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Nitrogen Fixation in , Forestry, , and the Environment Fixation: Origins, Applications, and Research Progress

VOLUME 4 Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment

Edited by

Dietrich Werner Philipps-University, Marburg, Germany and William E. Newton Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U.S.A. A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN-10 1-4020-3542-X (HB) ISBN-10 1-4020-3544-6 (e-book) ISBN-13 978-1-4020-3542-9 (HB) ISBN-13 978-1-4020-3544-5 (e-book)

Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. www.springeronline.com

Background figure caption: “A seed crop of (Trifolium hirtum) in flower near Moora, Western Australia. Photograph courtesy of Mike Davies, Senior Technical Officer, Pasture Research Group of Agriculture WA and reproduced with permission.” Vol. 4-specific figure caption: “First year cultivation from a field trial in Puerto Rico with both inoculated and non-inoculated rows. Photograph courtesy of R. Stewart Smith, Nitragin Company, USA, and reproduced with permission.”

Printed on acid-free paper

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Printed in the Netherlands TABLE OF CONTENTS

Series Preface...... ix Preface...... xiii

ListofContributors...... xvii

Chapter 1. Production and Biological Nitrogen Fixation of Tropical D. Werner ...... 1 1.Introduction...... 1 2. Phaseolus sp. and Vigna sp.()...... 2 3. Arachis hypogaea (Groundnut,)...... 7 4. Cicer arietinum (Chickpea)...... 8 5. Cajanus cajan (Pigeonpea) ...... 9 6. Mucuna pruriens (Velvetbean)andOtherLegumes...... 10 Acknowledgements...... 12 References...... 12

Chapter 2. Nitrogen Fixation by Soybean in North America S.G.Pueppke...... 15 1. Soybean: Pathways to North America and EstablishmentasaCrop...... 15 2.SoybeanProductioninNorthAmerica...... 17 3. Major Soybean Cropping Systems ...... 20 4. Biological Nitrogen Fixation by Soybean in North America . . . . . 20 5.Perspectives...... 21 Acknowledgements...... 22 References...... 22

Chapter 3. The Importance of Nitrogen Fixation to Soybean Cropping in South America M. Hungria, J. C. Franchini, R. J. Campo and P. H. Graham...... 25 1.Introduction...... 25 2. Taxonomy, Origins, and Importance of Soybean ...... 25 3.BiologicalNitrogenFixation...... 29 4. Economic Importance of Biological Nitrogen Fixation (BNF) inSouthAmerica...... 32 5.CropManagementinSouthAmerica...... 34 6.FinalConsiderations...... 38 Acknowledgement...... 39 References...... 39

Chapter 4. Production, Regional Distribution of Cultivars, and Agricultural Aspects of Soybean in India S. K. Mahna ...... 43 1.IntroductionandHistoricalBackground...... 43

v vi 2. All-India Area Coverage, Productivity, and Production of Soybean between 1970-2003 ...... 45 3. All-India State-wise Area Coverage, Productivity, and ProductionofSoybean...... 46 4.RegionalDistributionofSoybeanCultivars...... 49 5.RegionalAgriculturalAspectsofSoybeanCultivation...... 59 Acknowledgements...... 64 References...... 64

Chapter 5. Soybean Cultivation and BNF in China J. E. Ruiz Sainz, J. C. Zhou, D.-N. Rodriguez-Navarro, J. M. Vinardell and J. E. Thomas-Oates ...... 67 1.Summary...... 67 2. Soybean Cultivation in China: Historical Aspects andCurrentSituation...... 68 3.Nitrogen-FixingBacteriathatNodulateSoybean...... 75 4.TheSoybeanGermplasmCollectioninChina...... 81 5. Soybean in and in Continuous Cultivation ...... 82 6.Conclusions...... 84 Acknowledgement...... 85 References...... 85

