M., Frankow-Lindberg, B., Ghesquiere, A., Guckert, A., Guinchard, M. P., Helgadottir, A., Luscher, A., Nolan, T., Nykanen-Kurki, P., Nosberger, J., Parente, G., Puzio, S., Rhodes, I., Robin, C., Ryan, A., Staheli, B., Stoffel, S., Taube, F., and Connolly, J. (2001). Overwintering and growing season dynamics of Trifolium repens L. in mixture with Lolium perenne L. A model approach to plant–environment interactions. Ann. Bot. 88, 683–702. Ward, C. Y., Jones, J. N., Lillard, J. H., Moody, J. E., Brown, R. H., and Blaser, R. E. (1996). Effects of irrigation and cutting management on yield and botanical composition of selected legumes–grass mixtures. Agron. J. 58, 181 –184. Weeda, W. C. (1967). The effect of dung patches on pasture growth, botanical composition and pasture utilization. NZ J. Agric. Res. 10, 150–159. Whitehead, D. C. (1995). “Grassland Nitrogen”. CAB International, Wallingford, UK. Widdup, K. H., and Turner, J. D. (1983). Performance of 4 white clover populations in monoculture and with ryegrass under grazing. NZ J. Exp. Agric. 11, 27–31. Willatt, S. T., and Pullar, D. M. (1983). Changes in soil physical properties under grazed pastures. Aust. J. Soil Res. 22, 343–348. Woledge, J., Tewson, V., and Davidson, I. A. (1990). Growth of grass/clover mixtures during winter. Grass Forage Sci. 45, 191 –202. Woodﬁeld, D. R., and Caradus, J. R. (1996). Factors affecting white clover persistence in New Zealand pastures. Proc. NZ Grassl. Assoc. 58, 229–236. Woodﬁeld, D. R., Clifford, P. T. P., Cousins, G. R., Ford, J. L., Baird, I. J., Miller, J. E., Woodward, S. L., and Caradus, J. R. (2001). Grasslands Kopu II and Crusader: new generation white clovers. Proc. NZ Grassl. Assoc. 63, 103 –108. Woodward, S. L., MacDonald, K. A., Carter, W. A., Eerens, J. P. J., and Crush, J. R. (2001). Milksolids production from different combinations of perennial ryegrass and white

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SEED-FILL DURATION AND YIELD OF GRAIN CROPS* Dennis B. Egli Department of Agronomy, University of Kentucky, Lexington, Kentucky 40546-0312, USA

I. Introduction II. Seed Filling: Deﬁnition and Measurement III. Variation in Seed-Fill Duration A. Temperature B. Water Stress C. Assimilate and Nutrient Supplies D. Flower and Fruit Development E. Photoperiod F. Plant Growth Substances G. Genetic IV. Regulation of Seed-Fill Duration A. Regulation by the Seed B. Regulation by the Plant C. Senescence V. Seed-Fill Duration and Crop Productivity A. Yield B. Future Yield Improvement VI. Conclusions References Time, as expressed through seed-ﬁll duration, is an important component of the productivity of grain crops. Seed-ﬁll duration is inﬂuenced by environmental conditions and there is genetic variation in most crops. Seed-ﬁll duration is regulated by the leaf’s ability to supply assimilate to the developing seed (i.e., leaf senescence) and by the ability of the seed to use this assimilate for continued growth. Seed-ﬁll duration in many crops increases as temperature decreases below 308C. Water stress during seed ﬁlling accelerates leaf senescence and shortens the seed-ﬁlling period. The response of seed-ﬁll duration to alterations in source – sink ratios is inconsistent and probably depends on whether the supply of assimilate to the seed is affected and if the seed can respond to changes. In some cases increases in harvest index are a result of lengthening the * Published with the approval of the Director of the Kentucky Agricultural Station Experiment Station, University of Kentucky, Lexington, Kentucky as paper number 03-06-107.

243 Advances in Agronomy, Volume 83 Copyright q 2004 by Elsevier Inc. All rights of reproduction in any form reserved. DOI 10.1016/S0065-2113(04)83005-0

244

D. B. EGLI seed-ﬁlling period. Environmental variation in seed-ﬁll duration is frequently associated with yield in most crops. Direct selection for long seedﬁlling periods may increase yield and, conversely, selection for higher yield in many crops resulted in longer seed-ﬁlling periods. Seed-ﬁll duration can have a relatively low heritability and is somewhat difﬁcult to measure, and it has not been widely used by plant breeders in cultivar improvement programs. However, given the intractable nature of crop growth rate, lengthening the seed-ﬁlling period may be the most promising avenue to higher yields, but new approaches to manipulate it will probably have to be devised. q 2004 Elsevier Inc.

I. INTRODUCTION Yield of a grain crop is determined by the rate (on an area basis) and duration of dry matter accumulation by the seeds. A model consisting only of rate and duration of growth may be too simplistic to be of much value as it combines all of the production processes, e.g., photosynthesis, respiration, translocation, nutrient uptake, etc., into the total seed growth rate. Such a model, however, has one advantage, it clearly describes yield as a rate expressed over a speciﬁc period. The same model, with the same weaknesses, can be applied to the accumulation of biomass by a plant community, but, while the seed growth rate is related to the growth rate of the community, there may be no relationship between the duration of growth of the community and that of the seeds. Yield or biomass would be increased if plant communities, or the seed fraction, would grow faster or for a longer period, barring any offsetting changes in either factor. Increasing crop growth rate (Evans, 1993) or photosynthesis (Horton, 2000) has proven difﬁcult and, if radiation use efﬁciency in non-stress environments is near its maximum (Sinclair and Horie, 1989), future increases may be modest. What then are the prospects of creating crops that grow longer? Time is a valuable component of any environment as the potential productivity is determined by the solar radiation available when temperatures are suitable for plant growth (de Wit, 1967). In environments with short growing seasons there is usually little time unused when the crop matures, but in many temperate environments and in most tropical environments there may be a substantial amount of time and potential productivity that are not used by a single grain crop. A crop that grew for a longer time would use more of this wasted potential (Duncan, 1969). Historically, research in crop physiology emphasized plant processes associated with the rate of growth, with less attention given to the duration of growth. This emphasis, however, seems to be shifting and my objective in this

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review is to encourage this shift in emphasis to duration, and more speciﬁcally, to the duration of seed growth. I will investigate the variation in seed-ﬁll duration in grain crops, the factors responsible for this variation, the mechanisms regulating seed-ﬁll duration, the relationship between seed-ﬁll duration and yield and the potential for manipulating seed-ﬁll duration to increase crop productivity. Crops that are harvested for seed or grain include C3 and C4 species, grasses and legumes, and they produce seeds with different morphology and a substantial range in oil, protein and starch concentrations. In spite of this variation, however, the characteristics of seed growth are remarkably uniform (Egli, 1998), so I will include all grain crops in this review, seeking to identify the characteristics of seed-ﬁll duration that are common to all species.

II. SEED FILLING: DEFINITION AND MEASUREMENT Seed-ﬁll duration—the time from the beginning of seed growth until the seed reaches maximum dry weight at physiological maturity (PM)—can be deﬁned for an individual seed, a plant or a plant community. All three levels share the same fundamental deﬁnition, but they are not measures of the same quantity. The seed-ﬁll duration of a plant or a plant community will typically be longer than that of an individual seed (measured by sampling seeds from ﬂowers that were pollinated at the same time) because all seeds on a plant or in a plant community do not start or stop growing at the same time (Tollenaar and Daynard, 1978; Simmons et al., 1982; Jongkaeuwattana et al., 1993; Egli, 1994). Seed-ﬁll duration at any of these levels can be used to estimate relative genetic or environmental effects within a species, but comparisons among levels can be misleading. Seed-ﬁll duration can be estimated from a complete seed-growth curve derived from repeated determinations of seed dry weight on an individual seed, plant or plant community basis. A regression model that accurately describes seed dry weight as a function of time can be used to estimate the beginning and end of seed growth providing an estimate of seed-ﬁll duration. The logistic model has been used successfully (Loss et al., 1989; Darrock and Baker, 1995; Santiveri et al., 2002), as have cubic polynomial models (Jones et al., 1979; Gebeyehou et al., 1982). A variety of deﬁnitions of seed-ﬁll duration have been used with these models including the time from 5 to 95% or 10 to 90% of maximum seed dry weight (Johnson and Tanner, 1972) or from anthesis or zero dry seed weight to the maximum (Gebeyehou et al., 1982; Mou et al., 1994). Alternatively, the effective ﬁlling period (EFP)—ﬁnal seed weight divided by the rate of seed growth during the linear phase of growth (Daynard et al., 1971; Daynard and Kannenberg, 1976)—can be used to avoid determining when seeds start and stop growing.

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Both of these methods can be applied at all levels, but the growth curve method requires extensive sampling and selection of the appropriate regression model to produce acceptable estimates. The EFP avoids dealing with the nonlinear lag phases of seed growth and requires fewer samples. Plant growth stages can also be used to estimate seed-ﬁll duration at the individual plant or plant community levels. The time between growth stages R5 (beginning of seed ﬁll) and R7 (an estimate of PM, Fehr and Caviness, 1977) is widely used in soybean [Glycine max (L.) Merr.] (Egli et al., 1984; Agudelo et al., 1986). The period between anthesis or silking and PM is popular in many cereals (Daynard and Kannenberg, 1976; Darrock and Baker, 1995). PM is usually estimated from a visual appraisal of seed or plant characteristics (Egli, 1998). Growth stage estimates may not be as precise as those based on seed growth curves [e.g., R5 – R7 is not the same for indeterminate and determinate soybean cultivars (Agudelo et al., 1986; Pfeiffer and Egli, 1988)], but they have the advantage in that they are non-destructive and easy to determine. Munier-Jolain et al. (1993) and Ney et al. (1993) used seed moisture levels, which are closely associated with the stage of seed development (Fraser et al., 1982), to estimate seed-ﬁll duration in soybean and pea (Pisum sativum L.). Comparisons of published estimates of seed-ﬁll duration can be misleading because of the variety of methods used to estimate it, species variation in growth characteristics and environmental effects. However, comparisons of published estimates of seed-ﬁll duration provide some useful general information about the length and variation of this important growth stage. The variation in seed-ﬁll duration among species, based on whole-plant growth characteristics, is frequently less than the variation within a species in a summary of ﬁeld trials (Table I). A similar summary of EFP also found more variation within than between species (Egli, 1981). The longer seed-ﬁll duration of maize (Zea mays L.) (Table I) represents an apparent exception to this pattern, however, this advantage was not evident in comparisons of EFP (Egli, 1981), so it could be longer simply because the anthesis to beginning seed growth period is longer in maize than in other species. There are no obvious relationships between known plant characteristics and seed-ﬁll duration in Table I. The seed-ﬁll duration of C4 crops [sorghum (Sorghum bicolor L.), pearl millet (Pennisetum glaucum (L.) Leeke)]—discounting any advantage for maize—is not longer than C3 crops. Species with high seed protein [soybean, groundnut (Arachis hypogea L.)] or oil [sunﬂower (Helianthus annuus L.), groundnut] concentrations do not have shorter seed-ﬁll durations than cereals [wheat (Triticum aestivum L.), rice (Oryza sativa L.)] that produce seeds with low protein and oil concentrations. The seed-ﬁlling period represents less than half of the total growth cycle of most crops. It represented only 26 –41% of the total growth cycle of a group of soybean cultivars from Maturity Groups 00 through V (Zeiher et al., 1982; Egli, 1994). The proportion was only 32 – 38% in pearl millet (Craufurd and Bidinger, 1988), but it was slightly larger in maize (44% for six hybrids)

