The Rhizosphere
BOOKS IN SOILS, PLANTS, AND THE ENVIRONMENT
Editorial Board Agricultural Engineering Robert M. Peart, Universityof Florida, Gainesville Animal Science Harold Hafs, Rutgers University, New Brunswick, New Jersey Crops Mohammad Pessarakli, Universityof Arizona, Tucson Imgation and Hydrology Donald R. Nielsen, Universityof California, Davis Jan Dirk van Elsas, Research Institutefor Plant Microbiology Protection, Wageningen,The Netherlands Plants L. David Kuykendall, U.S. Departmentof Agriculture, Beltsville, Maryland Soils Jean-Marc Bollag, Pennsylvania State University, University Park, Pennsylvania Tsuyoshi Miyazaki, Universityof Tokyo
Soil Biochemistry, Volume 1, edited by A. D. McLaren and G. H. Peterson Soil Biochemistry, Volume 2, edited by A. D. McLaren and J. SkujinS Soil Biochemistry, Volume 3, edited by E. A. Paul and A. D. McLaren Soil Biochemistry, Volume 4, edited by E. A. Paul and A. D. McLaren Soil Biochemistry, Volume 5, edited by E. A. Paul and J. N. Ladd Soil Biochemistry, Volume 6, edited by Jean-Marc Bollag and G. Stotzky Soil Biochemistry, Volume 7, edited by G. Stotzky and Jean-MarcBollag Soil Biochemistry, Volume 8, edited by Jean-Marc Bollag and G. Stotzky Soil Biochemistry, Volume 9, edited by G. Stotzky and Jean-MarcBollag Soil Biochemistry, Volume IO,edited by Jean-Marc Bollag and G. Stotzky Organic Chemicals in the Soil Environment, Volumes l and 2, edited by C. A. I. Goring and J. W. Hamaker Humic Substances in the Environment, M. Schnitzer and S. U. Khan Microbial Life in the Soil: AnIntroduction, T. Hattori Principles of Soil Chemistry, Kim H. Tan Soil Analysis: Instrumental Techniques and Related Procedures, edited by Keith A. Smith Soil Reclamation Processes: Microbiological Analyses and Applications, edited by Robert L. Tate Ill and Donald A. Klein Symbiotic Nitrogen fixation Technology, edited by Gerald H. Elkan Soil-WaterInteractions:Mechanisms and Applications,Shingo lwata and Toshio Tabuchi with Benno P.Warkentin
Soil Analysis: Modem Instrumental Techniques, Second Edition, edited by Keith A. Smith Soil Analysis: Physical Methods, edited by Keith A. Smithand Chris E. Mullins Growth and Mineral Nutrition of Field Crops, N. K. Fageria, V. C. Baligar, and Charles Allan Jones Semiarid Lands and Deserts: Soil Resource and Reclamation, edited by J. SkujinS Plant Roots: The Hidden Half, edited by Yoav Waisel, Amram Eshel, andUZi Kafkafi Plant Biochemical Regulators, edited by Harold W. Gausman Maximizing Crop Yields, N. K. Fageria Transgenic Plants: Fundamentals and Applications, edited by AndrewHiatt Soil Microbial Ecology: Applications in Agricultural and Environmental Management, edited by F. Blaine Metting, Jr. Principles of Soil Chemistry: Second Edition, Kim H. Tan Water f l o w in Soils, edited by Tsuyoshi Miyazaki Handbook of Plant and Crop Stress, edited by MohammadPessarakli Genetic Improvement of Field Crops, edited by Gustavo A. Slafer Agricultural Field Experiments: Design and Analysis, Roger G. Petersen Environmental Soil Science, Kim H. Tan Mechanisms of Plant Growth and Improved Productivity: Modern Approaches, edited by Amarjit S. Basra Selenium in the Environment, edited by W. T. Frankenberger, Jr., and Sally Benson Plant-Environment Interactions, edited by Robert E. Wilkinson Handbook of Plant and Crop Physiology, edited by MohammadPessarakli Handbook of Phytoalexin Metabolism and Action, edited by M. Daniel and R. P. Purkayastha Soil-Water Interactions: Mechanisms and Applications, Second Edition, Revised and Expanded, Shingo Iwata, Toshio Tabuchi, and Benno P. Warkentin Stored-Grain Ecosystems, edited by Digvir S. Jayas, Noel D. G. White, and William E. Muir Agrochemicals from Natural Products, edited by C. R. A. Godfrey Seed Development and Germination, edited by Jaime Kigel and Gad Galili Nitrogen fertilization in the Environment, edited by Peter Edward Bacon Phytohormonesin Soils: Microbial Production andFunction, William T. Frankenberger, Jr., and Muhammad Arshad Handbook of Weed Management Systems, edited by Albert E. Smith Soil Sampling, Preparation, and Analysis, Kim H. Tan Soil Erosion, Conservation, and Rehabilitation, edited by Menachem Agassi Plant Roots: The Hidden Half, SecondEdition, Revised and Expanded, edited by Yoav Waisel, Amram Eshel, and Uzi Kafkafi Photoassimilate Distribution in Plants and Crops: Sourcesink Relationships, edited by Eli Zamski and ArthurA. Schaffer Mass Spectrometry of Soils, edited byThomas W. BouttonandShinichi Yamasaki
Handbook of Photosynthesis, edited by Mohammad Pessarakli Chemical and Isotopic Groundwater Hydrology: The Applied Approach, Second Edition, Revised and Expanded, Emanuel Mazor Fauna in Soil Ecosystems: Recycling Processes, Nutrient Fluxes, and Agricultural Production, edited by Gero Benckiser Soil and Plant Analysis in Sustainable Agriculture and Environment, edited by Teresa Hood and J. Benton Jones, Jr. Seeds Handbook: Biology, Production, Processing, and Storage, B. B. Desai, P. M. Kotecha, and D. K. Salunkhe Modern Soil Microbiology, edited by J. D. van Elsas, J. T. Trevors, and E. M. H. Wellington Growth and Mineral Nutrition of Field Crops: Second Edition, N. K. Fageria, V. C. Baligar, and Charles Allan Jones Fungal Pathogenesis inPlants and Crops: Molecular Biology and Host Defense Mechanisms, P. Vidhyasekaran Plant Pathogen Detection and Disease Diagnosis, P. Narayanasamy Agricultural Systems Modeling and Simulation, edited byRobert M. Peart and R. Bruce Curry Agricultural Biotechnology, edited by Arie Altman Plant-Microbe Interactions and Biological Control, edited by Greg J. Boland and L. David Kuykendall Handbook of Soil Conditioners: Substances That Enhance the Physical Properties of Soil, edited by ArthurWallace and Richard E. Terry Environmental Chemistry of Selenium, edited by William T. Frankenberger, Jr., and Richard A. Engberg Principles of Soil Chemistry: Third Edition, Revised and Expanded, Kim H. Tan Sulfurin the Environment, edited by Douglas G. Maynard Soil-Machine Interactions: A Finite Element Perspective, edited by Jie Shen and Radhey La1 Kushwaha Mycotoxins in Agriculture and Food Safety, edited by Kaushal K. Sinha and Deepak Bhatnagar Plant Amino Acids: Biochemistry and Biotechnology, edited by Bijay K. Singh Handbook of Functional Plant Ecology, edited by Francisco I. Pugnaire and Fernando Valladares Handbook of Plant and Crop Stress: Second Edition, Revised and Expanded, edited by Mohammad Pessarakli Plant Responses to Environmental Stresses: From Phytohormones to Genome Reorganization, edited by H. R. Lerner Handbook of Pest Management, edited by John R. Ruberson Environmental Soil Science: Second Edition, Revised and Expanded, Kim H. Tan Microbial Endophytes, edited by Charles W. Bacon and JamesF. White, Jr. Plant-Environment Interactions: Second Edition, edited by Robert E. Wilkinson Microbial Pest Control, Sushi1 K. Khetan Soil and Environmental Analysis: Physical Methods, Second Edition, Revised and Expanded, edited by Keith A. Smith and Chris E. Mullins
The Rhizosphere: Biochemistry and Organic Substances at the Soil-Plant Interface, Roberto Pinton, Zen0 Varanini, and Paolo Nannipieri
Additional Volumes in Preparation
Woody Plants and Woody Plant Management: Ecology, Safety, and Environmental Impact, Rodney W. Bovey Handbook of Postharvest Technology, A. Chakraverty, Arun S. Mujumdar, and G. S. V. Raghavan Metals in the Environment, M. N. V. Prasad
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The Rhizosphere Biochemistry and Organic Substances at the Soil-Plant Interface
edited by
Roberto Pinton Zeno Varanini University of Udine Udine, Italy
Paolo Nannipieri University of Florence Florence, Italy
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Preface
The research on plant-soil interaction is focused on the processes that take place in therhizosphere,thesoilenvironmentsurroundingtheroot.Many of these processescancontrolplantgrowth.microbialinfections,andnutrientuptake. The rhizosphere is dominated by organic compounds released by plant roots and microorganisms. Furthermore, stable componentsof soil organic matter. namely, humic and fulvic substances,can influence both plantand microorganism metabolism. A variety of compounds are present in the rhizosphere, and they range from low-molecular-weight root exudates to high-molecular-weight humic substances. of these substances are becoming more and more The chemistry and biochemistry clear. and their study promises to shed light on the complex interactions between plant and soil microflora. The aim of this book is to provide a comprehensive andupdated overview of the most recent advances i n this field and suggest further lines of investigation. As an interdisciplinary approach is necessary to study such ;I complex matter. thebookpresents a goodopportunity to summarizeinformationconcerning agronomy, soil science, plant nutrition, plant physiology. microbiology, and biochemistry. The bookis therefore intended for advanced students, and researchers in agricultural,biological,andenvironmentalsciencesinterested i n deepening their knowledge of' the subject and/or developing new experimental approaches in their specific field of interest. The tirst chapter defines the spatial and functional features of the rhizosphere, which make this environment the primary site of interaction between soil, plant, and microorganisms. Among the multitude of organic compounds present in the rhizosphere those released by plant roots are the most important from a qualitative and quantitative point of view; furthermore, the relationships with soil components of any released compound need to be considered (Chapter 2). The release of these compounds strongly depends on the physiological status of the plants and is related to the ability of plantrootstomodifytherhizosphere in order to cope with unfavorable stress-reducing conditions. These aspects are disiii
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Preface
cussed in Chapter 3, with particular emphasis on water, physical, and nutritional stresses. A thorough analysis of how root exudates may influence the dynamics of microbial populations at the rhizosphere is provided in Chapter 4. However, the importance of the role played by biologically active substances produced by microbial populations cannot be underrated, and the organic compounds acting as signals between plants and microorganisms must be identified and characterized (Chapter 7). In this context the biochemistry of the associations between mycorrhizae and plants (Chapter 9) and the interaction between rhizohin and the host plant (Chapter IO) is also considered. It has long been recognized that both roots and microorganisms compete for ironattherhizosphere;a wealth of literatureisalreadyavailableonthis subject and many studieson the productionof siderophores by microbes are being carried out. Their potential use by plants and their relationship to other plantborne iron-chelating substances are still a matter of debate (Chapter 8). The fulfillment of the nutritional requirementof plants and microorganismsalso depends on the processes leading to mineralization and humification of organic residues (Chapter 6). The presenceof humic and fulvic substances can have a considerable effectonroothabitability,plantgrowth,andmineralnutrition(Chapter S). Knowledge of these aspects needs to be reconsidered at the rhizosphere (Chapters S and 6). The developmentof specific models can shed light on the events taking place at the rhizosphere (Chapter 1 l ) . Validation of the models and a betterunderstanding of these phenomena may come from the correct use and development of new experimental approaches (Chapter 12). We realize that the information in the book is still largely descriptive and that the interdisciplinary view of the causal relationships in the rhizosphere is still in its infancy. Nevertheless, we do hope that our efforts and the high-quality scientific contributionswill stimulate further interestin and work on this fascinating topic. Roberto Pitltotl Zerm V ~ m r l i r l i Pmlo Nmnipieri
Contents
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111
v rr
I.
The Rhizosphere as a Site of Biochemical Interactions Among Soil Components, Plants, and Microorganisms Roberto Pinton, Zerw V u r m i n i S Pm10 Nrrnnipieri
1
2 . Types, Amounts, and Possible Functions of Compounds Released into the Rhizosphere by Soil-Grown Plants
19
Nicholas C. Urerz
3. The Release of Root Exudates as Affected by the Plant’s Physiological Status Giirlter Neurnann and Volker Riirnheld
41
4. The Effect of Root Exudates on Rhizosphere Microbial Populations
9s
Melissu J . Brirnecornbe, Frms A. De Leij, c r r d J m x s M .
Lyr1ch
S.
Direct Versus Indirect Effects of Soil Humic Substances on Plant Growth and Nutrition Zerw Vnrclniwi and Roberto Pinton
6. Mineralization and Immobilization in the Rhizosphere Luigi Bndalucco m d Peter J . Kuikrnnrz
141
159 V
Contents
vi 7.
Organic Signals Between Plants and Microorganisms
197
Dietrich Wert1er
8. Function of Siderophores i n the Plant Rhizosphere D m i d Crorvlq 9.
10.
223
Mycorrhizal Fungi: A Fungal Comtnunity at the Interface Between Soil and Roots Frrrt1ci.y M . Mrrrtitl, Sihirr Pi.rotto, m d Pnolcr Bonfirtlte
263
Functional Ecology of the Rhizobium-Legume Symbiosis
297
Atdrcw S p u r t i t t i
I I.
Modeling the Rhizosphere PettJr R. Dcrrrtrh r r m l Tiitla Roose
12. Methodological Approaches to the Study of Rhizosphere Carbon Flow and Microbial Population D y n m i c s J . A I m W. Morgcrw m d JoIztt M . WI1ipp.s
327
373
41 l
Contributors
Luigi Badalucco Dipartimento di IngegneriaeTecnologieAgro-Forestali. Universit; di Palernlo, Palernlo, Italy
Paola Bonfante Dipartimento di Biologia Vegetale and Centro Micologia del Terreno-CNR, University o f Torino, Torino, Italy Melissa J. Brimecombe School of Biological Sciences, University of-Surrey, Guildford, Surrey, England
David Crowley
Department of Environmental Science, University of California-Riverside, Riverside, California
Peter R. Darrah Department of Plant Sciences, University of Oxford, Oxford, England
Frans A. De Leij School of BiologicalSciences,University
of Surrey,
Guildford, Surrey, England
Peter J. Kuikman Department of Water and the Environment, Alterra-Green World Research, Wageningen, The Nctherlands James M. Lynch
School of Biological Sciences, University Guildford, Surrey, England
of Surrey,
Francis M. Martin Department of Forest Microbiology.INRACenter
of
Nancy, Champenoux, France vii
viii
Contributors
J. Alun W. Morgan
Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick, England
Paolo Nannipieri DipartimentodiScienzaSuoloeNutrizionedellaPlanta, University of Florence, Florence, Italy Gunter Neumann InstitutfurPflanzenerniihrung,Universitat
Hohenheim,
Stuttgart, Germany
Silvia Perotto Dipartimento Biologia Vegetale and Centro Micologia del Terreno-CNR, University of Torino, Torino, Italy
Roberto Pinton DipartimentodiProduzioneVegetaleeTecnologieAgrarie, University of Udine, Udine, Italy Volker Romheld Institut fur Pflanzenerniihrung, Universitiit Hohenheim, Stuttgart. Germany
Tiina Roose Centre for Industrial and Applied Mathematics, University of oxford, Oxford, England
Andrea Squartini DipartimentodiBiotecnologieAgrarie,Universitidegli Studi di Padova, Padova, Italy
Nicholas C. Uren Department of Agricultural Sciences, La Trobe University, Bundoora, Victoria, Australia
Zen0Varanini DipartimentodiProduzioneVegetaleeTecnologieAgrarie, University of Udine. Udine, Italy Dietrich Werner Department of Biology, Philipps-Universitat Marburg, Marburg, Germany
John M. Whipps Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick, England
The Rhizosphere
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The Rhizosphere as a Site of Biochemical Interactions Among Soil Components, Plants, and Microorganisms Roberto Pinton and Zen0 Varanini University of Udine, Udine, Italy Paolo Nannipieri University of Florence, Florence, ltaly
1.
INTRODUCTION
Plant survival and crop productivity are strictly dependent on the capability of plants to adapt to different environments. This adaptation istheresult of the interaction among roots and biotic and abiotic components of soil. Processes at the basis of the root-soil interaction concern a very limited area surrounding the root tissue. In this particular environment, exchanges of energy, nutrients, and molecular signals take place, rendering the chemistry, biochemistry, and biology of this environment different from the bulk soil. The literatureon rhizosphere is extensive, as testimonied by several reviews and entire books devoted to the topic (l-S). Literature has been focused mainly on the beneficial (infections by mycorrhiza and N ? fixing microorganisms) and detrimental (infections by plant pathogens) microbial-root interactions, the role of rhizosphere in plant nutrition, the carbon economy of the rhizosphere, and the faunal-microbial interactions. In the last few decades, information has grownon the complex biochemical interactions in therhizosphere,althoughacomplete integrated view coming from interdisciplinary approaches has not yet been at-
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tained. Therefore, this book intends to discuss the most recent findings with the relative technical developments of the above-mentioned topics.