Chapter 6. Stress Factors Influencing Symbiotic Nitrogen Fixation M.J.Sadowsky...... 89 1.Introduction...... 89 2.ImportanceofSymbioticNitrogenFixation...... 89 3.SymbioticInteractionofLegumeswithRhizobia...... 90 4. Nodulation and Nitrogen-Fixation in the RhizobiaandBradyrhizobia...... 92 5.RhizobiaintheSoilEnvironment...... 94 6. Stress Factors in the Soil Environment that Influence N2 Fixation . . 95 7.ConcludingRemarks...... 101 References...... 102

Chapter 7. Nodulated Trees J. I. Sprent ...... 113 1.Introduction...... 113 2.Leguminosae...... 113 3.RhizobiathatNodulateLegumeTrees...... 133 4. Types of Nodule formed on Trees ...... 134 5. and Other Nutrient-Acquisition Systems ...... 135 6.MeasurementofNitrogenFixationbyTrees...... 136 7.RoleofLegumeTreesinNaturalandManagedSystems...... 137 References...... 139 vii Chapter 8. Nitrogen Fixing Trees with Actinorhiza in Forestry and R. O. Russo ...... 143 1.Introduction...... 143 2.GeneralCharacteristicsoftheActinorhizalSymbiosis...... 144 3. Host Botanical Families ...... 148 4.NitrogenFixationinActinorhizalTrees...... 149 5.MycorrhizalAssociationswithActinorhizalTrees...... 153 6.ActinorhizalTreesinAgroforestry...... 157 7. The Casuarina ...... 158 8.TheExperienceoftheCentralAmericaFuelwoodProject...... 159 9.TheCaseofAlnus acuminata inTropicalHighlands...... 161 10.OtherUsesofActinorhizalTrees...... 163 11.ConcludingConsiderations...... 164 References...... 165

Chapter 9. Molecular Ecology of N2-fixing Microbes Associated with Gramineous : Hidden Activities of Unknown T. Hurek and B. Reinhold-Hurek ...... 173 1.Introduction...... 173 2. The Problem of Identifying Key Diazotrophic Bacteria inGramineousPlants:TheClassicalApproach...... 174 3. The Significance of Diazotrophic Grass Endophytes ...... 176 4.TheSignificanceofCulture-IndependentMethods...... 179 5. The Significance of nifH-TargetedMethods...... 180 6. Limitations of nifH-TargetedMethods...... 181 7. Many Defy Cultivation ...... 183 8. Diazotrophic Grass Endophytes as Key Organisms for BNFinGramineousPlants...... 185 9.SummaryandOutlook...... 189 References...... 191

Chapter 10. Interactions of Arbuscular and Nitrogen-Fixing in J. M. Barea, D. Werner, C. Azcón-Guilar and R. Azcón ...... 199 1.Introduction...... 199 2.PurposeofReview...... 200 3.Nitrogen-FixingSymbioses...... 201 4.ArbuscularMycorrhiza...... 202 5. Interactions between AM Fungi and to Improve LegumeProductivityinAgriculture...... 205 6.Conclusions...... 214 Acknowledgements...... 215 References...... 215 viii Chapter 11. Inoculant Preparation, Production and Application M. Hungria, M. F. Loureiro, I. C. Mendes, R. J. Campo and P. H. Graham . . . 223 1.Introduction...... 223 2.StrainSelection...... 224 3.InoculantProduction...... 230 4.InoculantApplication...... 237 5.FactorsAffectingtheSuccessofInoculation...... 240 6.MainConclusions...... 245 Acknowledgements...... 246 References...... 246

Chapter 12. C. Fiencke, E. Spieck and E. Bock ...... 255 1.NitrificationasPartoftheNitrogenCycle...... 255 2. Two Physiological Groups of Bacteria Contribute to . .257 3.EcologyandDetectionofNitrifyingBacteria...... 258 4.MetabolismofNitrifyingBacteria...... 260 References...... 270