Table I Variation in Seed-Fill Duration Within and Among Crop Species

Maize

100

Wheat

120

Barley Rice

120 80

Sorghum Pearl millet Sunﬂower Soybean

120 110 200 380

Chickpea Cowpea Groundnut

230 250 310

Seed-ﬁll duration (days) Number of experiments

Number of genotypes

Method of estimation

Mean

Range

Reference

4 2 3 2 2 4 1 1 2 1 2 2 3 1 1 1

8 30 –35 6 11 16 9 5 29 19 2 5 27 13 3 38d 6

Silking to PM Silking to PM Silking to maximum seed mass Cubic polynomial Anthesis to maturity Anthesis to PM Cubic polynominal Broken stick-iterative regression Flowering to PM Flowering to maturity First anthesis to PM Growth stage R5–R7 Growth stage R5–R7 Sigmoid growth curve Flowering to maturity Curvilinear regression

58 65 51 36 37 31 35 26 35 28 41 33 41 37c 21 46

54–62 49–73 48–54 34–40 34–45 29–33 34–37 20–37 30–40 28–28 38–46 28–40 36–46b 34–39 15–30 35–53

Bolanos (1995) Daynard and Kannenberg (1976) Hanway and Russell (1969) Gebeyehou et al. (1982) Reynolds et al. (1994) Garcia del Moral et al. (1991) Jones et al. (1979) Kato (1989) Quinby (1972) Craufurd and Bidinger (1988) Villalobos et al. (1994) Egli (1994) Boerma and Ashley (1988) Davies et al. (1999) Dow el-madina and Hall (1986) Witzenberger et al. (1988)

PM, physiological maturity. Approximate concentrations from Sinclair and de Wit (1975), Hulse et al. (1980), Langer and Hill (1991) and Bewley and Black (1994). b Range based on cultivar means. c Irrigated. d Diverse genotypes from four countries grown in California.

SEED-FILL DURATION

Species

Protein concentrationa (g kg21)

a

247

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D. B. EGLI

(Hanway and Russell, 1969). The preliminary events in the yield production process (vegetative growth, ﬂowering and seed set), take up to twice as much time as the production of the seeds that make up economic yield. All crops do not suffer from such a limited yield production period—tuber growth in potato (Solanum tuberosum spp. tuberosum) continues for up to 120 days (Moorby and Milthorpe, 1975; Spitters, 1987; Vos and Groenwold, 1987; Burton, 1989). Storage root growth in sugar beet (Beta vulgaris L.) covers 90– 110 days (Milford and Watson, 1971; Fick et al., 1975) and cassava (Manihot esculenta Crantz) roots accumulate dry matter for up to 300 days (Howeler and Cadavid, 1983; Aleves, 2002). Compared to these root and tuber crops, the time allocated to yield production in grain crops is not long, which puts much more emphasis on the rate of growth when producing exceptionally high yields.

III. VARIATION IN SEED-FILL DURATION Seed-ﬁll duration, like most plant growth processes, has both an environmental and a genetic component. The length of the seed-ﬁlling period of a crop in a speciﬁc environment is determined by the genetic potential of the genotype and the environment. Environmental conditions during vegetative growth, ﬂowering or seed set could indirectly affect seed-ﬁll duration by altering other aspects of plant growth, but many environmental effects are a direct response to the environment during seed ﬁlling. We will focus on direct effects of the environment during seed ﬁlling.

A. TEMPERATURE Seed-ﬁll duration generally increases as temperatures decrease below 308C in many crops (Fig. 1) including maize (Tollenaar and Bruulsema, 1988; Muchow, 1990; Wilhem et al., 1999), spring and winter wheat (Spiertz, 1978; Vos, 1981; Al-Khatib and Paulsen, 1984; Wardlaw and Moncur, 1995; Gibson and Paulsen, 1999), sunﬂower (Chimenti et al., 2001), lentil (Lens culinais) (Summerﬁeld et al., 1989) and oat (Avena sativa L.) (Hellewell et al., 1996). There may be exceptions to this general trend, for example, there was little effect of temperatures between 20 and 308C on seed-ﬁll duration in soybean (Egli and Wardlaw, 1980) or rice (Chowdhury and Wardlaw, 1978). The seed-ﬁll duration of rice and wheat was the same at 36/318C (day/night temperature), but the duration of wheat was longer at temperatures below 33/288C (Tashiro and Wardlaw, 1989). The seed-ﬁll duration of a japonica rice cultivar did not change between 33/28 and 21/168C while that of sorghum more than doubled over the same range (Chowdhury and Wardlaw, 1978). The effect of shorter seed-ﬁll

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Figure 1 The relationship between temperature and seed-ﬁll duration for several crop species. Seed-ﬁll duration was estimated by the effective ﬁlling period and data from each source was averaged across genotypes, years or experiments where appropriate. The regression was signiﬁcant at P , 0:001: Maize—Tollenaar and Bruulsema (1988) and Wilhem et al. (1999); wheat—Vos (1981), Tashiro and Wardlaw (1989), Hunt et al. (1991) and Wardlaw and Moncur (1995); rice—Fujita et al. (1984) and Tashiro and Wardlaw (1989); sunﬂower—Chimenti et al. (2001).

durations at higher temperatures may be mitigated by an increase in seed growth rate (Chowdhury and Wardlaw, 1978; Spiertz, 1978; Wardlaw and Moncur, 1995) that reduces or prevents changes in ﬁnal seed size. Comparisons of relative temperature effects among experiments may be misleading because of variation in techniques, but comparisons in the same experiment suggest that species differences exist and are important. The response to temperature during seed ﬁlling may not be the same as during other reproductive growth stages. The simulation model CROPGRO uses a ﬂat temperature response between 25 and 358C for all reproductive growth stages in soybean, but the response to temperatures below 258C is less during seed ﬁlling than during early reproductive growth (Boote et al., 1998). The rate of development at 108C, relative to the maximum rate at 308C, was 0.2 during early reproductive development compared with 0.8 during seed ﬁlling. Including this differential response greatly improved the model’s predictive ability in cooler climates.

B. WATER STRESS Water stress during seed development shortens the ﬁlling period and reduces yield of many crop species, including wheat (Gallagher et al., 1976; Brooks et al., 1982; Nicolas et al., 1984; Yang et al., 2000; Ahmadi and Baker, 2001), rice

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(Yang et al., 2002), soybean (Meckel et al., 1984; Dornbos and Mullin, 1991; de Souza et al., 1997), maize (Jurgens et al., 1978; Quatter et al., 1987; Frederick et al., 1989; NeSmith and Ritchie, 1992), barley (Hordeum vulgare L.) (Aspinall, 1965; Brooks et al., 1982), pearl millet (Bieler et al., 1993) and chickpea (Cicer arietinum L.) (Davies et al., 1999). Seed-ﬁll duration was frequently shortened without any effect on individual seed growth rate (Nicolas et al., 1984; Quatter et al., 1987; NeSmith and Ritchie, 1992; Bieler et al., 1993) indicating that individual seed growth rate is less sensitive to water stress than seed-ﬁll duration. The effect of water stress on wheat depended on N availability with the largest response occurring at adequate N levels (Frederick and Camberato, 1995). Water stress accelerates leaf senescence (Asana et al., 1958; Sionit and Kramer, 1977; Aparicio-Tejo and Boyer, 1983; Whitﬁeld et al., 1989; de Souza et al., 1997; Davies et al., 1999) which is probably the primary cause of the shorter seed-ﬁlling period. The accelerated leaf senescence could not be reversed by re-watering soybean plants after 3 – 5 days of stress (Brevedan and Egli, 2003) suggesting that relatively short stress periods may have a disproportionate effect on yield. Water stress before seed ﬁlling seems to have no effect on seed-ﬁll duration (Jordan, 1983; Frederick and Hesketh, 1994).

C. ASSIMILATE AND NUTRIENT SUPPLIES Seeds depend on the mother plant for assimilate, and any alteration of the assimilate supply could affect seed-ﬁll duration. It is not surprising that reducing the assimilate supply to near zero by complete defoliation shortened the seedﬁlling period in maize (Jones and Simmons, 1983; Hunter et al., 1991), sorghum (Rajewski and Francis, 1991) and soybean (Vieira et al., 1992). Partial defoliation or shade treatments designed to produce more modest reductions in assimilate did not consistently affect seed-ﬁll duration. Shade treatments that reduced irradiance by 45 – 63% had no effect or lengthened seedﬁll duration in soybean (Egli et al., 1985; Andrade and Ferreiro, 1996; Egli, 1999). The seed-ﬁlling period of sunﬂower was shortened by 45% shade in 1 of 2 years, but this treatment had no effect on maize (Andrade and Ferreiro, 1996). Partial defoliation shortened seed-ﬁll duration in grain sorghum (Rajewski and Francis, 1991) and in one experiment with soybean (Munier-Jolain et al., 1998) but not in others (Egli and Leggett, 1976) or with maize (Frey, 1981). Pod or seed removal to increase the supply of assimilate to the remaining seed lengthened the seed-ﬁlling period of soybean (Konno, 1979; Egli et al., 1985; Munier-Jolain et al., 1996, 1998; Egli and Bruening, 2001), but not maize (Jones and Simmons, 1983; Kiniry, 1988) or wheat (Slafer and Savin, 1994). Reducing seed number slowed leaf senescence of maize in three of four comparisons (Borras et al., 2003). Reducing plant density at the beginning of seed ﬁlling to increase photosynthesis per plant had no effect on seed-ﬁll duration of maize or