II. THE COMPLEXITY OF THE RHIZOSPHERE It is well known that microbial metabolites can accumulate in the rhizosphere. These compounds, which include plant growth regulators, phytotoxins, antibiotics, and soil stabilizers, can affect the microbial activity and composition of microflora in the rhizosphere, as well as the activity of plant roots (6). Other compounds, such as enzymes, siderophores. and molecular signals, producedby both microorganisms and plants, can also affect the complex ecological interactions and the activity of plant roots and organisms of the rhizosphere. The functions of the compounds affecting the activity of specific microorganisms have been generally studied in axenic cultures, whereas those effective on plant physiology have been investigated in simplified systems, such as hydroponic cultures with single plant species. The environmental situation of the rhizosphere is much different. For this reason, this book discusses the distribution of these compounds in the rhizosphere. In particular, it is considered how this distribution changes along the root system (Chap.2) or by increasing the distance from the rhizoplane (Chap. 6) and how it is affected by microbial degradation or adsorption by soil colloids. In Chap. 2, Uren underlines that the “right conditions” must occur for biological activity of any compound in the rhizosphere. For example. the presence of colloidaladsorbingsurfaces,suchasclaysurfaces, intherhizospherecan markedly decrease the concentration of these substances to very low valuesmaking them ineffective. The physiological behavior of the plant can also be affected by compounds peculiar of the soil systems such as humic molecules. In Chap. 5, both direct and indirect effects of soil humic substances on plant nutrition are extensively discussed. According to Vaughan et al. (7), humic molecules, particularly those of low-molecular size, can positively affect plant growth and nutrition, through interactions with mechanisms of nutrient uptake and metabolic pathways. These effects are potential because the humic acids must be in solution. Most of these studies, however, have been carried out using humic acids prepared after extracting humic substances from soilby alkaline solutions. Needless to say, strong alkaline conditions (pH values higher than 12) do not occur in soil except for a few microsites of sodic soils (Chap. 5). Water-soluble humic substances reflect more the real conditions than humic substances solubilized in the classical way (8). In addition, plant cells have been shown to take up humic molecules (9). It has been suggested that organic acids, like those excreted from roots, could lead to the dissociation of the humic macrostructure and to the release of the more biologically active low-molecular-size humic fractions ( I O , I 1).
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The type and amounts of compounds released by roots in the rhizosphere are discussed in Chap. 2, whereas their influence on the soil microbiota and microbialprocessesresponsibleforthemineralizationofnativeorganicmatter, which withhold a considerable amount of nitrogen, phosphorus and sulphur in soil, are reviewed i n Chap. 6. Chap. 12 deals with the methodology for a better quantification of the carbon released by the plant, as well as for a more accurate measurement of microbial diversity in soil. Compounds released by roots and soil microbes can act as micronutrient-mobilizing substances. For example, siderophores are iron-chelating compounds secreted by microorganisms and graminaceous plants in response to iron deficiencies. Chap. 8 discusses the factors controllingsiderophoreproduction,aswell as the other compounds chelating iron in the rhizosphere. These compounds mediate competition for iron in the rhizosphere and may have a role in disease biocontrol. The possibility exists for plants to use microbial siderophores. Rhizodeposition includes lysates liberated by autolysis of sloughed cells and tissues, as well as root exudates released passively (diffusates) or actively (secretions) from intact cells. However. the process of exudation is poorly known from a physiological and molecular point of view (Chap. 3); active resorption of exudates byplantrootshasbeenshown(seeChaps. 3 and 1 l ) . Thus,the organic C released from root in the extracellular soil environment might be the result of the gross exudation rate minus the gross resorption rate. The effect of the physiological status of the plant on the release of exudates is discussed in Chap. 3. From 30 to 60% of the photosynthetically fixed carbon can be translocated to roots, and significant proportion is released into rhizosphere-in quantities depending on factors such as light intensity, temperature, nutritional status of the plant, stress factors, mechanical impedance, soil type, plant species, plant age, and microbiological activity of the rhizosphere (Chap. 3). Some of these factors are interrelated; for example, the effect of plant age can be explained by the fact that the root growth decreases with the plant age: the higher the root growth, the greater the amount of released root exudates (12). As much as 40% of the fixed C can be lost through rhizodeposition (13). Less studied is the N rhizodeposition, which can account for 20% of the total plant N (Chap. 4). Chapters 7 and 9 discuss specific exchange of molecular signals (the socalled “molecular cross talk”) betweenbeneficial microorganisms, such as rhizobia and mycorrhizas, and their host plants. Molecular cross talk seems to be a prerequisite mechanism for most of the plant infection by soil microorganisms (14). Only for a few microbial infections, however, the sequence and type of molecular signals involved have been characterized. Thus, there is the need for further studies to elucidate the unknown molecular cross talk between the most common rhizobacteria and fungi and the plant roots; it is also needed to better understand how molecular cross talk responds to the changing environmental conditions. The potential applications of these studies are important because the
Pinton et al.
4
manipulation of molecular cross talk could protect crops from damage caused by pathogens, or improve beneficialinfections to optimizenutrientuptake by plants.
111.
DEFINITION AND TERMINOLOGY
The term rhi,-.ospherr (from the Greek, meaning the influence of a root on its surrounding) was first used by Hiltner (IS) to indicate the zone of soil where root exudates released from plant roots can stimulate, inhibit. or have no effect on activitiesofsoilmicroorganisms. To be moreprecise,thesoillayersurrounding roots should be termed as Pctorhizo.sl)iler-e, whereas the root layer colonized or potentially colonizable by microorganisms should be indicated by onclo~i~i,7o.sl)her-. The two areas are separated by the root surface (rhi,-.opImc).The term r n ~ c o r r - l ~ i ~ o . s ~is~ Iused ~ c r eto indicate the soil surrounding a root infected by a mycorrhizal fungi. In this book the term rhizosphere is used to indicate the ec.?orhi,70sl~herc., as it normally does in the relative bibliography. Of the complex plant-microbe relationships, the positive effect on heterotrophical soil microorganisms, due to utilization of organic compounds released from plant roots, has been generally the most considered (Chap.4). The presence of available carbon can stimulate microbial growth: microbial cell numbers can reach 10"- 1 0 ' ? per gram of soil in the rhizosphere, whereas invertebrate number can be twice a s high in the rhizosphere than in the bulk soil (13,16). If the effect on either microbial number or changes in microbial diversity have been detnonstrated, i t has been more difficult to give experimental evidences for the rhizosphere effectin terms of carbon andnutrient flows within the rhizosphere environment (1 7).
IV. SOIL ENVIRONMENT,BOUNDARIES, AND MICROBIAL DIVERSITY The rhizosphere lacks physically precise delimitation( 1 8). The volume of rhizosphere depends on the rate of exudation and impact utilization of rhizodeposits (Chap. 6). The spatial and temporal distribution of exudates as well as their metabolism is related to the concentration of COz (Chap. 6). However, according to Darrah (Chap. I l ) , the layer of soil where microbial growth is affected by exudates can be 1-2 mm wide. Organic cornpounds released from plant roots have been categorized according to: (a) their chemical properties, such as stability (e.g., hydrolysis and oxidation), volatility, molecular weight, solubilityin water, etc. (Chap.2): (h) the modality of their release (exudates, secreted, or lysates): (c) the way of utilization
Biochemical
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by microorganisms; and (d) their function (phytohormones, ectoenzynies, phytoalexins, etc.). Low-molecular-weight compounds are readily assimilated by microbial biomass, whereas polymeric substrates such as proteins, nucleic acids, polysaccharides, etc., are first hydrolyzedby extracellular enzymes released from microorganisms and then the hydrolase-producing microorganisms can take up the monomers (see Chap. 4). The classification of rhizodeposition according to the modality of the release is difficult because processes involved in the release are not completely understood. On the other hand, the classificationbased on the exudate utilization by microorganisms is generally preferred because it’s more relevant to the microbial ecology the rhizosphere (Chap.4). As mentioned above, however, nlany processes can affect the fate of a metabolite in soil. Thus, the hydrolysis of more complex substrates by microbial extracellular hydrolases in soil can be affected by the interactions of substrates and enzymes with soil particles. As discussed by Burns ( 19) and Nannipieri et al.(20), the enzyme-producing microorganism will not always use the product of the reaction catalyzed by the released extracellular enzyme. Indeed, the reaction products can be adsorbed by soil colloids. I n addition, the enzyme-producing nlicroorganisnl must compete with opportunistic microorganisms that can utilize the reaction product or release proteases degrading extracellular hydrolases. Furthermore, microenvironmer~tal conditions (pH, temperature, moisture, etc.) surrounding the substrates may not be favorable to the enzyme reaction. It must be stressed that activity of enzymes in the rhizosphere requires the right set of environmental conditions, as does the activity of all biological compounds (Chap. 2). It is well established that enzyme activities in the rhizosphere are generally higher than in the bulk soil (19,21,22). Both beneficial and detrimental microbial infections occur becauseplant tissue maceration is carried out through the activity o f several cell wall-degrading exoenzymes releasedby the infecting microorganisms. Enzymes catalyzing the same reaction can be present in living plant and microbial cells, associated to cell debris or dead cells, adsorbed by clay particles or englobed by organic molecules (19-22). Thus. the examination of the soil ultrastructure by using a combination of histochemical and electron microscopic techniques, showed that phosphatase activity was prescntin intact plant cell walls and plant cell-wall fragments, intact microbial cells, and was also associated to amorphous organic Inatter (21). It was also shown that enzyme activity persisted for about 1 year within decomposing plant tissues. Root heterogeneitywithin m ; individualrootsystemmightaffectrhizosphere microbial population (23). Exudation is not uniformly distributed along the whole root system (Chaps. 4 and 1 1). The longitudinal gradient of exudation may reflect the gradient in microbial catabolismof root exudates as well as differences in root cell activity. Usually, apical root zones are characterized by a higher capacity for releaseof low-molecular-weight exudates, whereasbasal parts of the root system generally show higher microbial activity. The gradients of microbial
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population and exudates along the root axis can have important implications for the uptake of nutrients by plants; for example, the efficiency of root exudates released in response to nutrient deficiency can be much lower where microbial activity is higher. In the case of phytosiderophores (PS) it has been shown that graminaceous plants are able to cope with this cornpetion by releasing PS from root zones where microbial activity is lower, confining the exudation to a short period (Ramheld, 1991). Model calculations of effectivity of root exudates in nutrient acquisition have to consider the spatial separation of root exudation and microbial activity (see Chap. I l ) . As mentioned before and in Chaps. 4 and 6, the concentration of rhizodepositiondecreasesasthedistancefrom therhizoplaneincreases,whereasthe opposite generally occurs for the concentration of any plant nutrient in soil. In this context, the role of rhizospheric soil, rather thanthat of thebulk soil, is crucial for plant nutrition. It has alsoto be consideredthat very different situations of nutrient (24) and the nutritional status of can occur depending on the type plants (see Chap.3); furthermore, different portionsof the root system are characterized by differential nutrient-specific rates of uptake (25). All the above statements point to the necessity of reconsidering the concept of plant nutrient availability giving more importance to the situation occurring in the soil surrounding the root. Due to differences in rhizodeposition, different plant species but also different parts of the root system of the same plant, may have distinctive rhizosphere microfloras. Carbon source utilization tests based on the reduction of tetrazolium dye have been used to characterize microbial communities of terrestrial ecosystems. This approach, carried out by Biolog plates (Chap. 12), discriminated microbial communities from the same soil when sampled around different plant roots (26). No differences were observed between two different soils. The plant effect was mainly associated to the utilization profiles of carbohydrates, carboxylic acids, and amino acids, suggesting that plants may differ in the exudation of these compounds. It was suggested that the results probably reflected the Pseudormna.s carbon utilization profiles (26). Indeed the main disadvantage of the Biolog technique is to be culture dependent. In addition to Pseuclr~nzonns,Flavobac.teriurn, Alcaligenes,and A g r o h m t e r i u m species have been shown to be particularly stimulated in the rhizosphere due to the presenceof root exudates and lysates ( I p ) . The application of the PCR-DGGE technique by using universal primers for eubacteria on I-cm root segments sampled from different locations on ironstressed and nonstressed barley plants showed that microbial diversity (species richness and species evenness) was greater on the older than younger root parts (Chap. 8). Main bands species were analysed to assess the microbial species; Nitrosococcus was present in the older root parts, probably because nitrifying activity occurred in the oligotrophic environment of the mature rhizosphere. The cellulose degrader, Aureohacferium, was present in sites of lateral root emergence
Biochemical
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because in this location the sloughing of the root cortex tissues and damage to root cortical cells increase the amount of available cellulose. Both 'H-thymidineincorporationandradiolabeledleucineincorporation techniques have been recently used to determine bacterial activity and growth in the rhizosphere of barley seedling (28). Bacteria were initially released from the rhizosphere using homogenization and centrifugation before adding the labeled substrates. The cell incorporation rate was twice as high in the rhizosphere than in bulk soil. In addition, both the leucine and thymidine incorporation rates increased with the distances from the root tip (28).
V.
ROOT-MICROBES AND MICROBES-MICROBES MOLECULAR SIGNALS
As already mentioned, molecular cross talk seems to be the prerequisite mechanism for most of root microbial infections. Indeed the initial step of any root colonization involves the movement of microbes to the plant root surface; bacterial movement can be passive, via soil water flux,or active, via specific induction of flagellar activity by plant released compounds (chemotaxis) (Chaps. 4 and 7). Other important steps are adsorption and anchoring to the root surface. of If the rhizodeposition can affect, as previously stated, the composition rhizosphere microflora, microbial metabolites can also affect the rhizodeposition and the effects of microbes are species specific; metabolites from Arthrnhncter did not stimulate root exudation whereas did metabolites produced by Pseudomot1cr.s aenrgit?o.sn (29). Biological control of plant pathogens by rhizobacteria can be based on the productionof bacterial metabolites such as siderophores, antibiotics, and hydrogen cyanide (Chap. 4). In some cases, rhizobacteria promote plant growth due to the production of plant growth regulators like auxin derivatives (Chap. 4). The production of indolacetic acid by P.seudornoncr.s jl~rore.sce~~s M. 3. I . was increased when the rhizobacteria was grown in a medium containing maize root exudates (30). The molecular cross talk between the plant root and a specific microorganism or between two specific microorganisms depends on a continuous exchange of diffusable signal molecules which, once recognized by specific receptors, elict transductional processes, leading to a rapid activation of gene expression. Signals and receptor molecules involved in the cross talk between the specific plant and the specific microorganism have been detected. They concern the R/zi:ohiutn legume symbiosis (31, Chaps. 7 and IO), mycorrhizal infection (14. Chap. 9), the pathogenic microorganisms as inducers of plant defence response, the nonpathogenic organisms as inducers of plant defense responses and the Agrohcrcteriutnplant cell DNA transport. The most studied molecular cross talkisthat betweenrhizobiaandthe
Pinton et al.