Chapter 13. The : and its Relationship to N2 Fixation R. J. M. van Spanning, M. J. Delgado and D. J. Richardson ...... 277 1.Introduction...... 277 2.TheNitrogenCycle...... 279 3.Denitrification...... 281 4.BacterialRespiratoryNitrateReductases...... 282 5.NitriteReductases...... 296 6.NitricOxideReductases...... 301 7. Nitrous Reductase...... 306 8.LinkageoftheDenitrificationGeneClusters...... 307 9.BioenergeticsofDenitrification...... 310 10.RegulationofTranscriptionofDenitrificationGenes...... 312 11.RegulationofDenitrificationbyEnvironmentalFactors...... 320 12.DiversityofDenitrification...... 321 13. Yeast and Fungal Denitrification ...... 325 14.ConcludingRemarks...... 327 Acknowledgements...... 327 References...... 327

SubjectIndex...... 343 SERIES PREFACE

Nitrogen Fixation: Origins, Applications, and Research Progress

Nitrogen fixation, along with as the energy supplier, is the basis of all life on Earth (and maybe elsewhere too!). Nitrogen fixation provides the basic component, fixed nitrogen as , of two major groups of macromolecules, namely nucleic acids and . Fixed nitrogen is required for the N-containing heterocycles (or bases) that constitute the essential coding entities of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), which are responsible for the high-fidelity storage and transfer of genetic information, respectively. It is also required for the amino-acid residues of the proteins, which are encoded by the DNA and that actually do the work in living cells. At the turn of the millennium, it seemed to me that now was as good a time as any (and maybe better than most) to look back, particularly over the last 100 years or so, and ponder just what had been achieved. What is the state of our knowledge of nitrogen fixation, both biological and abiological? How has this knowledge been used and what are its impacts on humanity? In an attempt to answer these questions and to capture the essence of our current knowledge, I devised a seven-volume series, which was designed to cover all aspects of nitrogen-fixation research. I then approached my long-time contact at Kluwer Academic Publishers, Ad Plaizier, with the idea. I had worked with Ad for many years on the publication of the Proceedings of most of the International Congresses on Nitrogen Fixation. My personal belief is that congresses, symposia, and workshops must not be closed shops and that those of us unable to attend should have access to the material presented. My solution is to capture the material in print in the form of proceedings. So it was quite natural for me to turn to the printed word for this detailed review of nitrogen fixation. Ad’s immediate affirmation of the project encouraged me to share my initial design with many of my current co-editors and, with their assistance, to develop the detailed contents of each of the seven volumes and to enlist prospective authors for each chapter. There are many ways in which the subject matter could be divided. Our decision was to break it down as follows: , commercial processes, and relevant chemical models; genetics and regulation; genomes and genomics; associative, endophytic, and cyanobacterial systems; actinorhizal associations; leguminous symbioses; and agriculture, forestry, ecology, and the environment. I feel very fortunate to have been able to recruit some outstanding researchers as co- editors for this project. My co-editors were Mike Dilworth, Claudine Elmerich, John Gallon, Euan James, Werner Klipp, Bernd Masepohl, Rafael Palacios, Katharina Pawlowski, Ray Richards, Barry Smith, Janet Sprent, and Dietrich Werner. They worked very hard and ably and were most willing to keep the volumes moving along reasonably close to our initial timetable. All have been a pleasure to work with and I thank them all for their support and unflagging interest.