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soybean, but increased it in sunﬂower (Frey, 1981; Andrade and Ferreiro, 1996). Increasing plant density accelerated leaf senescence in maize, presumably shortening the seed-ﬁlling period (Borras et al., 2003). Exposing plants to atmospheres enriched with CO2 did not affect seed-ﬁll duration in wheat (Wheeler et al., 1996) or lupin (Lupinus albus L.) (Munier-Jolain et al., 1998). Seed-ﬁll duration did not respond to increased irradiance in wheat (Soﬁeld et al., 1977) or maize (Schoper et al., 1982), but seed size increased. Higher individual seed growth rates in rice shortened the seed-ﬁlling period when there was no change in seed size (Kato, 1999). The variable response of seed-ﬁll duration to treatments designed to alter source –sink ratios is probably due to (1) failure of the treatment to alter the ratio, (2) failure to extend photosynthesis to support continued seed growth or (3) the inability of the seed to respond to the alteration. Treatments applied before seed ﬁlling or in the early stages of seed ﬁlling may affect seed number so that the supply of assimilate per seed does not change, making it unlikely that there will be a treatment effect (Andrade and Ferreiro, 1996). Altering source– sink ratios does not always affect leaf senescence, so the period when assimilate is available to the seed may not be changed (Crafts-Brandner and Poneleit, 1987; Egli, 1997). The ability of the seed to respond will control the response when the treatment changes the supply of assimilate. Soybean seed-ﬁll duration increased when seed sucrose concentrations increased (Egli and Bruening, 2001) but, in rice, an increase in seed growth rate reduced seed-ﬁll duration because seed size could not change (Kato, 1999). Predicting the effect of source– sink alterations depends, in part, on knowing ﬁrst, whether the treatment affected seed number and the supply of assimilate to individual seeds and, secondly, whether the seed can respond to a change in assimilate supply. The N supply to the plant during seed ﬁlling plays an important role in maintaining green leaf area during seed ﬁlling (Wolf et al., 1988; Banziger et al., 1994). Nitrogen stress accelerated leaf senescence (Boon-Long et al., 1983a; Hayati et al., 1995) and shortened seed-ﬁll duration in soybean without affecting seed growth rate (Egli et al., 1985). However, high levels of N in nutrient culture did not lengthen the seed-ﬁlling period (Egli et al., 1978). Increasing N fertilizer rates in the ﬁeld lengthened the seed-ﬁlling period of soybean and bush bean (Phaseolus vulgaris L.) (Thies et al., 1995), and sorghum (Kamoshita et al., 1998), but wheat responded only when water was not limiting (Frederick and Camberato, 1995; Yang et al., 2000). The yield response of maize to P and K fertilizer was related to an increase in seed-ﬁll duration (Peaslee et al., 1971).

D. FLOWER AND FRUIT DEVELOPMENT Flower morphogenesis occurs sequentially in reproductive structures (e.g., racemes, ears, panicles, capitulums) of most species, which results in sequential

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variation in the beginning of seed ﬁlling of individual seeds. Maturation of seeds on a plant is usually more uniform (Spaeth and Sinclair, 1984) leading to variation in seed-ﬁll duration that is related to the position of the developing seed in the reproductive structure. Seeds at the upper nodes of soybean plants had shorter seed-ﬁll durations than those at lower nodes (Spaeth and Sinclair, 1984) as did seeds developing from ﬂowers opening at growth stage R4.5 (27 days) compared with those opening at growth stage R1 (36 days) (Egli et al., 1987c). These ﬁndings were conﬁrmed by Gbikpi and Crookston (1981). Seeds from late-developing ﬂowers at the tip of the ear in maize had shorter seed-ﬁlling periods in some hybrids (Tollenaar and Daynard, 1978), but not in others (Frey, 1981). Seeds in tip positions in wheat spikelets also had shorter durations (Simmons et al., 1982; Hanft et al., 1986). However, late developing seeds at the base of rice panicles had longer seed-ﬁlling periods (Jongkaeuwattana et al., 1993). The timing of ﬂower development and fertilization probably causes more differences in seed-ﬁll duration than morphological position (Munier-Jolain et al., 1994), although the two are usually completely confounded.

E. PHOTOPERIOD Morandi et al. (1988) suggested that seed-ﬁll duration in soybean was sensitive to photoperiod. Kantolic and Slafer (2001) found that lengthening the photoperiod by 2 h signiﬁcantly increased the time from growth stage R6 to R7 in two of eight comparisons in the ﬁeld with soybean, but long days shortened seed ﬁlling in three of six groundnut cultivars (Witzenberger et al., 1988). No mechanism was proposed to account for these relationships. Photoperiod did not affect seed-ﬁll duration of maize (Tollenaar, 1999), and the seed-ﬁll duration of soybean was not sensitive to planting date (Egli et al., 1987a), suggesting that photoperiod may not be important in production ﬁelds.

F. PLANT GROWTH SUBSTANCES Plant growth substances may play a regulatory role in seed growth (Rock and Quatrano, 1995) and they are involved in regulating senescence (Gan and Amasino, 1997), but there is no direct evidence that they play a role in regulating the ability of seeds to continue growth. Plant growth substances could, however, play a role in determining seed-ﬁll duration through their effect on leaf senescence.

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G. GENETIC Signiﬁcant genotypic variation in seed-ﬁll duration has been documented for most grain crops including wheat (Chowdhury and Wardlaw, 1978; Gebeyehou et al., 1982; Van Sanford, 1985), barley (Garcia del Moral et al., 1991; Leon and Geister, 1994; Doﬁng, 1997), oat (Wych et al., 1982; Peltonen-Sainio, 1993), rice (Kato, 1999), maize (Daynard et al., 1971; Poneleit and Egli, 1979), sorghum (Sorrells and Meyers, 1982), common bean (P. vulgaris L.) (Sexton et al., 1994), sunﬂower (Villalobos et al., 1994) and soybean (Hanway and Weber, 1971; Gay et al., 1980). Maize hybrids had longer seed-ﬁll durations than inbreds (Johnson and Tanner, 1972; Poneleit and Egli, 1979). Plant breeders modiﬁed seed-ﬁll duration by direct-selection in winter (Mou et al., 1994) and spring wheat (Talbert et al., 2001), barley (Rasmusson et al., 1979; Metzger et al., 1984), soybean (Metz et al., 1985; Salado-Navarro et al., 1985; Smith and Nelson, 1987; Pfeiffer and Egli, 1988) and maize (Cross, 1975; Fakorede and Mock, 1978; Hartung et al., 1989). Estimates of heritabilities were highly variable in barley (near zero to 0.94; Rasmusson et al., 1979; Talbert et al., 2001) and soybean (2 0.20 to 1.02; Metz et al., 1984, 1985; Salado-Navarro et al., 1985; Smith and Nelson, 1987; Pfeiffer and Egli, 1988). Mou et al. (1994) reported heritabilities of 0.84 in wheat, but the hertibilities realized by Hartung et al. (1989) when selecting for long and short seed-ﬁll duration in maize were much lower (0.19 and 0.14, respectively). Plant breeders also inadvertently lengthened the seed-ﬁlling period when selecting for higher yield. Modern maize hybrids had longer seed-ﬁll durations than older hybrids (McGarrahan and Dale, 1984; Frederick et al., 1989; Bolanos, 1995). This advantage for modern hybrids was apparent in a wet year with high yield and a dry year with lower yields (Fig. 2), so it seemed to be independent of water stress. Breeding for higher yield also lengthened the seed-ﬁlling period in groundnut (Duncan et al., 1978), oat (Peltonen-Sainio, 1993), wheat (Austin et al., 1989; Loss and Siddique, 1994; Penrose et al., 1998) and soybean (McBlain and Hume, 1981; Boerma and Ashley, 1988; Shiraiwa and Hashikawa, 1995; Kumudini et al., 2001). Domestication increased the seed-ﬁlling period in wheat (Evans and Dunstone, 1970) and maize (Gardner et al., 1990). The EFP of Glycine soja, a wild relative of cultivated soybean, was 22 days compared with 30 days for adapted G. max L. Merrill genotypes (average of 18 genotypes with a range of 24 – 34 days, Egli, 1998, unpublished data). The potential seed-ﬁll duration is determined by the genetic composition of the cultivar and species, but the length expressed is determined by the environment. Environmental and genetic effects on seed-ﬁll duration have been documented in many grain crop species and probably occur in all plant species. The environment can affect seed-ﬁll duration by acting on the seed (e.g., temperature) or on the ability of the plant to supply assimilate to the seed (e.g., water stress), so control resides at the plant and seed level. Variation in seed-ﬁll

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Figure 2 Seed-ﬁlling duration of maize hybrids released between 1936 and 1982 in the USA. Includes one open pollinated genotype from 1930. Slopes of the linear regression equations were not signiﬁcantly different. From Cavalieri and Smith (1985).

duration is one way that genetic differences and environmental effects can inﬂuence yield.

IV. REGULATION OF SEED-FILL DURATION The mother plant supplies the raw materials (primarily sucrose, amino acids and mineral nutrients) that are converted into starch, protein and oil by the metabolic activity of the developing seed (Egli, 1998). Thus, the termination of seed growth and seed-ﬁll duration may be controlled by either the vegetative plant or the seed. This dual approach was useful in understanding the control of seed growth rate, where both the supply of assimilate and the characteristics of the seed seemed to play a role (Egli, 1998). There is ample evidence that the answer

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to the question—why does the seed stop growing?—can also be found at both levels. Many of the characteristics of seed growth, including regulation of seed growth rate are consistent across crop species (Egli, 1998), in spite of variation in seed morphology, size, shape, color and composition. Consequently, a single set of mechanisms may also regulate the termination of seed growth of all grain crop species.

A. REGULATION BY THE SEED Seed ﬁlling continues only as long as the mother plant supplies raw materials to the seed and the seed has the ability to convert the raw materials into storage compounds. If the regulatory mechanism that stops seed growth resides in the seed, the termination of seed ﬁlling may not depend on the absence of raw materials. Temporal measurements of canopy photosynthesis in soybean (Christy and Porter, 1982) and maize (Pearson et al., 1984) suggest that seeds matured when photosynthesis was still 10 – 20% of its maximum rate. Flag leaf photosynthesis was 80% of its highest level when wheat kernels reached their maximum dry weight (Soﬁeld et al., 1977) and seeds on partially depodded soybean plants matured when the plants were still green and photosynthetically active (Munier-Jolain et al., 1996). Soybean seeds that had their growth limited by physical restriction matured when leaves were still photosynthetically active with high levels of Rubisco and chlorophyll (Crafts-Brandner, 1995, personal communication) and mature pods can be found on soybean plants with green leaves (Egli, 1998). Banziger et al. (1994) reviewed several reports of wheat seeds maturing when plants still had green parts and the stem reserves were not exhausted. Stay-green maize genotypes had high carbohydrate levels in the stem when the seeds matured (Gentinetta et al., 1986). Death of grain sorghum plants occurred after the seed reached PM (Rajewski and Francis, 1991). Pods and seeds matured on pigeon pea [Cajanus cajan (L.) Millsp.], cowpea [Vigna unguiculata (L.) Walp.] and rice plants that still had the capacity to produce a ratoon crop or a second crop of pods (Bahar and De Datta, 1977; Sharma et al., 1978; Gwathmey et al., 1992; Turner and Jund, 1993). Sucrose concentrations in wheat and soybean seeds were near maximum levels at PM suggesting that assimilate availability was not limiting when seed growth stopped (Jenner and Rathjen, 1975; Egli and Bruening, 2001). These results from many crop species suggest that completion of leaf senescence and death of the vegetative plant are not an absolute prerequisite for termination of seed growth. Seeds cannot continue to accumulate dry matter without a source of raw materials, but having a source does not guarantee that growth will continue.