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, leguminousandnonleguminoushostplants (3 I , Chap. 7). Nodulation isa multistep process that involves specific bacterial gene expression. The process starts with multiplication of bacteria in the rhizosphere, followed by chemotaxis to plant exudates, adhesion of rhizobia to the root, and infection. The initiation of the process is based on the mutual exchange of molecular signals between the bacterium and thehost plant; this molecular communication is notyet completely known. The expression of nodulation genes (rzocl) in the bacteria is induced by alfalfa (Medicago sativa) exudates, such as the flavonoid luteolin (32). The nod expression was shown to require the m d D gene product that occurs after the flavonoidhasinteracted with the rzodD gene. The Havonoid-NodD complexes activate the transcription of rhizobia1 nodulation ( r l o d ) genes and represent a level of specificity, since NodD proteins vary in their ability to recognize different flavonoid molecules from different legume species. Enzymes encodedby the /IUC/ genes are responsible for the production and secretion of a family of lipo-chitinoligosaccharides (LCOs), signalling molecules called Nod factors, which initiate nodule formation (31). Nod factors have also been suggested to be involved in the inhibition of salicyclic acid-mediated defense mechanisms in legume (3 1 ); this could explain why rhizobia prevent the triggct-ing of the host defense response. Among the environmental parameters,high temperature, low soil moisture, pH, P content, and toxic elements, such as AI, negatively affect nodulation and the exchange of molecular signals in tropical soils (31). The decrease in nodulation due to either temperature or pH stress was almost completely annulled in bean and soybean rootsby adding the r m l gene-inducer isoHavone genistein ( 3 l ) . This r z o d gene-inducer positively affected nodulation in bean and soybean when added (40 PM) to seeds grown in an oxisol (dark red latosol) with a Btwfwhizoh i l r r r z and Rhi:obiur?l population of IO4 and lo6 cell g of soil, respectively (31). A successful competitioncan also dependon the abilityof the rhizobium to utilize specific compounds of plant exudates as nutrient sources. Mimosine, a nonproteinogenicaminoacid present in largequantities in the leguminoustreesand shrubs of the genus Leucmwa, provides a nodulation competive advantage to the tnimosine degrading Rhizohiunl strains (33). According to Hungria and Stacey (3 I ) more than 4000 flavonoids have been identified within the plant kingdom and someof them have been recognized ;IS1 1 0 d gene-inducers (Table l ) . In addition, root Havonoids have been suggested as molecular signals for the initiation and development of mycorrhizal infection (Chaps. 7 and 9). It is well known that survival and proliferation of microbial cells in the environment depend on the expression of advantageous phenotypes controlled by the genotype expression. It is becoming clear that such evolutionary pressure that transduce cnvironmental has resulted in a network of sensor mechanisms stimuli into gene expression and hence a phenotype complementary to the prevail-
'
Biochemical Interactions 9
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ing environmental conditions. Often these stimuli are mediated by molecular signalling. Thus, the so-called “Quorum sensing” involves the extracellular accumulation of a low-molecular-weight pheromone; this allows individual cells to sense when the minimum population unit or quorum of bacteria has been achieved for a concerted population response to be initiated (34). The best studied example of quorum sensing is the regulation of bioluminescence in PhotoBrrctrrilrrlI jischrri. The bacterium is dark when present at lowcelldensity,and it emits blue-green light when it reaches a certain concentration, as it occurs i n the gut of some fish species. In this case the bacterial species provides the host fish with a source of light, which can act as a mean of communication, attraction or defense, and in return the bioluminescent bacteria receive a suitable habitat. When the cell density is low, two regulators, genes lux1 and lux-!?, are transcribed at low level and there is insufficient accumulation of the pheromone signal N-(3-oxo)hexanoyl-L-homoserine lactone (OHHL) to elicitlux!?-dependenttranscription of the luxCDABE operon for visible bioluminescence. As the bacterial population increases, the level of OHHL reaches a critical level (34). Then, an OHHL-lu.x-R complex is formed and this probably activates transcription of the lrrxCDABE operonandthusthebioluminescence.SeveralN-acyl-L-homoserinelactones (AHLs), acting as molecular signals, have been isolated; they differ for the chain length and the nature of the substituent (Fig. l ) . These variations determine the specific biological property of the AHL (35). The AHL signals are involved in various phenotype expressions including synthesis and release of extracellular enzymes or antibiotics, conjugation, etc. (35). The synthesis of plant cell-wall degradingexoenzymesandthecarbapenemantibiotics by aplant pathogen Erwinio ccrrotovorn is controlled by the population density through a quorum sensing mechanisms; the pheromone involved in this case is the N-(3-oxo)-hexanoyl-L-homoserine lactone (OHHL) (36). The activities of plant wall-degrading enzymes synthesized and releasedby E. ccrrotovortr are responsible for the maceration of plant tissues and the liberation of nutrients; this release is at the basis for the phytopathogenicity of E. curotovorcr (37). The timing of exoenzyme production by E. car(mwwo is tightly regulatedso as to evade and overcome defense reactions from the planthost. In addition to the quorum component, a few components of the plant extracts can also induce exoenzyme biosynthesis (38). Specific components of the plant extracts canalso coactivatethe carbopenem biosynthesis. The coordinated productionof carbopenem antibiotics by E. carotovorn is essential to eliminate potential microbial competitors for the released plant nutrients
(36). Any bacterial species living in a mixed microbial population, such as that of the rhizosphere, may encounter not only the molecular signal produced by a cell of the same species but also molecular signals produced by cells of different species. The situation is made more complex by the presence of plant molecular signals, and by the fact that the same AHL molecule can be used to regulate the
11
Biochemical Interactions
II CH2 CH2CH,
I
H
II
0
N-(3-oxohexanoyl)-L-hornoserinelactone (OHHL) Photobacteriwnjscheri bioluminescence
Erwinia carotovora synthesis of carbopenem and exoenzymes
H
0
N-(3-oxooctanoyl)-L-homoserine lactone (OOHL) Agrobacterium tumefaciens conjugation
N-(3-oxododecanoyl)-L-hornoserine lactone (ODHL) Pseudomonas aeruginosa synthesis of exoenzymes and exotoxins
Figure 1 N-acyl homoserinelactonenucleotidesproducedby and their phenotype function (From Refs. 34 and 35).
some bacterial species
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expression of different biological processes in different bacterial species ( 3 5 ) . On the contrary, some bacterial species can produce multiple AHLs, each having different effects on the phenotype. In soil, the situation is even more complex because the molecularsignals caninteractwithsurfaceactivesoilparticles through electrostatic interactions, hydrogen, and van der Waals bonds. In addition. molecular signals with anionic groups can be adsorbed by inorganic soil colloids by aligand exchange mechanism, unless steric hinderance avoids the contact of the anions groupswith the exchangeable sites. Adenosine triphosphate (ATP)andadenosinediphosphate(ADP) butnot adenosinemonophosphate (AMP) were strongly adsorbed by clay minerals (39). It was suggested that the bulky adenosine group of AMP affected the anion exchangeby sterically hindering close contacts between exchangeable sites andthe phosphate groups. The sterical hindrance did not occur in ADP and ATP molecules because the terminal phosphategroupexchangingtheOHgroupswasfurther fromthe adenosine group. In conclusion. the behaviorof the molecular signalscan be markedly different in soil with respect to that observed in microcosm experiments involving only the host plant and the infecting microorganism or a mixed microbial population, both without soil particles. Studies are needed to compare the diffusion of molecular signals in the presence of clay and/or humic barriers.
VI.
ROOT SENSING OF ENVIRONMENTAL SIGNALS
Itis well known that chemical composition of rhizosphere solution can affcct plant growth. Particularly, uptake of nutrients may be considerably influenced by the ionic concentration of the rhizosphere solution (40). Despite the difficulty of defining the exact concentration of ions in the rhizosphere surrounding each root (or even root portion), it has been unequivocally demonstrated that plants have evolved mechanisms to cope withthe uneven distribution of ions in the root surrounding in order to provide adequate supply o f each essential nutrient (4 1 ). These mechanisms include expression of transporter genes in specific root zones or cells and synthesis of enzymes involved in the uptake and assimilation of nutrients (40,43). Interestingly, it has been shown that specific isoforrns of the H'-ATPase are expressed in the plasma membrane of cell roots; it has been proposed that the expression of specific isoforms in specific tissues is relevant to nutrient (nitrate) acquisition (44) and salt tolerance (45). These plant responses are largely controlled by the internal status of plant (40). On the other hand, it has been shown that the occurrence of nutrient-rich patches in the soil can trigger changesin root architecture and also in the capacity for nutrient acquisition (46,47). Recent results indicate that this behavior is dependent not only on internal (metabolic) signals but also on the capacity t o sense
Biochemical
13
the external nutrient. Zhang and Forde (48) demonstrated that roots of ArtrDit l o p s i s can detect local nitrate inducing root proliferation. The response involves the rapid specific activation of a gene with homologies to the MADS-box tranto know the molecularmechascriptionfactor;however,studiesarerequired nism(s) of nitrate-sensing in higher plants. Togetherwith sensing systems devoted to the maintenance of nitrate homeostasis within the root cell, it is reasonable to envision the presence of a mechanism able to monitor incoming nitrate. Consequently it can be hypothesized the existence of a finely tuned adjustment of root structure and physiology to environmental signal(s) coming from the rhizosphere. Sensing nutrients in therhizospheremaybefurthercomplicatedbythe presence of microorganisms. It is well known that mycorrhizal fungi can increase Pi uptake in infected plants by extending the Pi depletion area around the root and reaching P sources unaccessible to plant roots (49). The efficiency of these processes would conceivably rely on how fungal and plant processes are integrated to provide a soil-to-fungus-to-plant pathway of Pi uptake. Recently it has been shown that (a) a H'-ATPase gene is upregulated during mycorrhizal colonization in barley (50);(h) a fungal Pi transporter gene (GvPT) is highly expressed in the external mycelium (where the fungus can absorb Pi from the soil) but not i n the fungal structure within the root (SI); (c) a high affinity Pi transporter gene (LePTI) is overexpressed in P-starved tomato plants, whereas it is restricted in mychorrizal plants (52); LePTl transcripts appeared to be localized in cortical cells containing arbuscles ( 5 2 ) . All the evidence support the idea that plant and fungal transporters may be organizedin a way that wouldpromote one-way transfer of Pi in the direction of the root (52).
VII. CONCLUSION Most research on the rhizosphere environment has been forcedly descriptive in the past. Recent advances show that organic compounds present in the rhizosphere can have a specific role in plant-micro-organism-soil interactions. Moreover, it starts to be elucidated:
1. Whyplantsreleaseexudates 2 . Whatenvironmentalfactorsaffectexudation 3. How the release is regulated and affected by changes in microbial activity and composition
4. How changes in soil microbiota affect the
root exudation
These processes and relationships, however, need to be further investigated. Signal molecules exchanged between plants and microorganisms have been identified that favor beneficial plant colonization. Some compounds present in
14
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soil, e.g., humic molecules, and nutrients can affect root growth and metabolism through stimulation or inhibition of biochemical reactions or processes of root cells and triggering of specific signal transduction pathways; however, molecular information on how the plant senses the environmental condition of the rhizosphere is still lacking. Molecular analysis of the interaction between plants, microbes, and soil components may help us understand the causal relationships of events taking place in the rhizosphere:Nevertheless, due to the necessity to simplify the experimental approaches, we still do not have the complete picture that takes into account the relative weight of each factor.
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Rhizosphere the Compounds into Released
39
plarlt Nrttritiorl Jhr Srlstaincthle Food Productiorl nrrd E~~viro~rnre~rt, (T, Ando, K. Fujita, T. Mae, H. Matsumoto, S. Mori, and J. Sekiya, eds.). Kluwer Academic Pub-
lishers.Dordrecht.1997.p.301. 80. P. Hinsinger, How do plant roots acquire mineral nutrients? Chemical processes in the rhizosphere. A ~ JAgmn. . 64:225 (1998). 81. E. P. Delhaize, P. R. Ryan, and P. J. Randall, Aluminium tolerance in wheat (Triticum {testilwnr L.). 11. Aluminium-stimulated excretion of malic acid from root apices. Plant Physiol. 103:695 ( I 993). as an alumin82. D. M. Pellet, D. L. Grunes, and L. V. Kochian, Organic acid exudation ium-tolcrance mechanism in maize (Zeo rnctys L.). Plorrtct 196:788 (1995). 83. W. J. Horst, The role of the apoplast in aluminium toxicity and resistance of higher plants: a review. Z. Pjun:ewrnuhr. Bodcrlk. 158:419 (1995). 84. D. L. Jones, A. M. Prabowo. and L. V. Kochian, Kinetics of malate transport and decomposition in acid soils and isolated bacterial populations: the effect of microorganisms on root exudation of malate under AI stress. Plarrt Soil 182:239 (1996). 85. D. M.Pellet, L. A. Papernik, D. L. Jones, P.R. Darrah, D. L. Grunes. and L. V. Kochian, Involvement of multiple aluminium exclusion mechanisms in aluminium tolerance in wheat. P l m t Soil 192:63 (1997). 86. A. Huang and R. D. Graham, Efficient Mn uptake in barleyis a constitutive system. Plant Nutritiolr j?)r Sustctir~ctbleFood Prodrtctiou curd E ~ ~ ~ i r o r r r n(T, r r ~Ando, t K. Fujita, T. Mae, H. Matsumoto, S. Mori, and J. Sekiya. eds.), Kluwer Academic Publishers, Dordrecht. 1997, p. 269. 87. K. Venkatraju and H. Marschner, Inhibition of iron-stress reactions in sunflower by bicarbonate, Z. Pfkrrr:rnerniihr. Borlerrk. 144:339 (198 1). 88. E. Bar-Ness. Y. Hadar. Y. Chcn, V. Riimheld, and H. Marschner, Short-term effects of rhizosphere microorganisms on Fe uptake from microbial sidcrophoresby maize and oats. Plnrzt Physiol. 100:451 (1992). 89. N. von Wiren, S. Mori, H. Marschner, and V. Romheld, Iron inefficiency in maize mutant ysl (Zea nmqs L. CV.Yellow-stripe) is caused by a defect in uptake of iron phytosiderophores. Plwrt Phy.siol. 106:71 (1994). 90. N.vonWiren, V. Riimheld, T. Shioiri,and H. Marschner,Competitionbetween micro-organisms and roots of barley and sorghum for iron accumulated in the root apoplasm. Now Phytol. 13051 1 (1995). 91 G. W. Leeper, Relationship of soils to manganese deficiency of plants. Ncrtrrre 134: 972 ( 1934). 92. N.C . Urcn, Chemical reduction of an insoluble higher oxideof manganese by plant roots. J. P h t Nrrtr. 4 6 5 ( 198I ). 93. P. de Willigcn and M. van Noordwjik, Mathematical models on diffusion of oxygen to and within plant roots, with special emphasis on effects of soil-root contact: I. Platrt Soil 77215 ( 1984). 94. M. van Noordwijk and P.de Willigen, Mathematical models on diffusion of oxygen to and within plant roots, with special emphasis on effects of soil-root contact: 11. Plmrlt Soil 77:233 (1984). 95. N. C. Uren. Mucilage secretion and its interaction with soil, and contact reduction. Plum ard Soil 15.5/156:79 (1993).
40
Uren
96.
K. H. Park. K. Moody, S. C. Kim, and K. U. Kim, Allelopathic activity and determination of allelochemicals from sunflower (Helictrrthm mzt1uu.Y L.) root exudates. 11. Elucidation of allelochemicals from sunflower exudates. Koretrrz J. Weed Sci. 12:
97.
173 (1992). W. A. Ayers and R. H. Thornton, Exudation of amino acids by intact and damaged roots of wheat and peas. Plcurt Soil 28:193 ( 1 968).
3 The Release of Root Exudates as Affected by the Plant’s Physiological Status Gunter Neumann and Volker Romheld Universitat Hohenheim, Stuttgart, Germany
1.
INTRODUCTION
Apart from the function of plant roots as organs for nutrient uptake, roots are also able to release a wide range of organic and inorganic compounds into the root environment. Soil-chemical changes related to the presence of these compounds and products of their microbial turnover are important factors affecting microbial populations, availability of nutrients, solubility of toxic elements in the rhizosphere, and thereby the ability of plants to cope with adverse soil-chemical conditions (1). Organic rhizodeposition includes lysates, liberated by autolysis of sloughed-off cells and tissues, as well as root exudates, released passively (diffusates)oractively(secretions)fromintactrootcells(Table 1; seealso Chap. 2). In annual plant species, 30-60% of the photosynthetically fixed carbon is translocated to the roots, and a considerable proportion of this carbon (up to 70%) can be released into the rhizosphere (2,3), as pointed outin Chaps. 4,6, and 12. This rhizodeposition is affected by multiple factors, such as light intensity, temperature, nutritional statusof the plants, activityof retrieval mechanisms,various stress factors, mechanical impedance, sorption characteristics of the growth medium, and microbial activity in the rhizosphere. This chapter focuses on the release of water-soluble organic root exudates and highlights effects of the physiological status on root exudation and its significance for adaptations to adverse soil conditions and nutrient efficiency. Since the methods employed for collection and analysis of root exudates play an important role in the qualitative and quanti41
poundsof
Neumann and Romheld
42
Table 1 RootExudatesDetectedinHigherPlants ~~
Class Sugars Amino acids and amides
Aliphatic acids
Aromatic acids Miscellaneous phenolics Fatty acids Sterols Enzymes
Micellaneous
Arabinose, glucose, fructose, galactose, maltose, raffinose, rhamnose, ribose, sucrose, xylose All 20 proteinogenic amino acids, aminobutyric acid, homoserine, cysrathionine, mugineic acid phytosiderophores (mugineic acid, deoxymugineic acid, hydroxymugineic acid, epi-hydroxymugineic acid, avenic acid, distichonic acid A) Formic, acetic, butyric, popionic, malic. citric, isocitric. oxalic, fumaric. malonic, succinic, maleic, tartaric, oxaloacetic, pyruvic, oxoglutaric, maleic, glycolic, shikimic, cis-aconitic, trans-aconitic, valeric, gluconic p-Hydroxybenzoic, caffeic, p-coumaric, ferulic, gallic, gentisic, protocatechuic, salicylic, sinapic, syringic Flavonols, flavones, flavanones, anthocyanins, isoflavonoids Linoleic, linolenic, oleic, palmitic, stearic Campestrol, cholesterol, sitosterol, stigmasterol Amylase, invertase, cellobiase. desoxyribonuclease, ribonuclease, acid phosphatase, phytase, pyrophosphatase apyrase, peroxidase, protease Vitamins, plant growth regulators (auxins, cytokinins, gibberellins), alkyl sultides, ethanol, H'. K' nitrate, phosphate, HCOl
Soltrcr: Adapted from Ref. 274.
tative interpretation of measured exudate data, methodological aspects are also discussed in an introductory section.