ix x Nitrogen-fixation research and its application to agriculture have been ongoing for many centuries – from even before it was recognized as nitrogen fixation. The Romans developed the crop-rotation system over 2000 years ago for maintaining and improving soil fertility with nitrogen-fixing legumes as an integral component. Even though crop rotation and the use of legumes was practiced widely but intermittently since then, it wasn’t until 1800 years later that insight came as to how legumes produced their beneficial effect. Now, we know that bacteria are harbored within nodules on the legumes’ and that they are responsible for fixing N2 and providing these plants with much of the fixed nitrogen required for healthy growth. Because some of the fixed nitrogen remains in the unharvested parts of the crop, its release to the soil by mineralization of the residue explains the follow-up beneficial impact of legumes. With this realization, and over the next 100 years or so, commercial inoculants, which ensured successful bacterial nodulation of legume crops, became available. Then, in the early 1900’s, abiological sources of fixed nitrogen were developed, most notable of these was the Haber-Bosch process. Because fixed nitrogen is almost always the limiting nutrient in agriculture, the resulting massive increase in synthetic fixed-nitrogen available for has enabled the enormous increase in food production over the second half of the 20th century, particularly when coupled with the new “green revolution” crop varieties. Never before in human history has the global population enjoyed such a substantial supply of food. Unfortunately, this bright shiny coin has a slightly tarnished side! The abundance of nitrogen fertilizer has removed the necessity to forage legumes and to return animal to fields to replenish their fertility. The result is a continuing loss of soil organic matter, which decreases the soil’s tilth, its water- holding capacity, and its ability to support microbial populations. Nowadays, farms do not operate as self-contained recycling units for crop nutrients; are trucked in and meat and food crops are trucked out. And if it’s not recycled, how do we dispose of all of the animal waste, which is rich in fixed nitrogen, coming from feedlots, broiler houses, and pig farms? And what is the environmental impact of its disposal? This problem is compounded by inappropriate agricultural practice in many countries, where the plentiful supply of cheap commercial nitrogen fertilizer, plus farm subsidies, has encouraged high (and increasing) application rates. In these circumstances, only about half (at best) of the applied nitrogen reaches the crop plant for which it was intended; the rest leaches and “runs off” into streams, rivers, lakes, and finally into coastal waters. The resulting can be detrimental to marine life. If it encroaches on drinking-water supplies, a human health hazard is possible. Furthermore, oxidation of and fertilizers to progressively acidifies the soil – a major problem in many agricultural areas of the world. A related problem is the emission of nitrogen (NOx) from the soil by the action of on the applied fertilizer and, if fertilizer is surface broadcast, a large proportion may be volatilized and lost as ammonia. For urea in paddies, an extreme example, as much as 50% is volatilized and lost to the . And what goes up must come down; in the case of fertilizer nitrogen, it returns to Earth in the rain, often acidic in nature. This xi uncontrolled deposition has unpredictable environmental effects, especially in pristine environments like forests, and may also affect biodiversity. Some of these problems may be overcome by more efficient use of the applied fertilizer nitrogen. A tried and tested approach (that should be used more often) is to ensure that a balanced supply of nutrients (and not simply applying more and more) is applied at the right time (maybe in several separate applications) and in the correct place (under the soil surface and not broadcast). An entirely different approach that could slow the loss of fertilizer nitrogen is through the use of nitrification inhibitors, which would slow the rate of conversion of the applied ammonia into nitrate, and so decrease its loss through leaching. A third approach to ameliorating the problems outlined above is through the expanded use of biological nitrogen fixation. It’s not likely that we shall soon have plants, which are capable of fixing N2 without associated microbes, available for agricultural use. But the discovery of N2-fixing endophytes within the tissues of our major crops, like rice, , and , and their obvious benefit to the crop, shows that real progress is being made. Moreover, with new techniques and experimental approaches, such as those provided by the advent of genomics, we have reasons to renew our belief that both bacteria and plants may be engineered to improve