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The termination of seed growth when assimilate is still available requires a regulatory mechanism in the seed. Changes in tissue water status may play a regulatory role in plant development (Walbot, 1978; Adams and Rinne, 1980; McIntyre, 1987), so the widely observed desiccation of seed tissues that occurs as the seed approaches PM (Westgate, 1994; Egli and TeKrony, 1997; Saini and Westgate, 2000) may provide a mechanism. Desiccation occurs when cell expansion and water movement into the seed stops but dry matter accumulation continues (Ray, 1987; Egli, 1990, 1998). Cell expansion could be limited by physical restriction by seed or fruit structures (Boshankian, 1918; Murata and Matsushima, 1975; Scott et al., 1983), thus, the characteristics of the fruit or seed (i.e., its maximum potential volume) would play a role in triggering the termination of seed growth. The assimilate supply must also regulate cell expansion and seed desiccation since it can affect seed-ﬁll duration (Rajewski and Francis, 1991; Andrade and Ferreiro, 1996; Munier-Jolain et al., 1996, 1998; Egli and Bruening, 2001).

B. REGULATION BY THE PLANT Since a seed cannot grow for long without a photosynthetically active plant canopy, the progression of leaf senescence and seed-ﬁll duration tends to change in concert. Canopy photosynthesis begins to decline relatively early in the seedﬁlling period in most crops (Larson et al., 1981; Wells et al., 1982; Pearson et al., 1984; Hall et al., 1990; Gent, 1995) as leaf senescence reduces the photosynthetic capacity of the plant canopy. It seems ironic that soon after the preliminarily events of the yield production process (development of the canopy, ﬂowering and seed set) are completed and the plant ﬁnally begins to accumulate dry weight in the seeds—to actually produce yield—the productive capacity of the plant begins to decline as the photosynthetic machinery is destroyed and nitrogen is exported from the leaf. Manipulation of leaf senescence frequently results in corresponding changes in seed-ﬁll duration. Complete defoliation to artiﬁcially reduce photosynthesis to near zero results in a rapid cessation of seed growth (Jones and Simmons, 1983; Hunter et al., 1991; Rajewski and Francis, 1991; Vieira et al., 1992). Inadequate N supplies (Boon-Long et al., 1983a; Egli et al., 1985; Hayati et al., 1995) and water stress (Gallagher et al., 1976; NeSmith and Ritchie, 1992; de Souza et al., 1997; Davies et al., 1999) accelerated leaf senescence and shortened the seedﬁlling period. Variation in leaf senescence patterns is often associated with genetic differences in seed-ﬁll duration. Senescence progressed faster in a maize hybrid with a short EFP than in one with a long EFP (Crafts-Brandner and Poneleit, 1987). Hartung et al. (1989) selected for long and short seed-ﬁlling periods in a recurrent selection program with maize and changed leaf senescence, as indicated

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by photosynthesis and leaf N levels during seed ﬁlling, in concert with changes in seed-ﬁll duration (Crafts-Brandner and Poneleit, 1992). Longer seed-ﬁlling periods in modern soybean cultivars were associated with a slower decline in canopy photosynthesis, a good indicator of senescence (Wells et al., 1982). Leaf N concentration declined faster in a soybean genotype with an exceptionally short seed-ﬁll duration (Egli et al., 1987b), but differences among genotypes disappeared when leaf N concentration was related to the stage of seed development. Cultivars with delayed leaf senescence (stay-green types) frequently have higher yields (Tollenaar, 1991; Ma and Dwyer, 1998; Duvick and Cassman, 1999) which are probably a result of longer seed-ﬁlling periods (Frederick et al., 1989). Delayed senescence will increase seed-ﬁll duration only when the seed has the ability to continue growth and increase in size. The seed, in some situations, responds to treatments that simulated delayed senescence by increasing assimilate supply per seed (depodding, degraining, increasing photosynthesis after seed number is ﬁxed) by increasing seed size in soybean (Egli et al., 1985; Egli and Bruening, 2001), wheat (Fischer and Hille Ris Lambers, 1978; Winzeler et al., 1989), sorghum (Kiniry, 1988), sunﬂower (Steer et al., 1988; Charlet and Miller, 1993) and maize (Kiniry et al., 1990). Seed and fruit characteristics apparently limited seed size increases in some instances in maize (Jones and Simmons, 1983; Kiniry et al., 1990), barley (Scott et al., 1983; Dreccer et al., 1997), rice (Murata and Matsushima, 1975; Kato, 1999), wheat (Millet, 1986; Ma et al., 1995) and sunﬂower (Charlet and Miller, 1993). Developing soybean seeds spilt pods in environments that resulted in abnormally small pods (Egli, 1998), an example of potential physical restriction of seed growth when assimilate was available. Simply delaying senescence, therefore, may lead to a longer seed-ﬁll duration and higher yield in some crops or some environments, but in other situations the characteristics of the seed may also have to be changed. The characteristics of the seed and leaf senescence are clearly intertwined as seed ﬁlling must stop when the plant can no longer supply assimilate or when the seed can no longer accumulate dry matter. Leaf senescence, the process that ends the life of all annual grain crops, sometimes in spectacular fashion, has probably received more attention than the characteristics of the seed, but either can limit seed-ﬁll duration and yield.

C. SENESCENCE Leaf senescence comprises a series of events that result in cellular disassembly in the leaf and mobilization of the materials released (Thomas and Stoddart, 1980). Organelles and membranes maintain their integrity such that cellular compartmentation is maintained throughout the senescence process (Thomson and Platt-Aloia, 1987).

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Leaf senescence normally occurs during seed ﬁlling in all grain crops and the nitrogen released is exported to the developing seed (Wittenbach, 1979; Crafts-Brandner and Egli, 1987; Crafts-Brandner and Poneleit, 1987). Remobilized N can be a signiﬁcant source of N for the seed, with estimates of its contribution to the total seed N at maturity ranging from 11 to 100% in soybean (Egli et al., 1978, 1983; Israel, 1981; McBlain and Hume, 1981; Zeiher et al., 1982), 63 to 100% in wheat (Heitholt et al., 1990; Mi et al., 2000), 49 to 64% in sorghum (Borell and Hammer, 2000) and 41 to 69% in maize (Below et al., 1981; Rajcan and Tollenaar, 1999), while one cowpea cultivar averaged 60% (Peoples et al., 1983). Stay-green maize genotypes redistributed less N and, therefore, took up more N from the soil during seed ﬁlling than normal genotypes (Rajcan and Tollenaar, 1999). Soil N levels also inﬂuenced the contribution of remobilized N to total seed N (Pan et al., 1984). The importance of redistributed N to the N budget of developing seeds may have distorted our understanding of cause and effect relationships between leaf senescence and seed-ﬁll duration. The hypothesized need to remobilize N from the leaf to sustain seed growth was thought to accelerate senescence and limit seed-ﬁll duration (Sinclair and de Wit, 1975, 1976; Frederick and Hesketh, 1994). A presumed seed N “demand”—seeds cannot grow unless adequate N is available—was the driving force behind the remobilization of leaf N. Models based on this premise suggest that seed-ﬁll duration could be sustained only by (1) decreasing the total N requirement [total rate of N accumulation (g m22 day21) of the seeds] (2) by a larger pool of remobilizable N at the beginning of seed ﬁlling or (3) by taking more N from the soil during seed ﬁlling (Frederick and Hesketh, 1994; Saini and Westgate, 2000). The rate of N accumulation by the seeds, a function of total seed growth rate and seed N concentration, is important only if seeds demand N from the vegetative plant as a condition for growth. If seeds simply utilize the N made available by the mother plant, seed growth would not necessarily affect the rate of senescence, and variation in the supply of N may cause changes in seed N concentration. The in vitro seed growth rate was relatively insensitive to N availability in wheat (Barlow et al., 1983), maize (Singletary and Below, 1989) and soybean (Saravitz and Raper, 1995; Hayati et al., 1996). In planta seedgrowth rate is also insensitive to N stress during seed ﬁlling (Egli et al., 1985; Hayati et al., 1995). Seed N concentration is, however, sensitive to changes in N supply (Hayati et al., 1995, 1996). Hayati et al. (1996) found that 17 mM N in the media sustained maximum seed-growth rates, but 270 mM N produced the highest seed N concentration. The preponderance of evidence suggests that there is no active seed N demand; instead seeds seem to subsist on the N supplied by the vegetative plant. Abandoning the concept of seed N demand eliminates a primary mechanism by which seed growth could regulate senescence. Models that assumed an active seed N demand (Sinclair and de Wit, 1975; Frederick and Hesketh, 1994) predicted that changes in total seed growth rate

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and, consequently, the total seed N requirement would affect the rate of senescence. However, reducing seed number and total seed growth rate did not lengthen the seed-ﬁlling period in some experiments (Frey, 1981; Jones and Simmons, 1983; Kiniry, 1988) but it did in others (Konno, 1979; Egli et al., 1985; Munier-Jolain et al., 1996, 1998; Egli and Bruening, 2001). Reducing photosynthesis and total seed growth rate with shade also resulted in conﬂicting responses, sometimes seed-ﬁll duration increased and sometimes there was no change (Frey, 1981; Simmons et al., 1982; Egli, 1999). Increasing photosynthesis and total seed growth rate with elevated CO2 levels accelerated leaf senescence in barley and wheat (Fangmeier et al., 2000), but there was no effect when high radiation levels were used with soybean (Hayati et al., 1995). The high radiation levels increased total seed growth rate, but seed N concentration was reduced and the rate of senescence was not changed in non-nodulated soybean plants with and without N available to the roots (Hayati et al., 1995). There was also no obvious relationship between seed protein concentration, the concentration aspect of the total seed N accumulation rate and seed-ﬁll duration among species (Table I) (Egli, 1981) or among cultivars within a species (Fig. 3). Salado-Navarro et al. (1985), however, found that soybean genotypes with high seed protein concentrations had shorter seed-ﬁll durations and lower yields than genotypes with normal seed protein levels. Experiments with several species following a variety of treatment protocols did not establish a consistent relationship between total seed growth rate and seed-ﬁll duration, suggesting again that there may be no active seed N demand driving senescence.