II. COLLECTION OF ROOT EXUDATESMETHODOLOGICAL ASPECTS
A.
Collection Techniques with Trap Solutions
Water-soluble root exudates are most frequently collected by immersion of root systems into aerated trap solutions for adefined time period (Fig. 1A). The technique is easy to perform and permits kinetic studies by repeated measurements over time usingthe same plants. While it is possible to getafirst impression in response about qualitative exudation patterns and even quantitative changes to different preculture conditions, the technique also includes several restrictions that should betakeninto account for theinterpretation of experimentaldata.
43
Release of Root Exudates -
........... ,
0.2
C
Figure 1 Techniques for collection of root exudates. (A) Solution culture system (282); root exudates collected from the whole root system by immersion into aerated trap solutions under sterile conditions (optional). (B) Plant culture in solid media (vermiculite, sand); root exudates collected from the whole root system by percolation of the culture vessels with trap solution (9,l l). (C) Localized root exudate sampling from plants grown in solution culture. Exudates collected into trap solution inside of sealed plastic rings agar, ion-exchange straddlingthe root (28) or by applicationof sorption media (filter paper, resins) onto the root surface (l7,45). (D)Localized collection of rhizosphere soil solution from plants grown in soil culture. Rhizoboxes with removable front lids for plant culture (root windows under fieldconditions). Collection by insertion of microsuctioncups (made from HPLC capillaries, 1 mm in diameter) connected to a vacuum collection device (50) or by application of sorption media (filter paper, agar, ion-exchange resins) onto the root surface (27.45).
Application should be restricted to plants grown in nutrient solution, since removal of root systems from solid media (soil, sand) is almost certainly associated with mechanical damage of root cells, resulting in overestimation of exudation rates. On the other hand, it has frequently been demonstrated that the mechanical impedance of solid growth media leads to alterations in root morphology and of the mechanstimulates root exudationIn liquid (43). culture media, simulation ical forces imposed on roots of soil-grown plants may be achieved by the addition of small glass beads (5-7). Alternatively, exudate collection from plants grown
44Romheld
and
Neumann
in solid media (sand, vermiculite) may be performed by percolating the culture vessels with the trap solution for a defined time period (Fig. IS) after removal of rhizosphere products accumulated during the preceding culture period by repeated washing steps (8-1 l ) . For this approach, however, recovery experiments and comparison with results obtained from experiments in liquid culture are essential, since sorptionof certain exudate compounds to the matrix of solid culture media cannot be excluded. As a modification of the percolation technique, cartridges filled with selective adsorption media (e.g., XAD resin for hydrophobic compounds, anion exchange resins for carboxylates). which are installed in the tube below theplant culture vessel, can be employed for the enrichment of distinct exudate constituents (12,13). After adsorption to a resin, exudate compounds are also protected against microbial degradation. Trap solutions employed for the collection of water-soluble root exudates are nutrient solutions of the same composition as the culture media (8,10,1 I ) solutions of 0.5-2.5 mM C a S 0 4or CaCI2to provide Calf for membrane stabilization (14) or simply distilled water (9,lS- 17). Since the osmotic strengthof nutrient solutions is generally low, short-term treatments ( 1 -2 h) even with distilled water are not likely to affect membrane permeability by osmotic stress. Accordingly, comparing exudation of amino acids from roots of Brcmiccl nnpus L. into nutrient solution, 20 mM KCI, or distilled water, respectively, revealed no differences during collection periods between 0.5 and 6 h (18). In contrast, Cakamak and Marschner ( 1 9) reported increased exudation of sugars and amino acids from roots of wheat and cotton during a collection period of 6 h when distilled water instead of 1 mM CaSOJ was applied as the trap solution.Thus, for longer collection periods or for repeated measurements, only complete or at least diluted nutrient solutions should be employed as trap solutions in order to avoid depletion of nutrients and excessive leaching of Ca?+.Long-term exposure of plant roots to external solutionsof very low ionic strength is also likely to increase exudation ratesdue to an increasedtransmembraneconcentrationgradient of solutes (20,2 I ). Exudate collection in trap solutions usually requires subsequent concentration steps (vacuum evaporation, lyophilization) due to the low concentration of exudate compounds. Depending on the composition of the trap solution, the reduction of sample volume can lead to high salt concentrations, which may interferewith subsequentanalysis or mayevencauseirreversibleprecipitation of certain exudate compounds (e.g., Ca-citrate, Ca-oxalate, proteins). Therefore, if possible, removal of interfering salts by use of ionexchange resins priorto sample concentration is recommended. Alternatively, solid-phase extraction techniques may be employed for enrichment of exudate compounds from the diluted trap solution ( I 1,22). High-molecular-weight compounds may be concentrated by precipitation with organic solvents [methanol, ethanol, acetone 80% (v/v) for polysaccharides and proteins] or acidification [trichloroacetic acid 10% (w/v), per-
Exudates Release of Root
45
chloric acid S% (w/v) for proteins] (23). Alternatively, ultrafiltration of the trap solutions or even cultivation of plant roots enclosed in dialysis bags is possible (24). Mucilage polysaccharides adhering to the root surface have been collected by application of vacuum suction (2S), by abrasion with a soft brush and subsequent transfer to cellulose acetate filters (26), or simply by collection with forceps (27).
B. Localized Sampling Techniques in Solution and Culture Systems
Soil
In many plants, root exudation is not uniformly distributed over the whole root system.Considerablespatialvariationhasbeen reported for the exudation of carboxylates and protons in P-deficient oil-seed rape (28,29), the exudation of protons and phenolics in many dicotyledonous plant species in response to Fe deficiency (1.30), o r for the release of phytosiderophores in Fe-deficient barley (30). In all these cases, exudation was mainly confined to apical root zones. Various plant species adapted to low-fertile soils, such as members of the Proteaceae and Casuarinaceae but also white lupin, are characterized by the formation of cluster roots (proteoid roots) mainly under conditions of P deficiency or Fe deficiency (3 1.32). Exudation of large amounts of carboxylates and protons involved in the mobilization of mineral nutrients such as P and Fe (see Sec. 4) is mainly confined to these root clusters (22,33,34), and moreover to distinct stages during cluster root development ( 17,27,35,36). Intense root exudation, restricted to distinct root zones (root tips, proteoid roots), may enhance the mobilization efticiency due to localized accumulation of exudate compounds in the rhizosphere to a concentration level that is sufficient to mediate desorption and solubilization of mineral nutrients from the soil matrix (22,37). This enhanced mobilization effect may be further increased by a low density of microbial colonization (3840) and a high capacity for nutrient uptake in apical root zones. Longitudinal gradients of exudation along the roots may however also reflect gradients in microbial catabolization of root exudates, which is frequently expressed more in basal parts of the root system than in apical root zones ( I ,38,4 1 ). This may be attributed to the high intensity of cell division and cell elongation, which is restricted to the apical parts of the root. Assuming a growth rate of 1.5 cm d” for wheat roots, Jones et al. (42), calculated a residence time of the root apex in given soil zone of about S h, associated with continuous production of new cell material, which has to be newly colonized by soil microorganisms. However, recent reports suggest, that even in apical root zones rapid microbial colonization may be possible at least for some species of soil microorganisms (43,44). Variations in root growth rates in different plant species and under different environmental conditions are likely to explain variability in microbial colonization patterns in different root zones. Reliable evaluation of root exudation is possible only considering the spa-
46 Romheld
and
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tial variability along the roots. Also, the possibility of temporal changes in root exudation, such as transient release of organic acids in cluster rooted plant species (27,45) or diurnal variations in exudation of phytosiderophores in Fe-deficient barley (46) (see Sect. 4.3.2) and of citrate in P-deficient white lupin (36), has to be taken into account.
1. SolutionCultureSystems Spatial variation in root exudation has been investigated by separating distinct root zones of plants grown in hydroponic culture with small plastic rings (1.2 cm in diameter), which were sealedwith agar (28) or vacuum grease (35,47) and subsequently filled with trap solution for 1-2 h (Fig. 1C). Marschner et al. (48) used agarose sheets, which wereplaced onto the root surface, allowing diffusion of root exudates into the agarose layer. Neumann et al. (17) collected exudates from different root zones with a spatial resolution of 5 mm by incubating the roots for 3 h between double layers of filter disks (5 mm in diameter) made from moist chromatography paper (preparative quality) with a high soaking capacity (Fig. IC). Similarly, Kape et al. (49) used cellulose acetate filters with a high sorption capacity for hydrophobic compounds to investigate spatial variation of flavonoid exudation in soybean seedlings. For the selective adsorption of carboxylate anions released from roots of white lupin, Kamh et al. (45) applied anion exchange resins enclosed in dialysis bags or agar sheets, which were placed on the surface of different root zones (Fig. IC).
2. SoilCultureSystems Small sheets of chromatography paper (27) as well as resin bags or resin agar of distinct root zones were also used for sheets (45) applied onto the surface localized collection of compounds released into the rhizosphere soil solution from soil-grown plants, which were cultivated in rhizoboxes (Fig. ID). Giittlein et al. (50) reported the construction of microsuction cups made of HPLC capillaries ( 1 mm in outer diameter) and connected to a vacuum collecting device, which were inserted beneath the roots of soil-grown plants for collectionof rhizosphere soil solution (Fig. 1 D). This technique was also sucessfully employed underfield conditions by use of soot windows. Extraction of rhizosphere soil (22,34,51 3 2 ) is an approach that can provide information about long-term accumulationof rhizosphere products (root exudates and microbial metabolites) in the soil. Culture systems, which separate root compartments from adjacent bulk soil compartments by steel or nylon nets (52-54) have been employed to study radial gradients of rhizosphere products in the root environment. The use of different extraction media can account for different adsorption characteristics of rhizosphere products to the soil matrix (2234). How(> I O min) may ever, even extraction with distilled water for extended periods
Release of Root Exudates
47
lead to some contamination from damaged microbial cells and plant residues, which was not observed when centrifugation techniques were used for soil extraction (SS). A new approach to study root exudation of distinct compounds in soilgrown plants uses inoculationo f roots with genetically engineered reporter bacteria, which are able to indicate the presence of particular compounds by indicator reactions, such as production of ice-nucleation proteins. This technique has been employed to detect the release of amino acids from roots of soil-grown Avena hnrbnta (56).
C. Factors Affecting the Recovery of Root Exudates 1.
Microbial Activity
Organic compounds in root exudates are continuously metabolized by root-associated lnicroorganisms at the rhizoplane andin the rhizosphere. Microbial activity results in quantitative and qualitative alterations of the root exudate composition due to degradation of exudate compounds and the release of microbial metabolites.However,“C-labelingstudies with culturesystemsspatiallyseparating roots and microorganisms by using Millipore membranes demonstrated that root exudation can also be stimulated by microbial colonization of plant roots and by the presence of microbial metabolites (57,58). Thus, the use of axenic culture systems to avoid microbial degradation of exudate compounds ( 1 8,S9,60) may, on the other hand, underestimate exudation rates compared with those of nonsterile systems. Under nonsterile conditions, collection time isan important factor affecting the impact of microbial degradation on the recovery of root exudates. Recovery of amino acids in root exudates of Brassica n a p s L. grown in solution culture under nonsterile conditions remained constant during a collection period of 2.5 h but decreased by more than 90% during the next3.5 h due to microbial degradation (18). Similar results were obtained for microbial degradation of citric acid released from roots of white lupin into aerated distilled water (Fig. 2). Investigations on the fate of ‘‘C citrate and ‘‘C malate added to various soils at a realistic concentration level for root exudates (100-300 FM) revealed average half life times of 2-3 h and almost complete mineralizationwithin 48 h (42,61). Similarly, recovery of variousorganicacidsdetected in the rhizosphere soilsolution of Hukecr u d u l a t a (27) was little affected by microbial degradation during a 3-h incubation period in soils taken from the culture vesselsofthe plants, but recovery declined to zero after 20 h (Table 2). Rapid microbial degradation in the soil environment has been reported also for other constituents of root exudates, such as amino acids and sugars (29). Biodegradationof exudate compounds was found to be most expressed in soils high in organic matter, but it seems to be inhibited
48 Romheld
and
Neumann
P)
1 Y
removed from exudate solution
0
1
2
3
4
5
6
7
8
incubation time [ h ] Figure 2 Microbial degradation of citrate in aerated root washings of P-deficient white lupin 5.5 h after renloval of the root systems from the trap solution.
Table 2 RecoveryofWaterExtractableOrganicAcidsAppliedto African Soil Taken from the Culture Vessels of Hllkecr utzdularn"
a P-deficient West
Percent of carboxylate recovery after soil incubation 24-h nonsterile sterile nonsterile 24-h 3-h sterile Carboxylate 3-h Malic Citric Fumaric trans-Aconitic Lactic
46.9 32.5
? ?
88.1 ?
71.7 64.6
* Incubated under sterile
? ?
4.0 3.5 6.9 7.4 13.0
44.5 29.5 64.4 71.2 69.2
? ? ? ? ?
4.2 1.9 3.1 7.2 9.5
34.8 ? 12.8 t78.4 +63.1 5 10.2 5
1.7 11.1
1.6 0.8
5.7
0 0 0 0 0
(chloroform fumigation) and nonsterile conditions (organic acid application
according to the composition of root exudates).
Exudates Release of Root
49
when adsorption to the soil matrix occurs (61,62). Therefore, sorption materials such as ion-exchange resins ( 1 3,45) and reversed-phase materials (12) can be employed to remove certain compounds from the exudate solution in order to minimize microbial degradation during exudate collection. The application of various antibiotics such as rifampicirdtetracycline (63), cefatoxime/trimethoprim(64),orbacteriostaticcompoundssuch as Micropur (Roth, Karlsruhe, Germany) (65)used for root pretreatmentor addedto collection media is another strategy to prevent biodegradation during root exudate collection. However, depending on dosage and plant species, also phytotoxic effects of antibiotics have been reported (Table 3). Antibiotics in the soil environment
Table 3 Effect of Various Antibiotics on Phytosiderophore (PS) Concentrations in Root Exudates,' and PS Uptake in Fe-deficient Barley and Sorghum
Treatment/Plant species
Phytosiderophore concentration i n root exudates Fe-PS uptake [relativerate values]
Barley
100% antibiotics) (without Control Cefatoxime 36% [30 ppm] Trimethoprim [20 ppnl] 57% Cefatoxime 40% [60 ppm] Trimethoprim 140 ppml Rifampicin 99% 127%12.5 ppm] Tetracycline [6.25 ppm] 9 ppm] Rifampicin [25.0 Tetracycline [ 12.5 ppm] 87% Rifampicin n.d.h 150 ppm] Tetracyclin [ 25 ppm] Micropur 1/10 tablet L I 114 tablet L" 1 / 1 tablet L" 38%
10070
7470
1Yo
93%
125%
100%
112%
75%
75%
Sorghlm?
Micropur Control 1 / l 0 tablet n.d. L" ( 1 mgL- ') 114 tablet L" n.d.(2,5 mgL") 1 / 1 tablet L I (10 mgL")
100%
100%
n.d. n.d.
< 10%
15%
After a 4 h collection period Fe-PS uptake rates were determinedas a parameter for putative phytotoxic effects o f the antibiotica treatments. h 11.d. = not determined.
50 Romheld
and
Neumann
can rapidly stimulate the release of low-molecular-weight organic compounds from dead or damaged microbial cells (66), which may interfere with the determination of root exudates. On the other hand, the function of antibiotics can be affected by adsorption at the soil matrix and by microbial degradation due to the presence of resistant microorganisms (66). Thus, critical evaluation of root exudate data obtained with application of antibiotics is necessary. Addition of antibiotics to exudate solutions subsequent to the collection period can prevent proliferation of microorganisms and microbial degradationof exudate compounds during further sample processing (67).
2.
Sorption at the Soil Matrix
Root exudate compounds in soils are differentially affected by adsorption processes, depending on their charge characteristics and on ion-exchange properties of the soil matrix. The lack of charges prevents interactions of sugars with metal ionsboth in soilsolutionandatthesoilmatrix(68).However,adsorption of more hydrophobic exudate cornpounds such as flavonoids (69) and simple phenolics may be mediated by hydrophobic interactions with humic compounds; abiotic oxidation of phenolics and organic acids at surfacesof Fe and Mn oxyhydroxides has also been reported (70). The adsorption of charged compounds such as carboxylic acids, though largely dependent on the soil type and pH. generally tends to increase with the numberof negative charges available for anionic interactions with metal surfaces at the soil matrix (71), resulting in rapid removal of certain carboxylate species from the rhizosphere soil solution. Since metal complexation and ligand exchange are mechanisms involved in the mobilization of mineral nutrients (P, Fe) and exclusion of toxic elements (AI),the most effective organic chelators (e.g., citrate, oxalate, malate) for these elements frequently exhibit the most intense soil adsorption (22,71). In contrast, sorption of proteinaceous anlino acids and the related mobilizationof mineral nutrients in soils seems tobe comparably low, due to slow reaction kinetics with metal ions (68). However, the socalled phytosiderophores as nonproteinaceous, tricarboxylic amino acids behave differently and exhibit a fast reaction with amorphous iron hydroxides (ferrihydrite) in soils (72). 3.