biological nitrogen fixation, possibly through developing new symbiotic systems involving the major cereal and tuber crops. In the meantime, the major impact might be through agricultural sustainability involving the wider use of legumes, reintroduction of crop-rotation cycles, and incorporation of crop residues into the soil. But even these practices will have to be performed judiciously because, if legumes are used only as cover crops and are not used for grazing, their growth could impact the amount of cultivatable land available for food crops. Even so, the dietary preferences of developed countries (who eats beans when steak is available?) and current agricultural practices make it unlikely that the fixed-nitrogen input by rhizobia in agricultural will change much in the near-term future. A significant positive input could accrue, however, from matching rhizobial strains more judiciously with their host legumes and from introducing “new” legume species, particularly into currently marginal land. In the longer term, it may be possible to engineer crops in general, but cereals in particular, to use the applied fertilizer more efficiently. That would be a giant step the right direction. We shall have to wait and see what the ingenuity of mankind can do when “the chips are down” as they will be sometime in the future as food security becomes a priority for many nations. At the moment, there is no doubt that commercially synthesized fertilizer nitrogen will continue to provide the key component for the required by the next generation or two. So, even as we continue the discussion about the benefits, drawbacks, and likely outcomes of each of these approaches, including our hopes and fears for the future, the time has arrived to close this effort to delineate what we know about nitrogen fixation and what we have achieved with that knowledge. It now remains for me to thank personally all the authors for their interest and commitment to this project. Their efforts, massaged gently by the editorial team, have produced an indispensable reference work. The content is my responsibility and I apologize xii upfront for any omissions and oversights. Even so, I remain confident that these volumes will serve well the many scientists researching nitrogen fixation and related fields, students considering the nitrogen-fixation challenge, and administrators wanting to either become acquainted with or remain current in this field. I also acknowledge the many scientists who were not direct contributors to this series of books, but whose contributions to the field are documented in their pages. It would be remiss of me not to acknowledge also the patience and assistance of the several members of the Kluwer staff who have assisted me along the way. Since my initial dealings with Ad Plaizier, I have had the pleasure of working with Arno Flier, Jacco Flipsen, Frans van Dunne, and Claire van Heukelom; all of whom provided encouragement and good advice – and there were times when I needed both! It took more years than I care to remember from the first planning discussions with Ad Plaizier to the completion of the first volumes in this series. Although the editorial team shared some fun times and a sense of achievement as volumes were completed, we also had our darker moments. Two members of our editorial team died during this period. Both Werner Klipp (1953-2002) and John Gallon (1944- 2003) had been working on Volume II of the series, Genetics and Regulation of Nitrogen-Fixing Bacteria, and that volume is dedicated to their memory. Other major contributors to the field were also lost in this time period: Barbara Burgess, whose influence reached beyond the arena into the field of - cluster biochemistry; Johanna Döbereiner, who was the discoverer and acknowledged leader in nitrogen-fixing associations with grasses; Lu Jiaxi, whose “string bag” model of the FeMo- prosthetic group of Mo-nitrogenase might well describe its mode of action; Nikolai L’vov, who was involved with the early studies of -containing cofactors; Dick Miller, whose work produced new insights into MgATP binding to nitrogenase; Richard Pau, who influenced our understanding of alternative nitrogenases and how molybdenum is taken up and transported; and Dieter Sellmann, who was a synthetic inorganic chemistry with a deep interest in how N2 is activated on metal sites. I hope these volumes will in some way help both preserve their scientific contributions and reflect their enthusiasm for science. I remember them all fondly. Only the reactions and interest of you, the reader, will determine if we have been successful in capturing the essence and excitement of the many sterling achievements and exciting discoveries in the research and application efforts of our predecessors and current colleagues over the past 150 years or so. I sincerely hope you enjoy reading these volumes as much as I’ve enjoyed producing them.