Figure 3 The relationship between effective-ﬁlling period and seed protein concentration for 11 soybean genotypes grown in the ﬁeld at Lexington, Kentucky in 1998. Average size of seed from small-seeded genotypes was 96 mg seed21 compared with 251 mg seed21 for the large seeded genotypes. Egli and Bruening (1998), unpublished data.

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A large supply of N in the plant at the beginning of seed ﬁll, relative to that in the seed at maturity, may slow senescence and extend seed ﬁlling (Sinclair and de Wit, 1975; Triboi and Triboi-Blondel, 2002). Sinclair and Sheehy (1999) and Sheehy (2001) argued that a larger pool of remobilizable N was needed to increase yield of modern cultivars. There is no consistent relationship, however, between the size of the N pool and seed-ﬁll duration. The variation in seed-ﬁll duration among soybean cultivars was not related to the size of the potentially remobilizable N pool at the beginning of seed ﬁlling (Zeiher, 1980; Zeiher et al., 1982) (Fig. 4). Larger maximum vegetative mass and a larger N pool are usually associated with long total growth duration, but seedﬁll duration does not necessarily increase as total growth duration increases (Fig. 5) (Duncan, 1969; Egli, 1994, 1998). Stress during vegetative growth frequently reduces plant size without reducing yield (Frederick and Hesketh, 1994; Jiang and Egli, 1995). There are reports of a signiﬁcant association between total leaf N at the beginning of seed ﬁll and seed-ﬁll duration (Shibles and Sundberg, 1998) or yield (Loberg et al., 1984), but, in aggregate, the results do not consistently support a model where seed-ﬁll duration is determined by the size of the remobilizable N pool. Variation in pool size and the contribution of remobilized N must cause offsetting changes in the supply of N from the nodules or from the soil solution. This uptake during seed ﬁlling must be essentially zero when the contribution from remobilizable N approaches 100%, but it must remain high when remobilizable N makes only a small contribution. Variation in N ﬁxation

Figure 4 The relationship between potentially remobilizable N and seed-ﬁll duration (growth stage R5 –R7) for eight soybean cultivars from maturity groups III to V grown in the ﬁeld in 1978 (Zeiher, 1980). Potentially remobilizable N is the total N in the leaves, petioles, stems and pod walls at beginning of seed ﬁll (growth stage R5). Potentially remobilizable N was correlated ðr ¼ 0:99; n ¼ 8; signiﬁcant at P ¼ 0:001Þ with the mass of the vegetative plant at beginning of seed-ﬁll.

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Figure 5 The relationship between the length of the total growth cycle (planting to PM) and the duration of seed ﬁlling. (A) The effective ﬁlling period of four soybean cultivars grown in the ﬁeld in 1989 (X) and 1990 (W) (Egli, 1993). (B) The days between growth stage R5 and R7 of eight soybean cultivars grown in the ﬁeld, 1978 (Zeiher et al., 1982). (C) Days from ﬂowering to maturity for 38 diverse cowpea genotypes grown in the ﬁeld at Riverside, California in 1983 (Dow el-madina and Hall, 1986). Two genotypes with seed-ﬁll durations near 30 day and one with a 15 day seed-ﬁll duration were not included in the regression analysis. (D) Days from silking to maximum seed mass averaged across 1 or 2 years and several plant densities for 11 maize hybrids grown in the ﬁeld (Hanway and Russell, 1969).

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patterns during seed ﬁlling in soybean that match these extremes has been documented (Harper, 1974; Lawn and Brun, 1974; Denison and Sinclair, 1985). If crops can obtain most of their seed N from uptake or ﬁxation during seed ﬁlling, the maximum rate of uptake/ﬁxation may provide no meaningful limitation to seed growth. Sheehy’s (1983) calculations supported this position by suggesting that potential uptake rates were much greater than the 0.5 g m22 day21 assumed by Sinclair and de Wit (1975) and provided no limitations to seed growth. Perhaps it is more reasonable to assume that the N supply would be in balance with the productive capacity of the plant canopy, i.e., crop growth rate. If this is true, the N uptake rate may not be limiting, but N availability in the soil or an unfavorable soil environment that inhibited nodule function could limit N supply and affect seed-ﬁll duration. Seed growth requires a continuous supply of assimilate from the mother plant, paradoxically, canopy photosynthesis starts declining early in seed-ﬁlling as the leaves senesce and usually reaches zero when the seeds stop growing. Thus, the seed-ﬁlling period is characterized by a steady decline in the supply of assimilate, while the products of senescence are frequently a major source of N for the seed. Senescence must be involved in the regulation of seed-ﬁll duration and much remains to be learned about this complex relationship, but describing leaf senescence as a straight-forward interaction of N supply and seed N need is probably a serious oversimpliﬁcation. Although debate continues on the exact metabolic mechanisms regulating the cessation of seed growth (Kermode, 1995; Saini and Westgate, 2000) and leaf senescence (Buchanan-Wollaston, 1997), it is clear that the characteristics of the seed and of senescence regulate seed-ﬁll duration. The duration of growth of a seed that normally reaches its maximum potential size will not increase if leaf senescence is slowed without changes in the characteristics of the seed and, conversely, there will be no beneﬁt derived from increasing the potential size of the seed if there is no delay in leaf senescence to provide assimilate for continued seed growth. Slowing senescence may, however, increase seed-ﬁll duration in those species whose seeds do not normally reach their potential size.

V. SEED-FILL DURATION AND CROP PRODUCTIVITY A. YIELD Yield of a grain crop is determined by the accumulation of dry matter by the 21 seeds. This accumulation has two components, a rate (g m22 ) and a land area day duration, and either or both can contribute to variation in yield. There is ample evidence from many crops that seed-ﬁll duration can be associated with yield.

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However, variation in total seed growth rate could also affect yield, or variation in seed-ﬁll duration could be compensated for by changes in total seed growth rate to maintain a constant yield. Since yield is a function of a rate and a duration it is unrealistic to expect that seed-ﬁll duration will account for all variation in yield. The plant’s environment affects seed-ﬁll duration, as described previously, and these effects are frequently translated into changes in yield. Water stress shortened seed ﬁlling and reduced yield of chickpea (Davies et al., 1999), soybean (Meckel et al., 1984; de Souza et al., 1997), maize (NeSmith and Ritchie, 1992) and wheat (Frederick and Camberato, 1995). A shorter seed-ﬁlling period played a role in yield reductions from N stress with wheat (Frederick and Camberato, 1995) and soybean (Egli et al., 1978), and P and K stress with maize (Peaslee et al., 1971). Nitrogen stress reduced leaf area duration and yield of maize (Wolf et al., 1988), probably as a result of shortened seed-ﬁll duration. The effects of sowing date and irrigation on yield of pinto beans (Phaseolus vulgarius L.) and ﬁeld beans (Vicia faba L.) were expressed through changes in leaf area duration which probably represented differences in seed-ﬁll duration (Husain et al., 1988; Dapaah et al., 2000). Wheat yield was closely associated with leaf area duration across trials involving planting date, seeding rates and N fertilizer rates (Fischer and Kohn, 1966). Seed-ﬁll duration is sensitive to temperature, and this variation frequently translates into changes in yield. Artiﬁcially lowering night temperature increased yield of wheat maize, and soybean, apparently as a result of a longer seed-ﬁlling period (Peters et al., 1971). Lower temperatures and longer seed-ﬁlling periods increased yield of oat (Hellewell et al., 1996) and wheat (Wardlaw et al., 1980). Lower temperatures and longer seed-ﬁll durations contributed to larger yields at higher elevations (Cooper, 1979). However, compensatory effects of solar radiation (Muchow, 1990) or seed-growth rate (Chowdhury and Wardlaw, 1978) minimized changes in seed size and yield in other situations. Long seed-ﬁlling periods may be partially responsible for exceptionally high yields in some environments with moderate temperatures (Duncan et al., 1973; Muchow et al., 1990; Sinclair and Bai, 1997; Hall, 2001). Yield is frequently associated with genetic differences in seed-ﬁll duration. Positive associations between seed-ﬁll duration and hybrid or cultivar yields were found in maize (Daynard and Kannenberg, 1976; Bolanos, 1995), wheat (Gebeyehou et al., 1982; Penrose et al., 1998), barley (Leon and Geister, 1994; Doﬁng, 1997) and soybean (Hanway and Weber, 1971; Dunphy et al., 1979). The higher yields of maize hybrids compared with inbreds were associated with a longer seed-ﬁll duration (Johnson and Tanner, 1972; Poneleit and Egli, 1979). Dwyer et al. (1994), however, found a positive correlation between yield and seed-ﬁll duration in only two of nine combinations with early maturing maize hybrids while a positive association occurred in only 2 of 4 years with oat (Peltonen-Sainio, 1991). The yield advantage for tropical maize hybrids over open-pollinated cultivars was not associated with seed-ﬁll duration

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(Bolanos, 1995) and sorghum inbreds and hybrids had the same seed-ﬁll duration (Quinby, 1972). Yield variation among common bean cultivars was not associated with seed-ﬁll duration (Sexton et al., 1994). Egli et al. (1984) found a signiﬁcant correlation between soybean yield and seed-ﬁll duration among genotypes in only 1 of 3 years. Selection for long seed-ﬁll duration resulted in higher yields in soybean (Smith and Nelson, 1986) and maize (Cross, 1975; Crosbie and Mock, 1981). Metz et al. (1984) increased seed-ﬁll duration of soybean by selection but did not change yield in 10 of 12 comparisons and Hartung et al. (1989) successfully manipulated EFP in maize but seed growth rate compensated and there was no change in yield. New cultivars with higher yield had longer seed-ﬁlling periods than old cultivars in oat (Helsel and Frey, 1978), peanut (Duncan et al., 1978), soybean (Gay et al., 1980; McBlain and Hume, 1980; Boerma and Ashley, 1988; Kumudini et al., 2001) and maize (Russell, 1991). The increase in average maize yields in Indiana from 1950 to 1980 was associated with an increase in seed-ﬁll duration (McGarrahan and Dale, 1984). Gardner et al. (1990) reported that the low yield of one of two ancient maize cultivars was associated with a seed-ﬁlling period that was 15% shorter than a modern hybrid. Wheat yields increased substantially in the 19th and 20th centuries in England, but there was no change in seed-ﬁll duration (Austin et al., 1989). Lopez Pereira et al. (1999) also found no change in seed-ﬁll duration of sunﬂower during the 20th century in Argentina. Longer seed-ﬁll durations in crop simulation models were associated with higher soybean (Boote et al., 2001), wheat (Asseng et al., 2002) and rice (Kropft et al., 1995) yields. If the total growth duration was held constant, increasing the seed-ﬁlling period shortened vegetative growth and reduced plant size, and, eventually, light interception and yield (Boote and Tollenaar, 1994; Boote et al., 2001), illustrating the potential for complex interactions among plant growth characteristics. Rasmusson (1987) included a longer seed-ﬁll duration in his ideotype of a high-yielding barley plant. Historical yield improvement in some crops was associated with an increase in harvest index [yield/(vegetative biomass þ yield)] (see Evans, 1993 for an extensive review) which is usually attributed to an increase in partitioning of assimilate to seeds at the expense of vegetative plant parts. A higher harvest index could also result from a longer seed-ﬁlling period and higher yields assuming that vegetative biomass did not decrease (Egli, 1998). Measurements of harvest index and seed-ﬁll duration are rarely made in the same experiment, but there are reports of positive associations between the two variables in maize (Fakorede and Mock, 1978) and wheat (Sharma, 1994). Increasing seed-ﬁll duration in a soybean simulation model also increased harvest index (Boote et al., 2001). The contribution of changes in seed-ﬁll duration to the historical increase in harvest index remains to be determined, but all of the increase may not be due to changes in partitioning. Increasing the harvest index by extending the seed-ﬁlling period