RetrievalMechanisms
Carbon flow in the rhizosphere is not a strictly unidirectional process from root to soil. Active retrieval mechanisms for sugars and amino acids have been identitied i n plant roots, which were capable of recovering up to 90% of the exudates passively lost into the rhizosphere (21,61,73,74). Even the preferential uptake of organic nitrogen has been reported for plant species adapted to ecosystems such as arctic tundras, where the rate of nitrogen mineralization is generally low (75). These tindings arei n good agreement with recent reportson the molecular biolog-
Release of Root Exudates
51
ical characterization of root-specific transporters for amino acids (76) and small peptides (77) in higher plants. Similarly, induction of a reuptake system for phytosiderophores as Fe complexes has been reported in graminaceous plant species under iron-deficient conditions (16,39).In contrast, no such retrieval mechanisms could be identified for carboxylic acids (78). The ecological significance of retrieval mechanisms for plants maybe related to improved nitrogen and Fe acquisition and to limitation of carbon losses. Especially for long-term studies on root exudation in closed systems (e.g., sterile culture systems), the impact of selective reabsorption of exudate compounds has to be taken into account (74).
4.
Root Injury
Various techniques for collection of root exudates are associated with the risk of root injury by rupture of root hairs and epidermal cells or rapid change of the environmental conditions (e.g., temperature, pH, oxygen availability) during transfer of root systems into trap solutions, application of absorbtion materials onto the root surface, and preparation of root systems for exudate collection. The possible impact of those stress treatments may be assessed by measuring parameters of plant growth in plants either subjected or not subjected to the collection procedure (6) andby comparing exudation patterns after exposure of roots to the handling procedures with different intensity.
111.
MECHANISMS OF ROOTEXUDATION
A.
Diffusion
Release of the major low-molecular-weight (LMW) organic constituentsof root exudates suchas sugars, amino acids, carboxylic acids, and phenolics is a passive process along the steep concentration gradient that usually exists between the cytoplasm of intact root cells (millimolar range) and the external (soil) solution (micromolarrange). Directdiffusionthroughthelipidbilayer of theplasmalemnla (Fig. 3) is determined by membrane permeability, which depends on the physiological state of the root cell and on the polarity of the exudate compounds, facilitating the permeation of lipophilic exudates (79). At the cytosolic pH of approximately 7.1-7.4 ( l ) , more polar intracellular LMW organic compounds such as amino acids and carboxylic acids usually exist as anions with low plasmalemma permeability. A positive charge gradient, which is directed to the outer cell surface as a consequenceof a large cytosolic K' diffusion potential (80) and of plasmalemma ATPase-mediated proton extrusion (Fig. 3), promotes not only uptake of cations from the external solution but also the outward diffusion of carboxylate anions. Basedon studies on the permeability of lipid bilayers to polar substances by use of synthetic membrane vesicles and the flux density
52Romheid
and
Neumann PLAIMA
MEMBRANE
polysaccharides mudlage proteins
*
L W mWMs
rb !:anEZ%ms phytosiderophoras
H+
K+ K+
robn extrudq K* extruskq. electm-chemlcal p b n t i e l gradlent charge exudation balance for of carboxylates
adds amino phenolics
APOPLASM Figure 3 Model for mechanisms involved in the release of root exudates.
equation of Nobel et al. (til), diffusion-mediated basal exudation of amino acids or malate from plant roots has been calculated at a rate of approximately 0.3 nmol h-' cm" root length or 120 nmol h" g-l root fresh weight (68,71). This is in good agreement with experimental data for carboxylate exudation ratesof 0.5-0.9 nmol h" cm-' root length (100-380 nmol h"g" root fresh weight) determined for 5-mm apical root zones of wheat, tomato, chickpea, and white lupin grown in a hydroponic culture system (82). Jones et al. (68,78) suggested that root exudation of amino acids and sugars generally occurs passively via diffusion and may be enhanced by stress factors affecting membrane integrity, such as nutrient deficiency (e.g., K, P, Zn), temperature extremes, or oxidative stress (19,83,84).
B. Ion Channels Root exudation of extraordinary high amounts of specific carboxylates (e.g., citrate, malate, oxalate, phytosiderophores) in response to nutritional deficiency stress or AI toxicity in some plant species cannot simply be attributed to diffusion processes. The controlled releaseof these compounds, involved in mobilization of mineral nutrients and in detoxification of Al, may be mediated by more specific mechanisms. Inhibitory effectsby exogenous application of various anion chan-
Release of Root Exudates
53
ne1 antagonists indicate the involvement of anion channels (17,85-87) with a concomitant release of protons or K+, probably mediated by plasmalemma ATPase(17,34) or K' channels(88,87)respectively(Fig. 3). However,theinterpretation of inhibitor data obtained from experiments with complex tissues is difficult, since unspecific effects cannot be excluded ( 8 5 ) , and the future characterization of this kind of transport mechanisms requires a membrane physiological approach (e.g., patch clamp studies) and finally the cloning of ion channel genes. C.
Vesicle Transport
Vesicle transport is involved in root secretion of high molecular weight (HMW) compounds (88). The release of mucilage polysaccharides from hypersecretory cells of theroot cap is mediated by Golgi vesicles (Fig. 3). Subsequently the secretory cells degenerate and are sloughed off. Secretory proteins such as ectoenzymes (e.g., acid phosphatase, phytase, peroxidase, phenoloxidase) are synthesized by membrane-bound polysomes and cotranslationally enter the endomemof the endoplasmic branesystem by vectorialsegregationintothelumen reticulum (ER). While passing through the Golgi apparatus, they are separated from proteins destinated for the vacuolar compartment and are transportedto the plasmalemma by transfer vesicles (89,90). Processes involved in exocytosissuchasformation ofvesiclesandtheirfusionwiththeplasmamembranestrongly depend on extracellular and intracellular calcium levels ( I ). Vesicles have also been implicated in storage and release of LMW compounds such as phenolics (91,92) and phytosiderophores (93) in plant roots. but the characterization of mechanisms remains to be established (see Sect. 4.3.2).
W. NUTRITIONALFACTORS A.
Phosphorus
1. Root-Induced Phosphorus Mobilizationin Soils Phosphorus (P) is one of the major limiting factors for plant growth in many soils. Plant availability of inorganic phosphorus (Pi) can be limitedby formation of sparingly soluble Ca phosphates, particularly in alkaline and calcareous soils; by adsorption to Fe- and Al-oxide surfaces in acid soils; and by formation of Fe/ AI-P complexes with humic acids (94). Phosphorus deficiency can significantly alter the composition of root exudates in a way that is, at least in some plant species, related to an increased ability for mobilization of sparingly soluble P sources (29,3 1,7 1 ).
54
Neumann and Romheld
Increased root exudation of carboxylates (e.g., citrate, malate, oxalate) is a P-deficiency response, particularly in dicotyledonous plant species. Mobilization of Pi by exogenous application of carboxylates to various soils with low P availability has been demonstrated in numerous studies (22,94-97) and seems to be mediated by mechanisms of ligand exchange, dissolution, and occupation of P sorption sites (e.g., Fe/AI-P and Ca-P) in the soil matrix (22). Citrate and oxalate were found to be the most efficient carboxylates withrespect to P mobilization in many of these model experiments according to high stability constants for complex formationwith Fe, AI,and Ca (71 ). To mediate significant desorption of Pi. however, carboxylate concentrations in the millimolar range are required in the soil solution, associated with a carboxylate accumulation of > l 0 pmol g~ of rhizosphere soil (98,99). A similar concentration level in the rhizosphere has so far been reported for only a limited number of plant species such as Lupinus nlbus L. and members of the Proteaceae (22,27,31,45,52). For I cm apical root zones, where the most intense root exudation is frequently observed (see Sect. 2.2), the volume of rhizosphere soil solution can be calculated at about 30 pL cm ' root length at a distance of I mm from the root surface (100). Assuming a residence time of 5 h for the root apex i n a given soil zone (42), carboxylate exudation at a rate of 6 nmol h" cm" root length is required to reach a concentration level of I mM. Although the impact of microbial degradation (42) and the mechanical impedance of the soil matrix on root exudation ( 4 3 ) was not taken into account, this is similar to the exudation rates of citrate reported for proteoid roots of P-deficient Luyir1lr.s crll~lrsL. (17.35), which is a plant species with a proven ability for citrate-mediated P mobilization in the rhizosphere (22,33,34). In cluster-rooted plant species, carboxylate accumulation in the proteoid rhizosphere is further promoted by the high density of carboxylate-excreting root tips, which exhibit no more growth activity(3 l ) . Intense exudation of carboxylic acids in response to P deficiency has been reported also for oil-seed rape( 101), spinach (102), red clover (99). and chickpea (17,82). Striim et al. (10.3) have suggested that P (and Fe) mobilization, mediated by root exudation of citrate and oxalate, might be related to the abilityof various calcicole plant speciesto grow on calcareous soils. I n many studies, however. interpretation of data is biased by a lack of information about spatial variation of exudation along the root system and by suboptimal conditions during exudate collection (e.g., long-term collection under nonsterile conditions using inappropriate collection media-collection from root systems removed from solid substrates (see Sect. 2). Although in many soils with low P availability, significant desorption of sparingly soluble Pi fornls requires at least millimolar concentration levels of specific carboxylates (e.g., citrate, oxalate) in the soil solution, much lower concentrations (0. I 1nM) were necessary to reduce soil adsorption of Pi. which was applied simultaneously with carboxylates [ 100).Thus, competition of carboxylates with pi for P sorption sites in the soil matrix may be a mechanism that can,
Release of Root Exudates
55
to some extent, prevent soil-fixationof Pi after fertilizer application evenin plant species with moderate exudation rates.
2.
Physiology of CarboxylateExudation
Only limited information is available on the physiological basis of P deficiencyinducedrootexudation ofcarboxylates.Increasedcarboxylaterelease is frequently observed in later stages of P deficiency ( I 1,82). Major exudate compounds are malate, citrate, and also oxalate, especially in plant species where oxalate replaces malate as the major internal carboxylate anion, e.g. members of the Chenopodiaceae (99,102,104). Solution culture experiments have revealed that plant species with intenseP deficiency-induced carboxylateexudation-such asoil-seedrape (101). chickpea,andwhitelupin (82)--accumulatedorganic acids mainly in the root tissue and, moreover, i n the root zones where exudation was most intense (e.g., subapical root zones, proteoid roots). In contrast, root exudation of carboxylates even decreased in response to P deficiency in plant species such as SysimOr-ium cd$cirlalr ( I O I ) , wheat, and tomato (82) and was associated with predominant carboxylate accumulation in the shoots (Fig.4). The results suggest that accumulation of organic acids in the root tissue is a prerequisite for enhancedroot exudation of carboxylates underP-deficient conditions and may be determinedby shoot/root partitioningof carboxylates or of carbohydrates as related precursors. Differential ‘+‘CO2 pulse-chase labeling experiments with shoots and rootsof white lupin revealed predominant biosynthesisof carboxylates in the root tissue and particularly in proteoid roots under P-deficient conditions ( 1 l ) . This is i n good agreement with enhanced expression and in vitro activities of a specific set of glycolytic enzymes, such as sucrose synthase, phosphoglucomutase, fructokinase, PPi-dependent phosphofructokinase, and PEP carboxylase detected in P-deficient root tissues (Fig. 5 ) . These enzymes may operate as an alternativepathway of carbohydratecatabolismunderP-deficientconditions, which facilitates a more economic Pi utilization by Pi recycling. minimization of Pi consumption, and utilization of alternative P pools such as PPi ( 1 OS- 107). Phosohorus deprivation seems to be generally associated with increased shootto-root allocation of carbohydrates, at least in the initial stages of P deficiency (108-1 IO). The ability to maintain glycolytic carbohydrate catabolism under Pdeficient conditions is a prerequisite for the utilization of carbohydrates and thus also for the biosynthesis of carboxylates in the root tissue. However, Hoffland et al. (101) stated that at least a partof the carboxylates that accumulated in roots of P-deficient oil-seed rape was synthesized i n the shoot. Nonphotosynthetic C O z tixationviaphosphoenolpyruvatecarboxylase (PEPC) can contribute a substantial proportion of carbon(>30%) for the biosynthesis of carboxylates in roots of P-deficient plants (Fig. S) ( I 1,82,101,1 1 1 - 1 13). Thus, PEPC-mediated COz fixation may be interpreted as an anaplerotic carbon
Neumann and Romheld
56
d carboxylate
-10
1
l
l
1
I
I
I
I
25
E P 7
en
U
‘5
-m0
E,
20
accumulation inroot tissue
15
10
5
0 -5 -10
I
I
l
l
I
I
l
I
l
I
P defidency-induced root exudation of carboxylates
P
I
I
I
I
Tomato Syslm- Wheat Chick- Rape briurn P-
WhiteLupin
whde probold roots
roots
Figure 4 P deficiency-induced changes in tissue concentrations and root exudation of carboxylates in different plant species. The zero line represents the P-sufficient control. (Adapted from Refs. 82 and 102.)
-
activation
==m==
inhibition
Figure 5 Model of phosphorus (P) deficiency-induced physiologlcal changes associated with the release of P-mobilizing root exudates in cluster roots of white lupin. Solid lines indicate stimulation and dotted lines inhibition of biochemical reaction sequences or metabolic pathways in responseto P deficiency. For a detailed description see Sec. 4.1. Abbreviations: SS = sucrose synthase; FK = fructokinase; PGM = phosphoglucomutase; PEP = phosphoenolpyruvate; PEPC = PEP-carboxylase; MDH = malate dehydrogenase; ME = malic enzyme; CS = citrate synthase; PDC = pyruvate decarboxylase; ALDH = alcohol dehydrogenase; E-4-P= erythrose-4-phosphate; D A W = dihydroxyacetonephosphate; APase = acid phosphatase.
58 Romheld
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supply to compensate for carbon losses related with root exudation of carboxyl5 and 25% of photosynthetic net fixation of ates, which can make up between CO? ( 1 1,7 1 , l 12). Phosphorus deficiency-induced inductionof PEPC is regulated at the transcriptional and also at the posttranslational level by protein phosphorylation ( l 12- l 14). The cytosolic enzyme catalyzes the carboxylationof phosphoenolpyruvate (PEP) to oxaloacetate, which can be further converted to malate by enhanced expression of cytosolic malate dehydrogenase (MDH) (35,112,l 14). Moreover, the PEPC reaction results in liberation ofPi from PEP and may be therefore regarded as an alternative pathway for PEP catabolization via pyruvate kinase, which depends on the presence of ADP and Pi (106). Especially in dicotyledonous plant species such as tomato, chickpea, and white lupin (82,l 1 l), with a high cation/anion uptake ratio, PEPC-mediatedbiosynthesis of carboxylates may also be linked to excessive net uptake of cations due to inhibitionof uptake and assimilationof nitrate under P-deficient conditions (Fig. 5) (17.1 1 1.1 15). Excess uptake of cations is balanced by enhanced net release of protons (82,l I 1,116), provided by increased biosynthesis of organic acids via PEPC as a constituent of the intracellular pH-stat mechanism ( 1 17). In these plants, P deficiency-mediated proton extrusion leads to rhizosphere acidification, which can contribute to the solubilization of acid soluble Ca phosphates in calcareous soils (Fig. 5 ) (34,l 18,l 19). In some species (e.g., chickpea, white lupin, oil-seed rape, buckwheat), the enhanced net release of protons is associated with increased exudation of carboxylates, whereas in tomato, carboxylate exudation was negligible despite intense proton extrusion (82,120). In many plants, citrate and malate are the major carboxylate anions that accumulate in the root tissue in response to P deprivation (82,101, I 12,l 13). Increased citrate accumulation is frequently associated not only with enhanced activity of PEPC but also with a reduction in aconitase activity (82), which is involved in the turnover of citrate within the tricarboxylic acid (TCA) cycle. Thus, citrate accumulation is probably a consequence of both increased biosynthesis and reduced turnover ofcitrate under P-deficient conditions (Fig. 5). Citrate accumulation during proteoid root development in white lupin was also associated with reduced respiration and a concomitant decrease of soluble intracellular Pi (17,l 12). It was suggested that Pi limitation of the respiratory chain may induce a feedback inhibition of citrate turnover in the TCA cycle, in order to prevent excessive production of reducing equivalents (Fig. S) (17). Accordingly. in roots of Phaseol~rsvdgrrris L., P deprivation decreased the activity of the respiratory cytochrome pathway but increased the ratio of NADH/NAD and cyanide-resistant respiration ( 12 1 ). The P deficiency-induced inhibitionof nitrate assimilation in theroot (seeabove)mayalsocontribute toincreasedcitrateaccumulation tissue by downregulation of citrate conversion to 2-oxoglutarate (Fig. 5). which is an important acceptor for amino N as a product of NO3- reduction (122). From these findings, it may be concluded that increased accumulation of organic acids
Exudates Release of Root
59
and particularly of citrate in the root tissue is a P deficiency-induced metabolic disorder, and the release of high amounts of citrate and protons into the rhizosphere in some plant species might serve as a detoxification mechanism to prevent cytoplasmic acidosis. A similar mechanism hasbeen discussed for the detoxification of lactic acid, which accumulates in root tips of maize under hypoxic conditions ( 1 23) with a comparable intracellular carboxylate concentration (20-30 pmol g" root fresh weight) and similar exudation rates (2000-5700 nmol h" g" root fresh weight) as observed in proteoid roots of P-deficient white lupin ( 17,7 I , 123). Besides of increased root exudation of carboxylates and protons, some othcr strategies may also prevent excessive accumulation of organic acids in the root tissue of P-deficient plants. Increased contribution of the Pi-independent cyanide-resistant pathway to root respiration under P deprivation, which was detected i n roots of Phtrsrolus w l g n r i s L. ( 124), although not associated with ATP production, may enable the operation of the TCA cycle, and thus the turnover of citrate under P-deficient conditions. Similarly, an ATP-dependent citrate lyase, which is predominantly expressed in juvenile proteoid roots of white lupin during early stages of P deficiency-induced citrate accumulation, may be involved in the cleavage of citrate into oxaloacetate and acetyl-coA. In later stages of proteoid root development, the activity of citrate lyase decreases, probably due to ATP limitation, associated with increased accumulation and finally increased exudation of citrate (17) (Massonneau et al., unpublished). Another function of citrate lyase i n P-deficient root tissue may be the redistribution of acetyl-coA for the biosynthesis of lipids and phenolics (see Sect. 4.1.3.) under metabolic conditions favoring the accumulation of di- and tricarboxylates. Increased root to shoot translocation of carboxylates in P-deficient Ricinus conzmmi.v L. has been reported by Jeschke et al. ( 1 2S), and may be related to the higher storage capacity for carboxylates in the leaf vacuoles (17). It was suggested that root exudation of high amounts of carboxylates in some P-deficient plant species is mediated by anion channelswith a concomittant release of protons via plasmalemma H'-ATPase (Fig. 5 ) to maintain charge balance ( l 1,17,34). However, enhanced leakiness of membranes in response to P deprivation may also contribute to enhanced release of sugars, amino acids, and organic acids (84; see also Chap. 2). Schilling et al. (29) reported a shift i n the qualitative composition of sugars in root exudates of maize and pea, leading to a higher proportion of pentoses at the expense of glucose and sucrose under Pdeficient conditions. P deficiency-induced root exudation of sugars and amino acids has been related to increased mycorrhizal colonization (126,127).