William E. Newton Blacksburg, February 2004 PREFACE

Nitrogen Fixation in Agriculture, Forestry, Ecology and the Environment

Most grant applications that involve basic research on biological nitrogen fixation (BNF) emphasiz e the economic importance of this basic biological process. But just what is the economic value of BNF? In several chapters of this volume, the authors report their estimations of the amount of nitrogen fixed biologically. For example, the amount of nitrogen fixed each year by the legume-rhizobia symbiosis is around 70 million tonnes. If we assume that at least 70% of this amount is utilised in agricultural and agroforestry systems and so replaces commercial N- fertiliser, we can estimate that around 50 million tonnes of fixed nitrogen are made available to support agricultural production. In Europe, the price of fertiliser-N to the farmer at the beginning of 2004 was 650€ per tonne, which is equivalent to US$ 800. Putting these two figures together indicates just how enormous the credit made by legume-based BNF to agriculture is. Of course, this price varies significantly in other regions of the world and is closely linked to the prices of oil and gas. This fertiliser-oil linkage is the reason that funding for both the basic and applied aspects of BNF research has increased in periods with high oil and gas prices and decreased when energy prices are moderate. Because of their importance in worldwide agriculture, the volume starts with surveys of the current uses and benefits, plus the future potential, of legumes in around the World. In Chapter 1, production and BNF of many tropical legumes, as well as fodder and green- legumes, such as Mucuna,are described to illustrate their potential. The "Phaseomics" consortium and its research targets are also summarized here. This consortium involves forty-six research groups working with Phaseolus sp. and is offered as a model for future collaborative research projects focussed on a single crop. Chapter 1 also suggests that, in addition to the already established legume crops, such as Phaseolus, Vigna, Arachis, Cicer, Cajanus and Mucuna species, many other tropical legumes may become regionally important crops, depending on when more research funding becomes available to develop breeding programmes that include both crop protection and crop by BNF. Soybean is one of the four most important crops worldwide and it is the number-one oil and protein supplier for animal and human nutrition. The 2002 world production of 180 million tons exceeded the combined production of all other grain legumes, such as groundnuts, beans, pigeon and chickpeas. Production and BNF in in the four major growing regions for this crop are described in Chapters 2-5, with Chapter 2 considering North America, Chapter 3 covering South America, Chapter 4 describing the situation in India, and Chapter 5 giving an overview of the history and current soybean cultivation in China, the original home of the soybean. The regional aspects are especially emphasized in this extensive coverage to illustrate the importance of both plant breeding (and consequent plant production) and their concurrent adaptation to regional priorities, where world xiii xiv market and trade regulations are becoming increasingly important. The areas planted with soybeans are 57,000 km² in India, 83,000 km² in China, 290,000 km² in South America, and 300,000 km² in North America. All other areas add only about 70,000 km2, making a global total of about 800,000 km². This area planted to soybean is more than the combined land area of France and the UK. Other crop legumes are planted on about 700,000 km², which together with the soybean area, adds up to 1.5 million km² of the Earth’s land area planted to cultivated legumes. With such a large land area dedicated to their cultivation, it is not surprising that considerable research emphasis and, in many countries strict regulatory control, is given to the preparation, production, and application of rhizobial inoculants for legumes. This is one of the few areas where research on symbiotic nitrogen fixation has directly achieved a level of economic relevance (Chapter 11). The rhizobial strain-selection programmes for soybeans and common beans in Brazil are successful examples of continuous efforts to promote inoculation with the appropriate strains and the correct techniques. The pasture and grassland areas on the five continents total about 30 million km2, but we do not have an accurate estimate of what percentage is covered with legume species. If we assume 10% coverage, they would occupy an area of 3 million km². When we add the area covered with nodulated legume trees and shrubs (Chapter 7), which is larger than previously assumed, especially in Africa, and the more than 160 species of nitrogen-fixing trees and shrubs associated with actinorhiza (Chapter 8), we can make the cautious estimate that about 10 million km² of the land area of this planet are covered with symbiotic nitrogen-fixing species. This is more than the area of the United States of America, again highlighting the importance of these symbiotic species to humankind. Among the important influences on symbiotic nitrogen fixation with these crop and related species are soil-stress factors. The major stress factors, soil water, soil pH, soil temperature, and nutrient limitations, have been studied in detail and are described in Chapter 6. The various combinations of these factors, together with the large number of host plant-microsymbiont permutations, make it impossible still to predict precisely the amount of nitrogen that will be fixed by a single plant species in any region of the World. For example, more than forty such factors, which affect nitrogen fixation by actinorhizal plants in the field, are listed in figure3 of Chapter 8. Another fascinating research area involves the interaction of nitrogen- fixing symbioses with arbuscular mycorrhizae (AM) to give a tripartite association (Chapter 10). The AM is probably the oldest symbiosis of higher plants with microbes. In some phases of the tripartite interaction, the legume-rhizobia symbiosis and the AM symbiosis not only share the same plant host, but also share signal-transduction pathways (see also M. Parniske et al., (2002) Nature). A second BNF-research area of agricultural relevance and potential involves the endophytic associations of diazotrophs with cereals and other grasses. These associations appear less formalised than the rhizobia-legume symbiosis and, unfortunately, we still do have not enough field data to quantify their economic impact. Attempts to develop such data are often frustrated by the very complicated situation that exists. For example, many of these close endophytic associations of nitrogen-fixing microbes with grasses include microbial species that cannot be xv cultivated directly with the result that their presence can only be detected by indirect methods, such as in situ nifH mRNA expression (Chapter 9 and see also volume 5 of this series, Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial Associations). The volume closes with consideration of the other major processes in the Nitrogen Cycle and their relationship to nitrogen fixation. The economic impact of free-living nitrogen-fixing species is probably negligible because, before the nitrogen fixed by the bacterial cells has a chance to reach the plant, a large portion of it is likely lost and returned to the atmosphere as N2 by the combined processes of nitrification and denitrification. However, the ecological importance of the complete Nitrogen Cycle for the the quality of soils and soil functions can hardly be overestimated. Therefore, the final chapters of this volume cover these two important processes; nitrification is covered in Chapter 12 and denitrification in Chapter 13. Both of these integral parts of the Nitrogen Cycle have microorganisms as their essential components. The progress made in understanding the , biochemistry, and molecular genetics that undergird these two processes is as equally impressive as that made in the field of nitrogen fixation as described in the seven volumes of this series. We could not close this volume without thanking everybody involved with its production. We are especially grateful to all the authors of this volume for contributing their experience, considerable effort, and time (over and above their regular work load) to produce interesting, up-to-date, and extensively referenced chapters. We also wish to give special thanks to Mrs. Lucette Claudet in Marburg for her dedicated work on this volume. D. Werner wants to thank the EU for the International Development Project ICA 4-CT-2001-10057, related to several chapters of this volume.