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without decreasing vegetative mass, i.e., longer ﬁlling period results in later maturity, eliminates concerns that there is a minimum plant size that will stymie further increases in harvest index (Austin et al., 1980). The time that dry matter accumulates in the seed is only one aspect of yield, the rate of seed dry matter production per unit ground area, a function of assimilate production, partitioning, and sink size, i.e., the number of seed, is also important. It is not surprising, therefore, that yield is not always a function of seed-ﬁll duration. The frequency with which yield is associated with seed-ﬁll duration in many crops suggests, however, that it is an important component of the yield production process.

B. FUTURE YIELD IMPROVEMENT Food production may have to double in the next 20 years to meet the demands of an increasing world population (Penning de Vries, 2001). Higher yields will have to play an important role in this increase since most of the land suitable for crop production is already in use. Lengthening the seed-ﬁll duration contributed to past increases in crop yield and agricultural productivity—is there a role for it in the future? The potential productivity of any location is set by the solar radiation available when temperatures are suitable for plant growth (de Wit, 1967). The time available for crop growth accounts for much of the worldwide variation in potential productivity, much more than variation in daily solar radiation levels. In some tropical environments there are no constraints and crops can grow for 365 days, but, at the other extreme, the period when temperatures are suitable for plant growth is too short for grain crop production. Grain crops produce their yield in a relatively short time, seed-ﬁlling periods for most crops usually vary between 30 and 40 days (Table I), compared with ﬁlling periods of some root and tuber crops which can exceed 100 days (Milford and Watson, 1971; Moorby and Milthorpe, 1975; Howeler and Cadavid, 1983). The record yields compiled by Evans (1993) clearly illustrate this difference in potential—record yields of potato and cassava are 20 – 30 t ha21 compared with 10– 15 t ha21 for wheat and rice. Unrealistically high crop growth rates would be required to produce 20– 30 t ha21 yields with the short ﬁlling periods in grain crops. The limits of a short seed-ﬁlling period cannot be overcome by increasing the total growth duration of the crop. Yield of a single grain crop does not increase indeﬁnitely as the time used for crop growth increases (Egli, 1998). Cultivars with long growth cycles may produce more total biomass (Murata, 1981), but this does not always translate into higher yields because seed-ﬁll duration does not always increase in concert with the total growth cycle. Seed-ﬁll duration of cowpea and one set of soybean cultivars (Fig. 5A,C) increased signiﬁcantly in

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conjunction with the total growth cycle, but the increase (0.2 d per d) was much less than the increase in total duration. There was no association in maize or another set of soybean cultivars (Fig. 5B,D). Thus, grain crops are not very efﬁcient in translating time into yield as time increases beyond some minimum level because yield is limited by seed-ﬁll duration. Multiple cropping, which effectively stacks several seed-ﬁlling periods in one growing season, is the only alternative in climates with long growing seasons. Increasing global temperatures may lengthen the growing season, increasing the time available for plant growth (Penuelas and Filella, 2001; Olesen and Bindi, 2002), but it may be difﬁcult to translate this extra time into higher yields unless cultivars with longer seed-ﬁlling periods are available. The higher temperatures may also shorten the ﬁlling period (Fig. 1), creating the paradox of having more time for crop growth and less time to produce economic yield. Some researchers have suggested that short seed-ﬁll durations may be advantageous in environments with relatively short growing seasons (Daynard and Kannenberg, 1976; Alexander and Cross, 1983; Peltonen-Sainio, 1990; Whan et al., 1996). Bieler et al. (1993) suggested that a short seed-ﬁlling period may reduce the chances of water stress by limiting total water use. These advantages may be real, but combining short seed-ﬁll duration and high yield will require extremely high crop and total seed growth rates which may be difﬁcult to obtain. Seed-ﬁll duration is under genetic control and it is related to yield, characteristics that theoretically make it useful as a selection criteria in plant breeding programs emphasizing yield improvement. Unfortunately, there is little evidence that yield can be increased faster by selecting for a long seed-ﬁll duration than selecting for yield. Pfeiffer et al. (1991) working with soybean found that selecting parents based on seed-ﬁll duration provided no advantage and Salado-Navarro et al. (1985), also working with soybean, concluded that large genotype £ environment interactions limited its usefulness. These ﬁndings are discouraging given that it is possible to genetically manipulate seed-ﬁll duration (Fakorede and Mock, 1978; Rasmusson et al., 1979; Metzger et al., 1984; Metz et al., 1984, 1985; Salado-Navarro et al., 1985; Smith and Nelson, 1987; Pfeiffer and Egli, 1988; Hartung et al., 1989; Mou et al., 1994; Talbert et al., 2001) and that higher yield of improved cultivars of several crop species was, in some cases, a result of inadvertently lengthening seed-ﬁll duration (Duncan et al., 1978; Helsel and Frey, 1978; Gay et al., 1980; Russell, 1991; Kumudini et al., 2001). Seed-ﬁll duration is not widely used by plant breeders, perhaps it is the complexity of this trait, involving both the characteristics of the seed and the leaf senescence process, that limits its usefulness. In spite of its intractable nature, seed-ﬁll duration remains an attractive trait for future yield improvement. Considering yield as a simple function of a rate of growth expressed over a ﬁnite time leaves only two options in any quest for higher yield, either the total seed growth rate or the seed-ﬁll duration, or some

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combination of the two, must increase. Seed-ﬁll duration or the time aspect is, in many respects, a more attractive target than growth rate. Canopy photosynthesis and crop growth rate have been remarkably resistant to change (Evans, 1993). Substantial yield improvement has occurred in several crops without any change in crop growth rate (Evans et al., 1984; Gifford et al., 1984; Slafer et al., 1990). Some have argued that improvements in radiation use efﬁciency in non-stress environments are unlikely (Sinclair and Horie, 1989). There is, on the other hand, considerable evidence that longer seed-ﬁll durations have contributed to higher yields in many crops. Grain crops use only part of the growing season in many environments where they are grown, so there is substantial unused time available to extend seed growth. Signiﬁcant increases in seed-ﬁll duration would not require much additional time, a 20% increase in seed-ﬁll duration is only 6 days if the original seed-ﬁll duration was 30 days, a period that could be easily accommodated in many environments. There is evidence that a longer seed-ﬁlling period does not have to lengthen the total growth cycle if seed growth starts earlier (Metz et al., 1985). Early ﬂowering would probably shorten the vegetative growth period and reduce the maximum vegetative mass, but this may not affect yield since excess vegetative mass is normally produced by many crops (Egli, 1998). Lengthening the seed-ﬁlling period will require changes in the patterns of leaf senescence. Such changes were associated with genetic manipulation of seed-ﬁll duration in soybean and maize (Wells et al., 1982; Boon-Long et al., 1983b; Crafts-Brandner and Poneleit, 1992), so there is no reason to assume they cannot occur in the future. The concerns raised by the self-destruction concept (Sinclair and de Wit, 1975, 1976) in the 1970s appear to be unfounded although they continue to be expressed (Evans, 1993; Triboi and Triboi-Blondel, 2002). There is little evidence today that senescence provides a fundamental absolute limitation to lengthening the seed-ﬁlling period. There is in fact substantial variation in senescence patterns available in several plant species (Thomas and Howarth, 2000) and some may prove useful. But there is no doubt that the control of seed-ﬁll duration is complex, residing in the leaf and the seed. Manipulating it may be difﬁcult, perhaps requiring small changes in many plant characteristics which will surely complicate attempts to modify it at the molecular level.

VI. CONCLUSIONS Time, as expressed through seed-ﬁll duration, is an important component of crop productivity. Although it is certainly not the only characteristic of the crop that inﬂuences grain yield, it probably deserves more attention than it has received in the past. Seed-ﬁll duration is sensitive to the environment and genetic variation exists in most crops. Manipulation of the processes that regulate

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seed-ﬁll duration may make plants less sensitive to some environmental insults, such as high temperatures or drought stress, that may be more prevalent in the future. Crops with longer seed-ﬁll durations will be more efﬁcient in using long growing seasons—they will overcome the inefﬁciencies created by the failure of seed-ﬁll duration and yield to increase in step with the total growth cycle. The higher potential productivity in long growing seasons can only be used by growing more than one crop in the available time. A longer seed-ﬁlling period, however, would increase yield without the economic liabilities associated with multiple-cropping systems and make it possible to utilize some of this untapped potential in tropical and sub-tropical climates. The historical involvement of seed-ﬁll duration in yield improvement provides some encouragement for a role for this character in future yield increases. There seems to be no fundamental physiological barrier to increasing seed-ﬁll duration, although plant breeders have not found it as useful as expected, given that it is a heritable characteristic that is related to yield. Seed-ﬁll duration is a complex character involving the characteristics of the seed and leaf senescence, but a better understanding of its regulation, coupled with the new techniques available to manipulate plant characters, may make it possible to lengthen the seed-ﬁlling period and increase yield.