3.
Exudation of PhenolicCompounds
I n manyplants,Pdeficiencyalsoenhancesproductionand
root exudation of phenolic compounds (Fig.S) (27,3 1,128- 130). Increased biosynthesis of pheno-
60 Romheld
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lics under P-deficient conditions was suggested as another metabolic bypass reaction involved in liberation and recycling of Pi in P-starved cells (106). Antibiotic properties of certain phenolic compounds (e.g., isoflavonoids) in root exudates (69) may not only counteract infection by root pathogens but also prevent the microbial degradation of exudate compounds involved in P mobilization (31). Certain root flavonoids have been identified as signal molecules for spore germination and hyphal growth of arbuscular mycorrhizae, and flavonoids are likely to be important also as signaling compounds for the establishment of ectomycorrhizae ( 1 3 I , 132) (this subject is reviewed in Chap. 7). Phenolics may further contribute to P mobilization by reduction of sparingly soluble Fell1 phosphates (Fig. S) (3 l ) . The specific releaseof piscidic acid (p-hydroxyphenyl tartaric acid) from roots of P-deficient pigeon pea (CNjcrn~rscc+uz L.), which is a strong chelator for FeIII, has been related to enhanced mobilization of Fe-phosphates in Alfisols ( 133). However, considering the comparatively low exudation rates, piscidic acidmaybemorerelevantasasignalingcompoundfortheestablishment of microbial associations (e.g.. arbuscular mycorrhiza (AM), rhizobia).
4.
Root-SecretoryPhosphohydrolases
Enhanced secretion of acid phosphatases (APase) (24,52,134) and phytases ( 1 35) by plant roots and also by rhizosphere microorganisms (136) under P-deficient conditions may contribute to Pi acquisition by hydrolysis of organic P esters in therhizosphere(Fig. S), whichcancomprise up to 30-80% of thetotalsoil phosphorus. In many soils, however, the availabilityof organic phosphorus seems to be limited mainly by the low solubility of certain P forms, such as Caand Fe/Al-phytates, whichcan make up a major propotion of the soil-organic P( 137139). Beissner ( I 04) reported that oxalic acid in root exudates can contribute to some extent tophytatemobilization in soils.Similarly, in ;I P-deficientsandy soil, more Pi was liberated by simultaneous application of acid phosphatase and organic acids identified in rhizosphere soil solution of Hclkm ltrzcllrlrrrcr than by separate application of organic acids or acid phosphatase, respectively (Fig. 6). Another limiting factor for phosphatase-mediated P mobilization is the low mobility of the hydrolytic enzymes (APase, phytase),mainly associated with the root cell wall and with mucilage in apical root zones (140). An alternative function of root secretory acid phosphatases may be the rapid retrieval of phosphorus by hydrolysis of organic P, which is permanently lost by diffusion or from sloughed off and damaged root cells (Fig. S) (141).
B. Nitrogen and Potassium 1. NitrateAssimilation At least in some plant species, such as maize (142), Lupinus mg:Ir.sfjfO/iusL. (143) and tomato (144), root exudation of di- and tricarboxylic acids (mainly
Release of Root Exudates
61
-
B
0
1
H,O APase (control)
organic acids
organic acids + APase
Figure 6 Water-extractable Pi in a phosphorus-deficient sandy soil from Niger (West Africa) after separate or simultaneous addition of acid phosphatase and of organic acids detected in the proteoid-rhizospheresoil solution ofHukeu unduluru. Organic acids: malic 7.5 mM; citric 2 mM; fumaric 1 mM, t-aconitic 0.6 mM acid phosphatase: Wheat germ APase according to enzyme activity in rhizosphere soil [0.7 U g soil”].
malate and citrate) seems to be affected by the form of nitrogen supplied as nitrate or ammonium. Generally, exudation of the carboxylates is increased with increasing levels of nitratein the culture medium. This may be related to the function of carboxylates in intracellular pH stabilization. Nitrate reduction in roots and in the shoot is stimulated with increasing nitrate supply and results in the production of an equivalent amountof OH-, which is neutralized by increased biosynthesis of organic acids or released into the rhizosphere when produced in the root tissue (143,144). The carboxylate anions can be stored in the leaf vacuoles but are also retranslocated to the roots via phloem transport when the leaf storage capacity is limited. In the root tissue, the carboxylate anions are either metabolized by decarboxylation or can be released into the rhizosphere (1,144,145).
2. AmmoniumAssimilation Excess uptake of cations over anions as a consequence of increased ammonium supply is balanced by extrusion of protons and by synthesis of carboxylic acids
62 Romheld
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for pH stabilization in the root tissue. The remaining carboxylate anions are required as acceptors for ammonium assimilationin the roots, which is also associated with the production of protons and decarboxylation of organic acids ( I ) . As a consequence, tissue concentrations and root exudation of carboxylates decline with increased ammonium supply (144). High nitrogen concentrations inhibit the production and release of isoflavonoids from lupin roots. Compared with nitrate supply, exudation was strongly enhanced by ammonium application (146). Similarly, the well-known inhibitory effect of nitrogen on nodulation during establishment of the legume-rhizobium symbiosis is mainly caused by nitrate (1 47). In the short term, intense rhizosphere acidification induced by NH,' nutrition or low rates of NO3- supply may directly stimulate the release of phenolics and other LMW root exudate compounds as a consequence of an increased electrochemical transmembrane potential gradient but also due to acid-induced impairmentof membrane integrity (148) (Table 4). Since flavonoids have important functions as chemoattractants and nod-gene inducers for rhizobia (149), nitrogen effects on nodulation may be explained by differential exudation of these compounds depending on N form supply and the N nutritional status of the plants. Root flavonoids are involved also in pathogen and allelopathic interactions (69,l SO), and these processes might be similarly affected by nutritional modifications in root exudation.
3. Potassium Nutrition Only limited information is available on effects of potassium (K) supply on root exudation.Increasedexudation of sugars,organicacids, and aminoacidshas
Table 4 RcleaseofReducingRootExudates(e.g.. Phenolics) by Peanut Plants as Affected by Fe Nutritional Status and Short-Term (10 h) Supply of NH,'-I 1 mM1-containing nutrient solution
pH of the nutrient N form in solution nutrient after solution Control NO3 -N NH,'-N
IO h 5.5 3 .Y
a
Reducing substances in the nutrient solution [nmol caffeic acid equivalents 10 h"g" root fresh weight] +Fe
-Fe
5
28 I15
Y
Release of Root Exudates
63
been detected in maize as a response to K limitation (142). This may be related to a K deficiency-induced preferential accumulation of LMW N and C compounds at the expense of macromolecules (1). In contrast, Gerke (99) reported enhanced extrusion of protons but a reduction in carboxylate exudation in Kdeficient wheat, sugar beet, and oil-seed rape. Soil-extraction experiments with carboxylates, amino acids, and sugars revealed that only citrate applied in extraordinarily high concentrations [6 mmolg" soil] was effectivein K desorption. ThusK mobilization by root exudates was suggested to be of minimal importance (99).
C. Iron Although iron (Fe) is one of the major soil constituents (0.5-5%), where it is usually present in the oxidized state (FeIII). plant availability is severely limited by the IOW solubility of Fe-(hydr)oxides at pH levels favorable for plant growth. Therefore, plants need special mechanisms for aquiring Fe from sparingly soluble Fe forms to fit the requirements for growth, especially in neutral and alkaline soils, where the availability of Fe is particularly low (151). Mechanisms involved in iron acquisition by plants are also discussed in Chap. 8.
1. Strategy I Plants In dicotyledonousplantsand in nongraminaceousmonocotyledons(strategyI plants) (152), FeIII solubilization is usually mediated by rhizosphere acidification (Fig. 7A).by complexation with chelating compounds (Fig. 7B), and by reduction to Fe11 (Fig. 7C), which istaken up by the roots probably by a specific transporter for Fe11 (Fig. 7C)(153). These root responses are frequently confined to subapical root zones (30,48), and associatedwith distinct changesin root morphology, such asthickening of theroottips andformation of rhizodermaltransfercells ( 1 54,155). Rhizosphere acidification in response to Fe deficiency is most probably mediated by activation of the plasmalemma H+-ATPase (Fig.7A) ( 1 56,157). The reductive capacity is increased by enhanced expression of a plasmalemmabound reductase system with a low pH optimum (158,159), which is further activated by rhizosphere acidification (Fig. 7C) (148). A concomitant release of phenolic acids ( 160,161) and of carboxylates is responsible for Fe111 complexation (22,162,163),and to someextentalsofor Fe111 reduction in therhizosphere (Fig. 7B) (148). In some plant species, Fe deficiency also stimulates root excretion of flavins (164) with yet unknown functions. Phytohormones such as ethylene and indoleacetic acid have been implicated in the signaling of the coordinated strategy I responses to iron deficiency in dicotyledonous plants (165-167). Release of carboxylates and phenolics under Fe stress may be stimulated by a steeper electrochemical potential gradient due to enhanced net extrusion of protons and to theP-deficiencyreelevatedinternalcarboxylateconcentrations.Similar sponse, Fe deficiency-induced accumulation of organic acids in the root tissue
64Romheld
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Neumann Plasmamembrane
CvtoDlasm
Figure 7 Model for iron (Fe) deficiency-induced changes in root physiology and rhizosphere chemistry associated with Fe acquisition in strategy I plants. (Modified from Ref. 1.) A. Stimulation of proton extrusion by enhanced activity of the plasmalemma ATPase FeIII solubilization in the rhizosphere. B.Enhanced exudation of reductants and chelators (carboxylates,phenolics) mediatedby diffusion or anionchannels + Fe solubilization by Fen1 complexation and Fe111 reduction. C. Enhanced activity of plasma membrane (PM)-bound FeIIIreductase further stimulated by rhizosphere acidification (A). Reduction of Fe111 chelates, liberation of FeII. D. Uptake of Fe11 by a PM-bound Fe11 transporter.
(citrate, malate) is associated with increased activity of PEPC (168) and probably also with other as yet unknown metabolic modifications involved in enhanced accumulation of carboxylates. However, a reduction in citrate turnover in the TCA cycle due to inhibition of the Fe-dependent aconitase reaction, discussed in earlier studies, seems not to be a limiting factor (169). The accumulation of organic acids in the root tissue in response to Fe limitation not only provides protons for the H+-ATPase-mediated rhizosphere acidification but the related metabolism also provides electrons for the Fe deficiency-induced plasma membrane-bound reductase system. Oxidation of citrate via cytosolic aconitase and cytosolicNADP-dependentisocitratedehydrogenase (170,171) hasbeen reported to be a major direct or indirect electron source for reductase-mediated iron reduction, whereas other studies have suggested an importantofrole NADH (172) and even of ascorbate (173). Citrate anions are also involved in root-toshoot translocation of Fe via Fe-citrate in the xylem (168,174). Increased root exudation of carboxylates in response to Fe deficiency has been reported for chickpea (163) but not for sunflower (175) and seems to be very low compared with the rateof proton extrusion. Based on model calculations, Jones et(162) al. suggested that even the low background exudation of citrate inof roots unstressed
Release of Root Exudates
65
oil-seed rape might be sufficient for FeIII solubilization at a rate that would be high enough to meet the Fe requirements of the plant. However, in face of rapid microbial turnover of carboxylates in the rhizosphere (42), slow formation and low stability of Fe-citrate complexes at soil pH levels above 6.8 (162), the significance of such a mechanism for citrate-mediated Fe mobilization, especially in Fe-deficient calcareous soils with a high buffering capacity, remains questionable.
2. Strategy II Plants In contrast to strategy I plants, grasses are characterized by a different mechanism for Fe acquisition, with Fe-mobilizing root exudates as main feature. In response to Fe deficiency, graminaceous plants (strategyI1 plants) (39) are able to release considerableamountsofnon-proteinaceousaminoacids(Fig. SB), so called phytosiderophores (PS), which are highly effective chelators for Fen1 (Fig. 8)
PS blosynthesls (in vesicles?)
Fe (Me) mobllhatlon
anion channel7)
'
I
FOPS+ SID t PS + FeSlD
6
Figure 8 Model for root-inducedmobilizationofironand other micronutrients(Zn, Mn, Cu) in the rhizosphere of graminaceous (strategy 11) plants. (Modified from Ref. 1.) Enhanced biosynthesis of mugineic acids (phytosiderophores, PS) in the root tissue. (A) Biosynthesis of PS.(B) Exudation of PS anions by vesicle transportor via anion channels, . (C) PS-inducedmobilizationof Fe111 ' K charge-balancedbyconcomitantreleaseof (MnII, ZnII, CuII) in the rhizosphere by ligand exchange. (D) Uptake of metal-PS complexes by specific transporters in the plasma membrane. (E) Ligand exchange between microbial (M) siderophores (SID) with PS in the rhizosphere. (F) Alternative uptake of microelements mobilized by PS after chelate splitting.