Dietrich Werner Marburg, January 2005

William E. Newton Blackburg, January 2005 LIST OF CONTRIBUTORS

Rosario AZCÓN Rubens José CAMPO Departemento Microbiologia del EMBRAPA-CNPSO Soja Suelo y Systemas Simbioticos, Cx. Postal 231, Estacion Experimental del 86001-970, Londrina PR, Brazil. Zaidin, CSIC, Email: [email protected] C/ Profesor Albareda 1, Apdo 419, E-18008 Granada, María Jesús DELGADO IGEÑO Spain. Estación Experimental del Email: [email protected] Zaidín, CSIC, C/ Profesor Albareda 1, Concepción AZCÓN-AGUILAR E-18008 Granada, Spain. Departemento Microbiologia del Email: [email protected] Suelo y Systemas Simbioticos, Estacion Experimental del Claudia FIENCKE Zaidin, CSIC, Institut für Bodenkunde, C/ Profesor Albareda 1, Universität Hamburg, Apdo 419, E-18008 Granada, Allende-Platz 2, Spain. D-20146 Hamburg, Germany. Email: [email protected] Email: [email protected] hamburg.de José-Miguel BAREA Departemento Microbiologia del Julio C. FRANCHINI Suelo y Systemas Simbioticos, EMBRAPA-CNPSO Soja, Estacion Experimental del Cx. Postal 231, Zaidin, CSIC, 86001-970, Londrina PR, Brazil. C/ Profesor Albareda 1, Email: Apdo 419, E-18008 Granada, [email protected] Spain. Email: Peter H. GRAHAM [email protected] University of Minnesota, Dept. of Soil, Water, and Eberhard BOCK Climate, Fachbereich Biologie 1991 Upper Buford Circle, der Universität Hamburg, St Paul, MN 55108, USA. Institut für Allgemeine Botanik Email: [email protected] Mikrobiologie, Ohnhorststr. 18, D-22609 Hamburg, Germany. Mariangela HUNGRIA Email: [email protected] EMBRAPA–CNPSO Soja hamburg.de Cx. Postal 231, 86001-970, Londrina PR, Brazil. Email: [email protected]