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Index biodegradation chloropicrin 27 –8 1,3-dichloropropene 26–7 methyl bromide 22–3 methyl isothiocyanate 25–6 Biolog Gram-negative microtiter plate assay 8– 10, 14 biomarkers 11, 12 –14 biomass 6, 10–12 biosorbents 160 biotechnology 93 –5 biotic stress 63– 75 breeding strategies and programs cadmium in tobacco 153–4 seed-ﬁll duration 253, 264, 266 sorghum improvement 37–96 broomcorn 93 brown midrib sorghums 90, 91– 2 bulk density of soils 188, 189 –92, 198– 9 burial of plant (hoof action) see also treading effects 186 –8, 212

A 16S rRNA 12, 15–20 ABC see ATP binding cassette abiotic stress 75– 86 accumulation cadmium in tobacco 114–27 adaptation, sorghum improvement 60–3 adaptive cellular mechanisms 196 aeration, soil 185, 192–8, 199–200, 212 aerenchyma 193, 196 agricultural practices cadmium in tobacco 130–5 soil fumigants 20– 8 air-ﬁlled porosity 199–200 aluminium-toxicity tolerance 84–6 amended soils cadmium in tobacco 130–1, 157–8 non-biological cadmium stabilization 157– 8 sludges 130 –1 soil fumigants 19– 20, 21 amylopectin 89 animal excretion 183, 184, 205, 207–11 animal grazing see grazing animal stand-off areas 222–4 animal treading see treading effects anther dehiscence control 45–6 anthracnose resistance 59, 64– 6 antiporter proteins 140, 144–5 apple roots 21 arbuscular mycorrhizae 158–60 assimilate 250–1, 256, 260, 262 Atherigona soccata (shoot ﬂy) 72 atmospheric cadmium 113, 136 ATP binding cassette (ABC) transporters 140, 141 –2 autumn pasture management strategies 214–15

C cadmium fertilizers 133 tobacco 111–62 agricultural practices 130 –5 atmospheric 113, 136 breeding strategies 153 –4 climatic conditions 135–6 concentration in ﬁeld-grown leaves 120 –1 crop year variations 136–7 deposition on leaves 136 developmental stages 123 distribution within plants 114 –27 environmental cadmium occurrence 113 –14 reduction options 137 –61 root distribution 119 –20 root-to-shoot transport 117– 19 shoot distribution 119–20 soil cadmium remediation 154 –61 soil characteristics 127–30 stalk position versus accumulation 121 –2

B bacteria communities soil fumigant studies 14 gram-negative 11– 12, 14 gram-positive 11–12, 13, 14 soil cadmium remediation 159, 160–1 bacterial colony forming units (CFUs) 15 beringite 157 bioavailability of cadmium in soil 114 281

282

INDEX

sub-cellular localization 124 tolerance 115– 17, 146–52 variation within leaf 123 –4 variety differences 124 –6 transporters 139 –46 calcium channels 140, 145–6 carbon dioxide enrichment 251 carbon substrate utilization 8–10 cation antiporters 144–5 cation channels 145 –6 cation diffusion facilitator (CDF) family 140, 143–4 cation exchange capacity 128 cattle see also grazing feeding style 201 –2 grazing management strategies 213 soil bulk density 189 CDE see cation diffusion facilitator CFUs see bacterial colony forming units charcoal rot 64 chelate-assisted phytoremediation 155–6 chloroform fumigation 13–14 chlorophyll ﬂuorescence 82 chloropicrin (CP) biological process effects 5–8 degradation 27–8 microbial activities/composition effects 8–20 microbial communities/agricultural practice impacts 27– 8 mode of action 3–4 structure 4 chlorothalonil 13 Claviceps africana 70 climatic conditions, cadmium in tobacco 135–6 cold tolerance 83 collections, sorghum germplasm 57 Colletotrichum graminicola 64, 65 color, sorghum grains and plants 86–8 community-level carbon source utilization 8–10 compaction, soil 188–98, 222 competition 183, 209 continuous grazing management 213 controlled pollination mechanisms 42– 9 conversion programs, sorghum 56–9 CP see chloropicrin CROPPGRO 249 crop productivity, seed-ﬁll duration 262–7 crop rotation 135 crop year variations 136–7 cross-pollination methods 43 –9

cultivars grass species 218–19 seed-ﬁll duration 253, 263 –4 sorghum improvement 40 –2, 52 tobacco cadmium accumulation 124– 6 white clover 216–18 Curvularia lunata 66, 67 cutting management practices 211, 214, 224 cytoplasmic–genetic male sterility 46–9 D 1,3-D see 1,3-dichloropropene decision making, technical based 224–7 deer 201 defoliation nitrogen ﬁxation in legume based pastures 183, 184, 200–5, 206, 216 seed-ﬁll duration 250, 256 degradation chloropicrin 27–8 1,3-dichloropropene 26 –7 methyl bromide 22 –3 methyl isothiocyanate 25–6 dehydrogenase activities 5 denaturing gradient gel electrophoresis (DGGE) 13, 14, 15–20, 23 –4 desiccation 256 detoxiﬁcation, cadmium in tobacco 146–52 development stages cadmium accumulation in tobacco 123 seed-ﬁll duration 246 sorghum drought stress 76–81 sorghum temperature stress 83 –4 DGGE see denaturing gradient gel electrophoresis 1,3-dichloropropene (1,3-D) biological process effects 5–8 degradation 26–7 microbial activities/composition effects 8–20 microbial communities/agricultural practice impacts 26–7 mode of action 3 –4 structure 4 diet selection 201–2 diffusion, oxygen in soil 199–200 dinoterb 15 disease resistance 63– 72 distribution, cadmium within tobacco plants 114–27

INDEX DNA molecular marker technology 153–4 downy mildew resistance 59, 68–9 drainage 222 drought stress see water stress dung pats 205, 207 E EDTA 155–6 effective ﬁlling period 245–6, 259 emasculation hand 43–4 hot-water 44–5 plastic bag 45–6 emergence sorghum cold tolerance 83 sorghum drought stress 77 environmental cadmium occurrence 113–14 environmental effects on seed-ﬁll duration 248 –50, 252, 253–4, 263 enzyme activities 5–6 ergot 70–1 Escherichia coli O157:H7 23, 28 ethanol production 89 excretion 183, 184, 205, 207–11 exotic germplasm–sorghum conversion 56–9 exudates, root 138–9 F fallowing 135 family-based sorghum population improvement programs 51 farmer study groups 224 –5, 226 farm-scale management practices 212, 221–8 fatty acids 10, 11–13, 14 feeding styles 201 feed pad system 223–4 feeds, supplementary 222–4 fenpropimorph 3 –4 fertilizers 133 –4, 219–21 ﬁngerprinting 15 –20 ﬂag leaf photosynthesis 255 ﬂooding 193 ﬂower development 251– 2 forage sorghum 89–92 fruit development 251–2 fumigants, soil 1– 29 fungi biomarkers 11–12 soil cadmium remediation 158–60

283

Fusarium moniliforme sensu lato 64, 66, 67 fusarium stalk rot 64 G genetics bacteria for soil cadmium remediation 160 cadmium phytoextraction 156 –7 male sterility 44 microbial community structure 15 –20 seed-ﬁll duration 253 –4, 263 genomic analysis, sorghum improvement 62, 67, 68, 80, 93–5 genotypes, sorghum improvement 37–96 germination 76, 77, 83 germplasm 56–9 glutamine-binding protein 149 g-glutamylcysteine synthetase 148, 149 glutathione 146–52 glutathione synthetase 148 goats 201 –2 grain see also seed-ﬁll duration color 86–8 mold resistance 59, 66– 8 quality 86–9 gram-negative bacteria 11– 12, 14 gram-positive bacteria 11–12, 13, 14 grass-legume pasture systems 181– 228 grass selection 216 –19 grazing animal excretion 183, 184, 205, 207–11 defoliation 183, 184, 200–5, 206, 216 diet selection 201 –2 intensity 184 nitrogen ﬁxation impact 181– 228 restricted 222–4 strategies to minimize impacts 211 –21 treading effects 183, 184–200, 212, 222 green bug (Schizaphis graminum ) 74– 5 growth stages see development stages guinea corn 41 H hand emasculation 43 –4 harvest index 264 –5 head smut resistance 59, 69– 70 heat tolerance 82–4 herbage rejection 203, 205, 207, 209, 212 herd houses 223

284

INDEX

hoof action see also treading 186 –8 hot-water emasculation 44–5 hybrids cytoplasmic–genetic male sterility 46–9 seed-ﬁll duration 253, 254, 263–4 sorghum improvement 41–2, 49, 52–6 hydrous iron oxide 157 hydrous manganese oxide 157 hyperaccumulators 154–5 I improvement methodology, sorghum 49–59 inbred line development 52 insect pest resistance 72–5 intensive grazing 184 iron 130 iron oxide 157, 158 irrigation cadmium in tobacco 134 –5 seed-ﬁll duration 263 J johnsongrass 95 K Klebsiella mobilis 160 L landraces, sorghum 40 late-summer pasture management strategies 214–15 LCT1 see low afﬁnity cation transporter leaf ﬁring ratings 82 leaves see also tobacco, leaves size of white clover 212, 216, 218 legume-based pasture systems 181–228 lenticels 193, 196 ligand protein 148 light competition 209 lignin 92 liming 132 lithospheric cadmium concentration 113 lodging disease 64 logistic model, seed-ﬁll duration 245 low afﬁnity cation transporter (LCT1) 140, 145

M Macrophomena phaseolina 64 macroporosity test 222 maize hybrids 253, 254 management farm-scale practices 212, 221–8 nitrogen ﬁxation in legume-based pastures 181–228 manganese oxide 157 manure-amended soil 19 –20 marker-assisted technology 153 –4 MATE see multidrug and toxic compound extrusion maturity, sorghum hybrids 60 –2 MeBr see methyl bromide mechanical impedance 188 –92, 212 metallothioneins 146– 52 metam sodium 3, 4, 25 methyl bromide (MeBr) biological process effects 5–8 degradation 22–3 microbial activities/composition effects 8–20 microbial communities/agricultural practice impacts 22–4 mode of action 3 –4 structure 4 methyl isothiocyanate (MITC) biological process effects 5–8 degradation 25–6 microbial activities/composition effects 8–20 microbial communities/agricultural practice impacts 25–6 mode of action 3 –4 structure 4 microorganisms see also biodegradation biomass 6, 10 –12 soil cadmium remediation 158–61 soil fumigant effects 1–29 mid-spring pasture management strategies 215–16 mid-summer pasture management strategies 215–16 MITC see methyl isothiocyanate molecular approaches cadmium reduction in tobacco 137–53 soil microbial community structure 15–20 morphology, white clover 216 –18 MRP see multidrug-resistance-related proteins

INDEX multidisciplinary on-farm study groups 224 –5, 226 multidrug-resistance-related proteins (MRP) 141 –2 multidrug and toxic compound extrusion (MATE) family 140, 145 mutagenized populations 154 Mycobacteria, soil fumigants 29 mycorrhizae 158–60

285 O

organic acids 138– 9 organic amendments 21 oxygen diffusion in soil 199–200 reduction in compacted soils 192–8, 199 –200 P