66 Romheld
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(46,176). This release takes place predominantly in subapical root zones (30). Unlike FeIII-citrate, FeIII-PS chelates are stable even at high soil pH levels > 7 ( 162,177,178). Due to the formation of high-affinity FeIII-PS complexes (Fig. 8C), there is only minimal competition by chelation with Ca2+, Mg?+ and AI3+, which are usually present in high concentrations in many soils (15 1). However, recent studies indicate that sulfate, and especially phosphate, applied as fertilizers at high rates, may inhibit PS-promoted FeIII dissolution, mainly by displacement of PS from the surface of Fe (hydr)oxides (179). Unlike strategyI plants, where Fe111 reduction is a prerequisite for iron uptake, strategy I1 involves a specific transport system for FelII-PS complexes, located at the plasma membrane in roots of graminaceous plants (Fig 8D) (1 80). The uptake system requires metabolic energy (46) and is highly specific with respect to FeIII as metal ligand and to PS as organic chelators as well ( 1 S l ) . FeIII chelated by synthetic or microbial siderophores is not recognized by this FeIII-PS transporter (39). However, Fe111 complexes with certain microbial siderophores, such as rhizoferrin, can improve Fe uptake i n graminaceous plant species via exchange chelation with phytosiderophores (Fig. 8E; 186). From the ecological point of view, strategy I1 has advantages over strategy I, especially in well-buffered calcareous soils with high pH, since Fe mobilization and Fe uptake by strategy I1 is less dependent on the external (soil) pH than strategy I ( I 80). After entering the cytosol, the behavior of FeIII-PS complexes is still unof - 102 mV suggests that Fe liberation is known, but the reduction potential possible via reduction by common physiologically available reductants such as NAD(P)H (-320 mV) and glutathione (-230 mV)(15 I ) . Tolerance to Fedeficiency in differentgraminaceous plantspeciesis roughly related to the amount of PS exuded under Fe-deficient conditions (barley > wheat > oat > rye > maize > sorghum > rice), although considerable genotypical variation exists within each single plant species ( 1 50,18 I , 182). The ability to accumulate high amounts of PS in the rhizosphere (up to 1 mM in Fedeficient barley) (39) seems to be associated also with a distinct diurnal rhythm of exudation, which is restricted to several hours after onset of the light period (46), and with restriction of PS release to subapical root zones (30). Temporal and spatial concentration of PS exudation may be a strategy to counteract microbial degradation (39,40,183),dilution by diffusion into the bulk soil(184), and immobilization by sorption of Fe to phospholipids during FePS uptake ( 1 85). In maize and sorghum, with a comparatively high susceptibilityto Fe deficiency chlorosis, PS are continously released at a relatively low rate (1 86) in the subapical root zones (Neumann, unpublished). The molecular mechanism of PS exudation is still not clear. Biosynthesis of PS seems to be regulated by the intracellular iron level ( I 87). Synthesis in the root tissue increases when Fe supply is limited even before iron deficiency c h h -
Release of Root Exudates
67
rosis appears and rapidly declines after reapplication of iron ( 188- 192). PS are derived from nicotianamine, which is ubiquitous in higher plants with putative functions in regulating the physiological availability of Fe and/or transport of copper and other micronutrients in the xylem (13,194). Nicotianamine is synthesized from L-methionine via trimerization of S-adenosyl-methionine (Fig. 8A) in a reaction sequence similar to ethylene biosynthesis (Yang cycle), with continuous recycling of L-methionine (195). I n graminaceous plants, PS formation proceeds by transamination and hydroxylation of nicotianamine to deoxymugineic acid (DMA). DMA is either released as PS into the rhizosphere (e.g., maize) or converted to higher hydroxylated PS derivatives such as mugineic acid (MA), hydroxymugineic acid (HMA), epi-hydroxy mugineic acid (epi-HMA), distichonic acid A and avenic acid A, which were identified as PS in barley, rye, and oat (1 8 I , 189,190,196,197). Several genes involved in the biosynthetic pathway have been cloned (191,198-200). In roots of Fe-deficient barley, biosynthesis proceeds throughout the whole daywith a decline of the internal PS levels during the period of release ( 1 5 1,201). Inhibitory effectsof KCN and DCDD andof low root-zone temperatures suggest that PS biosynthesis and the exudation process are highly dependent on metabolic energy (67,178,202). Low root carbohydrate concentrations under low light conditions and a stimulation of PS release at high light intensities may indicate the importanceof a continuous supplyof photosynthates from the shoot to the roots for the biosynthesis of PS in the roots (203). Ultrastructural investigations in Fe-deficient barley roots revealed, particularly in epidermal cells, the formation of large ER vesicles with attached ribosomes andfilled with fibrous materials priorto the release of PS. When PS exudation was terminated, these large vesicles disappeared. Therefore, a function of vesicles for storage and/or transport of PS (Fig. 8) has been implicated in the mechanism of diurnal PS exudation in barley (93). Accordingly, PS release was almost completely inhibited during the period of root exudate collectionby shortterm (2-h) application of the vesicle transport inhibitor brefeldin A in barley but not in maize (Table 5 ) , where diurnal variation in PS exudation is not detectable ( 1 86). However, in both plant species, PS release was also inhibited by shortterm application of various anion-channel antagonists (Table 5 ) (87), suggesting involvement of anion channels in PS exudation, which is associated with a concomitant equimolar release of Kt as counterion (Fig. 8) (87,178). Since vesicle transport in higher plants is usually implicatedin the secretion of polysaccharides and proteins ( M ) , another putative function of the vesicles in barley roots may be synthesis and transport of proteins to the plasmamembrane, which regulate the diurnal exudation of PS or of the channel proteins themselves. Mori (202) suggested that biosynthesis and release of PS in barely might be triggered by diurnal changes in temperature and not by light signals, but this could not be confirmed by the findings of Kissel (67). Studies including mutants such as the ys3 maize line, which is defective in PS release but not in biosynthesis of PS,
Neumann and Romheld
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Table 5 Effect of Anion-ChannelAntagonists(Anthracene-9-carboxylicacid, ethacrynic acid; each 100 FM) and of Brefeldin A (Exocytosis Inhibitor; 45 PM) on Release of Phytosiderophores from Roots of Fe-Deficient Barley and Maize
Anthracene-9carboxylic Ethacrynic Brefeldin-Aacid acid Barley EpiHMA Inmol h" g
I
F W I
SE M,dlLe '.
DMA [nmol h" g" FW] SE
87.42a 12.8
6.03b 4.9
70.9:l 6.8
30.73a 8.2
0.3b 0.3
9.69c
52.56a
4.4
11.9
8.48b 6.6
Inhibitors applied duringthe 2-h period ofexudate collection intodistilled water. starting at the beglnning of the light period. I n each row, significant differcnces are indicated by diffcrcnt characters.
could be a powerful tool to elucidate the mechanisms and Romheld, unpublished).
of PS exudation (Basso
D. Other Micronutrients and Heavy Metals Mobilization of micronutrients such as Zn, Mn, Cu, and CO and of heavy metals (Cd, Ni) in soil extraction experiments with root exudates isolated from various axenically grown plants is well documented (61,204-206) and has been relatcd to the presence of complexing agents.
1.
Role of Phytosiderophores
Formation of stable chelates with phytosiderophores occurs with Fe but also with Zn, Cu, CO, and Mn (Fig. 8) (39,207,208)andcanmediatetheextraction of considerable amounts of Zn, Mn, Cu, and even Cdi n calcareous soils (204,209). There is increasing evidence thatPS release in graminaceous plants is also stimulated i n response to Zn deficiency (210-212), but possibly also under Mn and Cu deficiency (2 13). Similar to Fe deficiency, the tolerance of different graminaceous plant species to Zn deficiency was found to be related to the amount of released PS (2 1 1,2 12), but correlation within cultivarsof the same species seems to be low (214). It is, however, still a matter of debate as to what extent PS release is a specific response to deficiencies of the various micronutrients. Gries et al. (2 13) reported that exudation of PS in Fe-deficient barley was about 1530 times greater than PS release in response to Zn, Mn, and Cu deficiency. In contrast, PS exudation in Zn-deficient bread wheat was i n a similar range as PS
Release of Root Exudates
69
release under Fe deficiency in barley (205,21 l ) . Walter et al. (215) demonstrated thatZn deficiency-induced PSrelease in bread wheat is probably anindirect response, caused by impaired iron metabolism; this is also supported by the data of Rengel and Graham (216). In contrast. Gries et al. (2 17) suggested that there was a specific response to Cu deficiency in Hordeivmus europaeus L. Root uptake rates of PS complexes with Cu, Zn, and CO were found to be much lower than uptake of FelII-PS chelates( I 5 1 ) but may still be sufficientdue to a lower demand for micronutrients (217).In contrast, based on studies with the maize y s l mutant, which is defective in Fe-PS uptake, v. Wiren (218) proposed two pathways of Zn uptake in grasses, including uptake of the free Zn cation and uptake of the Zn-PS complex via the Fe-PS transport system (Fig. 8F).
2.
Role of Carboxylates, Rhizosphere pH, and Redox State
Mobilization of micronutrients (Mn, Zn, Cu), heavy metals (Cd), and even uranium i n the rhizosphere has been also related to rhizosphere acidification and to complexation with organic acids (e.g., citrate) in root exudates (219-224). This view is further supportedby intense mobilizationof Mn, Zn, Cu, and Cd observed i n soil extraction experiments with leachates from rhizosphere soil or with organic acid mixtures according to the root exudate composition of plant species such as Lrrpirlus trlbus, Htlketr uruiuiatu, and Spinuceu oleruceu under P-deficient conditions, where exudationof carboxylates and protons is particularly expressed (27,33,34,102). However, only limited information exists about the plant availability and uptake of the metal-carboxylate complexes. Solution culture experiments in the presence of complexing agents revealed that plant uptake is correlated mainly with the activity of free uncomplexed metal ions in solution (225of the 229). This implies that utilization of chelated metals requires liberation metal ligands from the carboxylate complex, which may be mediated by rhizosphere acidification and reduction of metal species such as Mn and Cu (99,227). Similar to Fe acquisition in strategy I plants, Mn mobilization in the rhizosphere of soil-grown plants is a result of the combined effects of rhizosphere acidification, complexation with organic ligands and reduction of Mn oxides ( l). Phenolics and organic acids in root exudates (especially malate) are involved i n both complexation and reductionof Mn (230,23l ) . In cluster-rooted plant species such as Llrpirurs a1bu.s and members of the Proteaceae, particularly intense exudation of organic acids and phenolics in response to P deficiency is frequently also associated with enhanced Mn mobilization in the rhizosphere and accumulation of high or even toxic Mn levels in the shoot tissue (27,3 1,232). Similarly, Mn in flax grown i n toxicity was indirectly induced by the iron-deficiency response a calcareous soil high in extractable Mn but low in Fe (233). Besides mobilizing effects of plant root exudates, Mn availability in the rhizosphere is also strongly affected by the activity of microorganisms involved in Mn oxidation and Mn
bition
70Romheld
and
Neumann
reduction, which,in turn,depend on root exudates as a carbon source (1). Utilization of Cu complexes with humic acids and citrate has been reported for red (234).The authors suggested that clover, especially under P-deficient conditions this was the result of liberation of complexed Cu in the rhizosphere, mediated by an increased reductive capacity of the roots, which was also identified as an adaptive mechanism of Cu deficiency in Pisum sativum (227). Despite increased citrate accumulation in roots of Zn-deficient rice plants, root exudation of citrate was not enhanced. However, in distinct adapted rice cultivars, enhanced release of citrate could be observed in the presence of high bicarbonate concentrations in the rooting medium, a stress factor, which is frequently associated with Fe and Zn deficiency in calcareous soils (235)(Hajiboland, unpublished). This bicarbonate-induced citrate exudation has been related to improved Zn acquisition in bicarbonate-tolerant and Zn-efficient rice genotypes (Fig. 9) (235).Increased exudation of sugars, amino acids, and phenolic compounds in response to Zn deficiency has been reportedfor various dicotyledonous and monocotyledonous plant species and seems to be related to increased
elevated bicarbonate concentration in soil solution Increasein synthesis of citrate inroots root exudation citrateof citrate enhanced Zn mobilization in the rhizosphere improved root growth
/\
accumulation in roots disorder of root metabolism
of root growth
Exudates Release of Root
71
leakiness of membranes (19). Zinc has essential functions in the stabilization of membranes (236,237) and in preventing oxidative membrane damage as a metal component of superoxide dismutase, which is part of the free-radical scavenging system of higher plants (238). It is, however, as yet unknown whether this kind of Zn deficiency-induced root exudation has any impact on mobilization of Zn or other micronutrients in the rhizosphere. Comparatively high mobility of Cd in soils associated with high rates of uptake and accumulation in some plant species is an important aspect from the ecotoxicological point of view. Cd mobilizationin soils can be mediated by rhizosphere acidification (223) butto some extent alsoby complexation with carboxylates (99,221) or phytosiderophores (209). A comparison of high and low Cdaccumulating genotypes of durum wheat revealed higher levels of carboxylates in the rhizosphere soil of the Cd accumulator (222). Based on these findings, it was concluded that plant availability of Cd may be increased by complexation with root-derived carboxylates. In contrast, Gerke (99) suggested that carboxylate complexation of Cd might decrease plant availability, since only freeCd?+ seems in soilto be taken up by plant roots (229,239). Wallace (225) demonstrated that, plant systems, solubility and transport to the root uptake sites are likely to be the limiting steps in uptake of cationic microelements by plants.
V.
ALUMINIUM TOXICITY
Aluminium toxicity is a major stress factorin many acidic soils. At soil pH levels below 5.0, intense solubilization of mononuclear AI species strongly limits root growth by multiple cytotoxic effects mainly on root meristems (240,241 ). There is increasing evidence that AI complexation with carboxylates released in apical AI concentrationisawidespread rootzones in response toelevatedexternal mechanism for AI exclusion in many plant species (Fig. 10).Formation of stable AI complexes occurs with citrate, oxalate, tartarate, and-to a lesser extentalso with tnalate (86,242,243). The AI carboxylate complexes are less toxic than free ionic AI species (244) and arenot taken up by plant roots (240). This explains the well-documented alleviatory effects on root growth in many plant species by carboxylate applications (citric, oxalic, and tartaric acids) to the culture media in presence of toxic AI concentrations (8,244,245) Citrate, malate and oxalate are the carboxylate anions reported so far to be released from AI-stressed plant roots (Fig. lo), and AI resistance of species and cultivars seems to be related to the amount of exuded carboxylates (246,247) but also to the ability to maintain the release of carboxylates over extended periods (248). In contrast to P deficiency-induced carboxylate exudation, whichusually increases after several days or weeks of the stress treatment (72, I 13), exudation of carboxylates in response to AI toxicityisafastreactionoccurringwithinminutestoseveralhours
72Romheid
and
,mg nbrane
ApoDlasm
Neumann
Rhizosphere
Figure 10 Model for mechanisms involved in aluminium (AI) exclusion and detoxification at the root apex. (A) Enhanced solubilization of mononuclear AI species from AI oxides and AI silicates in the soil matrix at pH < 5.0. (B) Al-induced stimulation of K+. carboxylate exudation via anion channels, charge-balanced by concomitant release of (C) Formation of Al-carboxylate complexes in the apoplasm; restricted root uptake and lower toxicity of complexedAI. (D) AI complexation in the mucilage layer (polygalacturonates) and with Al-binding polypeptides. Increased accumulation of Al-chelating carboxylates in the mucilage layer due to limited diffusion.
(85,86,249);it rapidly drops to baseline levels when AI is removed from treatment solutions (83,85).Al-induced carboxylate exudation can reach a level that is comparable to the extraordinary high exudation of citrate in P-deficient white lupin (74), even in plant species without increased carboxylate exudation under P-deficient conditions, such asCassia tom (249), wheat (82,242),or potato (Neumann, unpublished). Carboxylate releasein Al-stressed plants can be blocked by anion channel antagonists (85,86), suggesting the involvement of an anion channel in the plasma membrane. The release of carboxylate anions may be balanced by an equimolar releaseof K+ (85). Patch-clamp studies with protoplasts isolated from root tips of wheat revealed the Al-induced activation of a Cl- channel with characteristics similar to those of Al-mediated release of malate, but with a slower response to the A1 treatment in some experiments. It remains to be established whether this anion channel also shows permeability toward malate (250). In elec-
Release of Root Exudates
73
trophysiological studies, an AI-induced membrane depolarization, which was not caused by malate efflux, was observedin roots of AI-tolerant but not in Al-sensitive genotypes of wheat (251). The authors suggested that this depolarization, together with other as yet unknown factors, might be involved in gating of a voltage-dependentmalatechannel in roottipsofAI-tolerant wheatlines.Although the release of carboxylates in response to AI treatments occurs almost instantaneously, inhibitory effects of cycloheximide suggest that intact protein synthesis is required for carboxylate exudation (85). During short-term exposure to A1 stress (2-3 h ) the internal concentrations of carboxylates in root tips of wheat (242) and buckwheat (86) remainedat a constant level, and there were no differences in AI-tolerant and AI-sensitive genotypes (242).In root tips of wheat, the activities of enzymes involved in biosynthesis of malate (PEP carboxylase; NAD malate dehydrogenase) were also not changed (85). It was concluded that the capacity for carboxylate accumulation in the root tissue is not a limiting factor for Al-induced exudation of carboxylates, at least in short-term experiments, and genotypical variations cannot be explainedon basis of differences in the capacity for biosynthesis of carboxylic acids (85,242). However, intense exudationof carboxylates over longer periods as a prerequisite for an efficient AI detoxification (248) would probably require also increased rates of biosynthesis. Accordingly, AI treatments over 16 h enhanced both the exudation and the internal concentration of citrate in root tips of potato and sunflower (Neumann, unpublished). This is in line with findings of De la Fuente et al. (252), who reported inductionof AI tolerance in transgenic tobacco and papayaby constitutive cytosolic expression of a bacterial citrate synthase gene from Pseudot~zorzasaeruginosu, which resulted in higher rates of citrate accumulation in the root tissue and higher ratesof citrate exudation. Genetic analysisof the AI-tolerance trait and AI-inducible root exudation of malate in hexaploid wheat revealed a predominant control by one single gene, which may be involved in the signaling of the exudation process (247). Also, other constituentsof root exudates havebeen implicated in binding of AI and thus in AI detoxification in the rhizosphere (Fig. IO). Examples are the release of AI-binding polypeptides (253,254) and of phosphate anions in wheat (246). A high binding capacity for A1 has also been demonstrated for mucilage, which is preferably released in apical root zones (26,255).AI complexation in the mucilage may be attributedto the presenceof polycarboxylates (polygalacturonic acids) as integral constituents( I ) but also to accumulation of LMW carboxylates and phosphates in the highly viscid mucilage layer (Fig. 10) (256). Unlike carboxylic acids, the release and also the production of phytosiderophores in roots of both AI-sensitive and AI-tolerant wheat cultivars was rapidly inhibited in response to AI treatments and seemsto be responsible for AI-induced iron chlorosis in wheat (257). Increased root exudation of amino acids in response to Cd toxicity has been reported for lettuce and white lupin grown in a hydroponic culture system under
Neumann and Romheld
74
axenic conditions (60). Under similar culture conditions, a transient release of organic acids (citric, maleic) was observed after addition of high Cu concentrations [50 PM] to the culture mediumof sunflower (258), suggestingthat stimulation of root exudation may be triggered by toxic levels of various metal species, probably as a consequence of impaired integrity of the plasma membrane.