xvii xviii Thomas HUREK Barbara REINHOLD-HUREK Allgemeine Mikrobiologie Allgemeine Mikrobiologie Fachbereich 2 Biologie/Chemie, Fachbereich 2 Biologie/Chemie, Universität Bremen, Universität Bremen, Postfach 33 04 40, Postfach 33 04 40, D-28334 Bremen, Germany D-28334 Bremen, Germany. Email: [email protected] Email: breinhold@uni- bremen.de M. F. LOUREIRO UFMT/FAMEV, David J. RICHARDSON Av. Fernando Correa s/n Centre for Campus Universitário, Spectroscopy and Biology, 78000-900 Cuiabá, MT, Brazil. School of Biological Sciences, Email: [email protected] University of East Anglia Norwich NR4 7TJ, U.K. S. K. MAHNA Email: [email protected] Department of , Maharshi Dayanand Saraswati University, D.N. RODRIGUEZ-NAVARRO K-22, Gandhi-Nagar, Naka Centro de Investigación y Madar, Ajmer - 305001, India. Formación Agraria “Las Torres Email: [email protected] y Tomejil”, Apartado Oficial, E-41200-Alcalá del Río, Sevilla, IIeda C. MENDES Spain Embrapa Cerrados. Email: Cx. Postal 08223, 73301-970 dulcenombre.rodriguez@untade Planaltina, DF, Brazil. andalucia.es Email: [email protected] Ricardo RUSSO Institute for Forestry and Natural William E. NEWTON Resources, EARTH University, Department of Biochemistry, P.O. Box 4442-1000, Costa Rica Virginia Polytechnic Institute and Email: [email protected] State University, Blacksburg, VA 24061, USA. José Enrique RUIZ SAINZ Email: [email protected] Departamento de Microbiologia Facultad de Biologia, Steven G. PUEPPKE Universidade de Sevilla Associate Dean for Research, Apartado 1095, 41080 Sevilla College of Agricultural, Spain. Consumer and Environmental Email: [email protected] Sciences, 211 Mumford Hall, University of Illinois, Urbana, IL 61801, USA Email : [email protected] xix Michael J. SADOWSKY Jane E. THOMAS-OATES Dept. of and Soil Department of Chemistry, Science, University of University of York, Heslington, Minnesota, Borlaug Hall, York, YO10 5DD, U.K. 1991 Upper Buford Circle, Email: [email protected] St. Paul, MN 55113, USA. Email: [email protected] J. M. VINARDELL Departamento de Microbiologia Rob J.M. van SPANNING Facultad de Biologia, Department of Molecular Cell Universidade de Sevilla Physiology, Faculty of EarTh Apartado 1095, E-41080, Sevilla and Life Sciences, Free Spain. University, De Boelelaan 1087, Email: [email protected] 1081 HV Amsterdam, The Netherlands. Dietrich WERNER Email: [email protected] FG Zellbiologie und Angewandte Botanik, Eva SPIECK Fachbereich Biologie, Institut für Allgemeine Botanik, Philipps-Universität Marburg, Abteilung Mikr obiologie, Karl-von-Frisch-Strasse, Universität Hamburg, D-35032 Marburg, Germany. Ohnhorststr. 18, D-22609 E-mail: [email protected] Hamburg, Germany. marburg.de Email: [email protected] J. C. ZHOU hamburg.de Department of Microbiology Huazhong Agricultural Janet I. SPRENT University, Shi Zi Shan Street, 32 Birkhill Avenue, Wormit, P.O. Box 430070, Wuhan Fife DD6 8PW, U.K. People’s Republic of China. Email: [email protected] Email: [email protected]