N natural resistance-associated macrophage protein (Nramp) family 140, 142 –3 Nicotiana tabacum see tobacco nitrilotriacetate 156 nitrogen fertilizers 134, 219–21 ﬁxation in legume-based pastures 181 –228 animal excretion 205, 207–11 animal grazing 183, 184, 200–5, 206, 216 animal treading 184 –200 defoliation 183, 184, 200– 5, 206, 216 farm-scale management practices 221– 8 grass selection 216 –19 pasture management 211–16 restricted grazing 222– 4 soil compaction 191–2, 196–8 soil management 222 strategies to minimize grazing impacts 211 –21 supplementary feeding 222–4 technically based decision making 224 –7 white clover cultivars 216 –18 seed-ﬁll duration 251, 257, 258 –62, 263 transformation 7 nitrogenase activity 204–5, 206 nodulation 191–2, 196 –8 non-biological soil amendments 157– 8 non-target soil organisms 1– 29 nopaline synthase promoter 150 Nramp see natural resistance-associated macrophage protein nutrients cycling 2 seed-ﬁll duration 250 –1 stress 84–6 nutritional value 86

participatory farmer study groups 224 –5, 226 pasture management strategies 211 –21 pasture tolerance to treading 186, 198–200 Peronosclerospora sorghii 68 pH 127–8 phosphate fertilizers 133–4 phospholipid fatty acid proﬁles (PLFA) 10, 11– 13, 14 photoperiod seed-ﬁll duration 252 silage hybrids 92 sorghum germplasm collections 57, 58 sorghum–sudangrass hybrids 91, 92 photosynthesis nitrogenase activity 204 seed-ﬁll duration 255, 256, 257, 267 phylogenetic trees 18–19 physical impacts on soil 184 –200 phytoavailability enhancement 155 phytochelatins 146 –52 phytochelatin synthase 147 phytoextraction 138, 154–7 phytoremediation 154–7 phytosiderophores 138–9 phytotoxicity 115 –17 pigmented testa 86, 88 plant growth substances 252 plants color/sorghum end use 87, 88 density/seed-ﬁll duration 250–1 height/grain yield relationship 62 seed-ﬁll duration regulation 256 –7 treading effects 186 –8, 212 plastic bag emasculation 45 –6 PLFA see phospholipid fatty acid proﬁles pollination control 42–9 population improvement programs for sorghum 51– 2 porosity, air-ﬁlled 199–200 post-ﬂowering drought stress 78, 79–81

286

INDEX

potentially remobilized nitrogen pool 260 poured crossing 45–6 pre-ﬂowering drought stress 77–9, 80–1 productivity, seed-ﬁll duration 262–7 protein grain sorghum 89 seed protein concentrations 259 proton antiporters 144–5 P-type ATPase 140, 143 puddling 198 pugging 188, 225, 226 pure-line sorghum cultivars 41, 49, 52 Q QLTs see quantitative trait loci quantiﬁable assessment tools 221 quantitative trait loci (QLTs) sorghum grain mold resistance 67, 68 sorghum grain yield 62 sorghum improvement biotechnology 94–5 sorghum post-ﬂowering drought stress 80 staygreen 80 quinone proﬁle analysis 10, 11, 13 R races of sorghum 39 –40 randomly ampliﬁed polymorphic DNA (RAPD) markers 153–4 regression model, seed-ﬁll duration 245 remediation, soil cadmium 154– 61 resistance disease 59, 63–72 insects 72–5 multidrug-resistance-related proteins 141–2 natural resistance-associated macrophage protein family 140, 142 –3 respiration 6 restricted grazing 222–4 rhizosphere cadmium reduction in tobacco 138, 139 colonizers 21, 23, 28 –9 16S rRNA 12, 15 –20 roots cadmium in tobacco 117 –20, 138 –9 exudates 138 –9 penetration impedance 185

root-to-shoot cadmium transport 117– 19 soil compaction effects 189–91, 193 –6 rotational grazing 202–3, 213, 216 S Saccharomyces cerevisiae 143, 147 sacriﬁce paddocks 223 saline soils 84 Schizaphis graminum (green bug) 74 –5 seed-ﬁll duration 243–68 assimilate 250–1 crop productivity 262–7 deﬁnition 245–8 environmental effects 248–50, 252, 253–4, 263 ﬂower development 251 –2 fruit development 251–2 future yield improvements 265–7 genetic variations 253–4, 263 measurement 245–8 nitrogen 251, 257, 258–62, 263 nutrient supplies 250 –1 photoperiod 252 photosynthesis 255, 256, 257, 267 plant growth substances 252 regulation 254– 62 senescence 250, 251, 256– 62 temperature 248 –9, 263 variation within/between species 246, 247 water stress 249–50, 263 yield 244, 253 –4, 262 –7 seed-growth curves 245, 246, 247 seedlings sorghum cold tolerance 83 –4 sorghum drought stress 77 seeds moisture levels 246 number reduction 250 seed-ﬁll duration regulation by seeds 255 –6 selective breeding seed-ﬁll duration 253, 264, 266 white clover 216 senescence 250, 251, 256 –62 shade 209, 224, 250 Shannon–Weaver index of diversity 13, 17 –18, 19, 20–1 sheep see also grazing feeding style 201– 2 grazing management strategies 213 soil bulk density effects 189

INDEX shoot ﬂy (Atherigona soccata ) 72 shoots cadmium distribution in tobacco 119 –20 root-to-shoot cadmium transport 117–19 soil compaction effects 189 –91, 193– 6 silage sorghum 90 –2 silking 246, 247 sludge amendments 130–1 smuts resistance 69–70 sodium methyldithiocarbamate 3 soil aeration 185, 192 –8, 199 –200, 212 air-ﬁlled porosity 199–200 amendments 19–20, 21, 130– 1, 157–8 bulk density 188, 189– 92, 198–9 cadmium concentrations in soil 113 –14 concentrations in tobacco leaves 127–30 microorganism effect on plant uptake 158 –61 remediation 154–61 stabilization 157–8 cation exchange capacity 128 characteristics 127–30 compaction nitrogen ﬁxation 191–2, 196–8 root growth 189 –91, 193 –6 shoot growth 189–91, 193–6 soil management 222 treading effects 188 –98 fumigants agricultural practices 20–8 biological processes 5–8 microbial activities/composition effects 8 –20 mode of action 3–4 non-target soil organisms 1–29 soil microbial communities impact 20– 8 speciﬁc microbial population effects 28 –9 health indicators 2, 4 management 212, 222 microbial communities fumigant effects 1 –29 non-biological amendments 157–8 nutrient stress 84–6 pH 127–8 physical condition indicators 222 phytoremediation 154–7 remediation 154–61 salinity 84

287

treading effects 184 –200, 212 waterlogging 185 somaclonal variation 154, 155 sorghum abiotic stress 75–86 biotechnology 93– 5 biotic stress 63–75 broomcorn 93 controlled pollination mechanisms 42 –9 conversion program 56–9 forage sorghum 89–92 germplasm collections 57 grain quality 86 –9 historical developments 40 –2, 49 improvement 37– 96 methodology for improvement 49–59 races 39–40 sweet sorghum for syrup 92– 3 taxonomic classiﬁcation 39 trait-based breeding 59– 93 trait variations 39–40 uses 38 world production 38 yield and adaptation 60–3 sorghum midge (Stenodiplosis sorghicola ) 72– 4 sorghum–sudangrass hybrids 90–1, 92 source–sink ratios 251 sowing date 263 space competition 209 Sporisorium relianum 69 spring pasture management strategies 215– 16 16S rRNA 12, 15–20 stabilization, cadmium in soil 157 –8 stalk lodging resistance 64 stalk position 121 –2 stalk rot resistance 64 stand-off areas 222 –4 staygreen 79, 80, 81 steel shots 157– 8 Stenodiplosis sorghicola (sorghum midge) 72– 4 sterility 46–9 stocking rates 202, 210 stolon fragmentation and burial 187 strategic grazing 211 strawberries 21–2, 28 stress abiotic stress 75–86 biotic stress 63–75 nutrient 84–6

288

INDEX

temperature stress 82–4 water 75–81, 249–50, 263 Striga hermonthica 71 striga resistance 71–2 sub-cellular localization 124 substrate-induced respiration 6 sub-tropical sorghum adaptation 62 sucrose 255 sulfur reductive assimilation pathway 152 summer pasture management strategies 214 –16 supplementary feeding 222–4 sward composition modiﬁcation 201–2 height 201 sweet sorghum 92–3 syrup 92 –3 T tannins 86, 87 tannin sorghums 86, 87 taxonomic classiﬁcation of sorghum 39 technical based decision making 224– 7 Telone see 1, 3-dichloropropene temperate sorghum adaptation 62 temperature seed-ﬁll duration 248–9, 263 sorghum improvement 82–4 stress 82 –4 testa, pigmented 86, 88 testcrossing 54 –5 Thomas phosphate basic slags 157 tillage 135, 222 tobacco cadmium reduction 111 –62 leaves cadmium distribution 119–20, 123–4 cadmium in ﬁeld-grown 120 –1 external factors affecting cadmium concentration 127–37 stalk position versus accumulation 121–2 virus interactions 117 tolerance cadmium in tobacco 115 –17, 146 –52 pasture treading 186, 198 –200 phytochelatin role in cadmium tolerance 151–2 total growth cycle 260, 261, 265–6 total organic carbon in the microbial biomass (biomass C) 10

total seed growth rate 263 trait-based breeding 59–93 transgenic sorghums 95 transport, cadmium root-to-shoot in tobacco 117–19 transporters, cadmium 139–46 treading effects mechanical impedance 188– 92, 212 nitrogen ﬁxation in legume-based pastures 183, 184 –200, 212, 222 plant damage and burial 186– 8, 212 signiﬁcance of effects 198–200 soil aeration 192 –8, 199 –200, 212 soil compaction 188 –98 soil management 222 waterlogging of soil 192– 8 trichloronitromethane see chloropicrin Trifolium repens see white clover

U under grazing 216 urine 205, 207–11 V varieties cadmium accumulation in tobacco 124–6 sorghum 39–40 virus interactions 117 W water see also irrigation cadmium concentrations 113 waterlogged soils 185, 192–8, 222 water status, tissue 256 water stress drought stress screening 81 seed-ﬁll duration 249–50, 263 sorghum improvement 75 –81 waxy endosperm sorghums 89 wheeled agricultural machinery compaction 191 white clover (Trifolium repens ) cultivars 216–18 grass associations 181–228

INDEX Y year variations 136 –7 yellow endosperm sorghum 88 yield seed-ﬁll duration 244, 253–4, 262–7 sorghum improvement 53– 6, 60–3

289 Z

zeolites 157 zinc 129–30 zinc regulated transporter- and iron regulated transporter- like proteins (ZIP) family 140 –1

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