VI. TEMPERATURE Diffusion-mediated release of root exudates is likely to be affected by root zone temperature due to temperature-dependent changes in the speed of diffusion processes and modifications of membrane permeability (259,260). This might explain the stimulation of root exudation in tomato and clover athigh temperatures. reported by Rovira (261), and also the increase in exudation of sugars and amino acids in maize, cucumber, and strawberry exposed to low-temperature treatments (S-IO'C), which was mainly attributed to a disturbance in membrane permeability (259,262). A decrease of exudation rates at low temperatures may be predicted for exudation processes that depend on metabolic energy. This assumption is supported by the continuous decreaseof phytosiderophore release in Fe-deficient barley by decreasing the temperature from 30 to 5°C (67).
VII.
LIGHTINTENSITY
Since a large proportion of the organic carbon released into the rhizosphere is derived from photosynthesis ( 1 1 ), changes in light intensity are likely to modify the intensity of root exudation. Early workof Rovira (261) demonstrated changes in quantity and quality of amino acids in exudates of tomato and clover with decreasing light intensity. High light intensities strongly stimulated the release of phytosiderophores in roots of Fe- and Zn-deficient barley and wheat cultivars (203). In P-deficient white lupin, citrate release from proteoid roots followed a diurnal rhythm with exudation peaks during the light period (36). This behavior might reflect the diurnal variations in carbohydrate (sucrose) supplyby the shoot (263) as precursors for citrate biosynthesis.
VIII. WATER SUPPLY Drought stress increases the soil mechanical impedance on plant roots, which in turn can stimulate root exudation (1,4,5). Increased release of mucilage may contribute to the maintainanceof Zn uptake in dry soils by facilitating Zn transport to the root surface in mucilage-embedded soil particles (264). This effect in the roots, which is mightbe supported by watertransferfromthesubsoil
Exudates Release of Root
75
subsequently released into the dry top soil layer (hydraulic lift) (265). Increased root exudation of carbohydrates under conditionsof mild or severe drought stress has been reported for several conifer species (266-268) and was attributed to root damage and increased internal carbohydrate concentrations. High soil moisture levels orflooding are limiting factors for oxygen supply to the roots. Hypoxia causes a shift from aerobic respiration to fermentation of carbohydrates in the root tissue yielding ethanol, lactic acid, and alanine as the main end products, which can accumulate to phytotoxic levels (123,269,270). At least in some plant species, therelease of substantialamounts of lacticacid (123,270) and ethanol (269,271) into the root environment may be involved in detoxification of these metabolites. Also the exudation of sugars and amino acids is frequently enhanced under hypoxic conditions (271-273). Ethanol is a chemoattractant to plant pathogens (274), and increased exudation in response to hypoxia may support pathogenic infections, especially in the presence of other exudate compounds such as amino acids (271).
IX.
ELEVATEDCO,
The continuous riseof the atmospheric COr concentration during the last decades, mainly as a consequence of anthropogenic COz production, is likely to affect photosynthesis and plant biomass production (275). Numerous studies have demonstrated a stimulation of shoot and root growth in plants exposed to elevated COz concentrations (275,276), but the putative consequences for rhizodeposition and rhizosphere processes are still not clear. Loss of assimilated carbon from maize roots was not affected by increased atmospheric COz levels (277). Similarly. “CO? pulse-chase labeling experiments with Plcrntago Ic~~zcc~olut~~ seedlings revealed that carbon loss from the root system was not changed at elevated COz levels, although shoot-to-root allocation of assimilates was increased (278). In P-deficient white lupin, elevated C O z treatments did not affect the amount of citrate released from cluster roots, but citrate exudation started in earlier stages of proteoid root development, suggesting COz-induced modifications of root carbohydratemetabolism(36).Thephosphataseactivityassociated withtheroot surface of Awrln barbatc1 and Brortlm hordecrcw~swas also not changed by elevated COz levels (279) but increased on a base of root dry weight and root length in asepticallygrownwheatseedlingswhenthe COz treatmentwasassociated with P deficiency (280).
X.
FUTURERESEARCHPERSPECTIVES
During the last two decades, the functional characterizationof plant root exudates involved in rhizosphere processes has attracted increased attention. The role of
76 Romheld
and
Neumann
root exudates in plant-microbial interactions, nutrient acquisition, and plant adaptations to environmental stress or adverse soil-chemical conditionsis not only of scientific interest but also implicates obvious practical aspects associated with the need for production of healthy crop plants and for sustainable agricultural systems. From the methodological point of view, a critical reevaluation of the techniques employed for collection of root exudates and of the experimental results is indicated for many earlier studies in the light of recent findings demonstrating spatial variation of exudate release along the roots and strong effects of mechanical impedance, root injury, microbial turnover in the rhizosphere, and sorption at the soil matrix on recoveryof root exudate compounds. Further miniaturization of sampling procedures and analytical techniques (e.g., use of specific microprobes for specific compounds, reporter bacteria, microsuction cups, capillary electrophoresis,imageanalysis,andvideodensitometry)shouldenablenondestructive measurements of rhizosphere processes at a high scale of resolution. Much more informationis necessary about effectsof root exudates at realistic rhizosphere concentration levels on mobility and plant availabilityof nutrients and toxic compounds such asheavy metals and pesticides in soils under different environmental conditions.In this context it is also important to consider the possibility of synergistic effects of simultaneous chemical reactionsin the rhizosphere, which have been demonstrated, for example, for the Fe deficiency response in nongraminaceaous plants including increased redox potential and increased release of reductants and chelating compounds in the apical root zones stimulated by increased release of protons ( l ) . Other examples are possible effects of transient pH changesin the rhizosphere and the releaseof phenolic compounds counteracting the microbial degradation of root exudates involved in nutrient mobilization, increased availability of organic P forms for root secretory phosphatases and phytases mediated by a concomitant release of carboxylates, and effects of rhizosphere pH on metal complexation with organic ligands. Flavonoidsandotherphenoliccompoundsreleased byplantroots have important functions in plant-pathogenic interactions, feeding deterrence, nematode resistance, and allelopathic interactions; they also serve as signal molecules for the establishment of symbiotic associations (72,149). However, a detailed analysis of signaling pathways involved in these interactions is currently available in only a limited number of cases (see also Chap. 7). Attempts to manipulate root exudation of higher plants by use of genetic engeneering, breeding technologies, or modification of culture conditions to increase efficiency for nutrient acquisition, resistance to adverse soil-chemical conditions, or designing of plants for phytoremediation and phytomining strategies (281) requires a detailed knowledge of the physiological mechanisms involved in the regulationof root exudation andof the rhizosphere processes as well. Thus, for a better understanding of the complex interactions at the root-soil interface,
Release of Root Exudates
77
a multidisciplinary approach is necessary, integrating the fields of soil chemistry, soil microbiology, plant nutrition, plant physiology, and molecular biology.
ACKNOWLEDGMENTS The authors wish to thank manuscript.
Mrs. Heidi Jane Hawkins for critically reading the
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/72:279 ( 1995). V. Groleau-Renaud, S. Plantureux, and A. Guckert, lnfuence of plant morphology on root exudation of maize subjected to mechanical impedance in hydroponic conditions. Pltrrrt Soil 20/:231 (1998). 6. D. A. Barber and K. B. Gunn. The effect of mechanical forces on the exudation of organic substances by the roots of cereal plants grown under sterile conditions. NOH*Phytol. 73:39 ( 1974). 7. R. Schonwitz and H. Ziegler, Exudation of water soluble vitamins and some carbohydrates by intact roots of maize seedlings (Zecr rncrys L.) into a mineral nutrient solution. Z. P/~rr~~err/~hy.sio1. 1077 ( 1 982). 8. S. C. Miyasaka, J. G. Buta, R. K. Howell. and C. D. Foy. Mechanism of aluminium tolerance in snapbeans. Root exudationof citric acid. Plnrrt P h y i o l . W 7 3 7 (1991). 9. A.HulsterandH.Marschner,PCDD/PCDF-TransferinZuchiniund Tomaten. Verijfliwtl. P A 0 8 5 7 9 ( 1994). citric IO. T. Shepherd and H. V. Davies, Effect of exogenous amino acids, glucose and acid on the patternsof short-term amino acid accumulation andloss of amino acids in the root-zone of sand-cultured forage rape (Rrc~ssicccrlczprcs L.). Plrrrt Soil 158: 1 I I (1994). 1 1 . J. F. Johnson,D.L.Allan, C. P.Vance,and G. Weiblen,Rootcarbondioxide fixation by phosphorus deficientLupirrus nlhts. Contribution to organic acid exudation by proteoid roots. P l m t Phy.siol. 112: 19 ( 1996). 12. C. S. Tang and C. C. Young.Collectionandidentification of allelopathiccompounds from the undisturbed root system of Bigalte Lompograss (Herncrrthicc ccltiss i r w ) . P h t Physiol. 69:155 ( 1982). 5.
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13. W. Petersen and M. Bottger, Contribution of organic acids to the acidification in the rhizosphere of maize seedlings. Plarlt Soil 132: 159 (1992). 14. Y.OhwakiandH.Hirata,DifferencesincarboxylicacidexudationamongPstarved leguminous crops in relation to carboxylic acid contents in plant tissues and phospholipid levels in roots. Soil Sci. P l m t Nurr. 38:23S (1992). IS. D. S. Lipton, R.W.Blanchar,and D. G. Blevins,Citrate,malate,andsuccinate concentration in root exudates from P-sufficient and P-stressed Medicugo s d v u L. seedlings. Plrrrlr Physiol. 8.53 15 (1987). 16. N. v. Wirin, S. Mori, H. Marschner, and V. Romheld, Iron ineffeciency in maize mutant ysl (Zeo mrys L. CV Yellow-Stripe) is caused by a defect in uptake of iron phytosiderophores. Plant Physiol. 10671 ( 1994). 17. G. Neumann, A. Massonneau, E. Martinoia, and V. Riimheld. Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. P lnrltm 208373 ( 1999). 1x. T. Shepherd and H. V. Davies, Patterns of short-termamino acid accumulation and loss in the root-zone of liquid culture forage rape (Brussicu ~ ~ L L.). L S Plu~ztSoil 1.58:99 (1994). 19. 1. Cakrnak and H. Marschner. Increase i n membrane permeability and exudation in roots of zinc deticlent plants. J. P l m r Physiol. 132:356 (1988). 20. Z. Prikryl and V. Vancura. Root exudates in plants. VI. Wheat exudationas dependent on growth, concentration gradients of exudates and the presence of bacteria. Plcrrlt Soil .57:69 (1980). D. L. Jones and P. R. Darrah, Rc-sorption of organic compounds by roots of Zen r~~crys L. and its consequences in the rhizosphere 11. Plurzt Soil /.53:47 (1993). 22. J. Gerke, W. Romer, and A. Jungk. The excretion of citric and malic acid by proteoid rootsof Lupirzus alhus L.: cffectson soil solution concentrationsof phosphate.
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268. C. P. P. Reid and J. G. Mexal. Water stress effects on root exudation of lodgepole pine. S o i l . B i d . BiochertI. P417 (1977). 269. D. D. Davies, Anaerobic metabolism and the productionof organic acids. The Hiochtwtistr:\~of P h t s , Vol. 2, Academic Press, 1980, p. 58 1 . 270. J. Rioval and A. D. Hanson, Evidence for a large and sustained glycolytic flux to lactate in anoxic roots o f some members of the halophytic genus Lirrwrliwrt. Pltrrlt Phy.si0l. 101:ss3 (I 993). 271. A. J. M. Smucker and A. E. Erickson, Anaerobic stimulation o f root exudates and disease of peas. Pltrrit Soil Y9:423 (1987). 272. W. A. Ayers and R. H. Thornton, Exudation of amino acids by intact and damaged roots of wheat and peas. Pltrru S o i l 2R: 193 ( I 968). 273. R. L. Rittenhouse and M.G. Hale, Loss of organic compounds from roots.11. Effect of O? and COz tension on release of sugars from peanut roots under axenic conditions. Pltrrrt Soil 3.5:31 1 (lY7l). i n trees. in compari274. S. J. Grayston, D. Vaughan, D. Jones. Rhizosphere carbon flow son with annual plants: the itnportancc of root exudation andits impact on microbial activity and nutrient availability. Appl. Soil G o / . S:29 ( 1996). 7-75, G. Bowes,Facingtheinevitable:PlantsandincreasingatmosphericCO:.Annu. Rcv. Plant Physiol. Plant Mol. Bid. 44309 (1993). 276. H. H. Rogers, G. B. Runion, and S. V. Krupa, Plant responses to atmospheric COz enrichmentwithemphasis on rootsandthcrhizosphere. Errliron Pollrrr. 83:ISS ( 1994). The. Khizosphrre, (J. M. Lynch. ed.), Wiley 277. J. M. Whipps,Carboneconomy, InterscicncelJohn Wiley and Sons. Chichester, UK, 1990,p. 59. 278. A. Hodgc and P. Millard, Effect of elevated COzon carbon partitioning and exudate release fiotn P k t r l t c l p ) k u l c w d r r r t r seedlings. Physiol. Pitwt. 103:280 ( 199X). 279. Z. G. Cardon. Influence of rhizodeposition under elevated COz on plant nutrition and soil organic matter. Plrrrlt Soil 187:277 ( 1996). 280. D. J. Barrett, A. E. Richardson. and R. M. Gifford, Elevated atmospheric COzconcentrationsincreasewheatrootphosphataseactivitywhengrowthislimited by phosphorus. Alrsr. J. P h t Physiol. 25:87 (1998). 281. R.R. Brooks, M. F. Chambcrs. L. J. Nicks,and B. H.Robinson.Phytomining. T w r d s Plrrrlr Sci. 3350 ( I 998). 282. N. v. Wirin, V. Riimhcld, T. Shiori, and H. Marschner, Competition between mii n thc root croorganisms and roots of barleyandsorghumforironaccumulated apoplasm. Ntwv Phytol. 130:S I 1 ( 1995).
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The Effect of Root Exudates on Rhizosphere Microbial Populations Melissa J. Brimecombe, Frans A. De Leij, and James M. Lynch University of Surrey, Guildford, Surrey, England
1.
INTRODUCTION
In this chapter we review the current literature available on the influence of root exudates on rhizosphere microbial populations and the effects of plant, microbial and soil factors on the processes of rhizodeposition and microbial colonization and activity. We first give a brief overview and definitions of some of the main concepts relating to the rhizosphere and rhizodeposition.
A.
TheRhizosphere-ADefinition
Hiltner is often quoted as having said “If plants have the tendency to attract useful bacteria by their root excretions, it would not be surprising if they would attract uninvited guests, which like the useful organisms, adapt to specific root excretions.” In 1904, Hiltner attributed a “tiredness of soil” to the activities of harmful organisms in the rhizosphere of unthrifty plants and suggested that the healthy plants formed a protective bacteriorhiza. Since his early pioneering work on the “rhizosphere” (a term Hiltner used to describe specifically the interaction between bacteria and legume roots), our knowledge of the subject has greatly increased, and today perhaps a more appropriate definition of the rhizosphere is “the field of action or influence of a root” ( l ) . The rhizosphere is generally considered to be a narrow zone of soil subject to the influence of living roots, where root exudates stimulate or inhibit microbial populations and their activities. The rhizoplane or root surface also provides a 95
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1
H:
Ectnrhizosohere-
Root halr
Muclgel (plant and bacterlal mucilage)
Plant mucilage ...... ..... .. :.
... ..
Sloughed root cap cell
a.:o:/;!:::. ..._
:D. .
,..: . ...
Figure 1 Root region. (From Ref. 2.)
highly favorable nutrient base for many speciesof bacteria and fungi, and these two zones together are often referred to as thesoil-plant inte$ace. We can further define the “endorhizosphere” as the cell layers of the root itself, and the “ectorhizosphere” as the area surrounding the root (Fig. 1). The rhizosphere is thus an environment createdby the interactions between root exudates and microorganisms that may either utilize the organic materials released as nutrient sourcesor be inhibited by them. The plant-microbe relationship is more often based on the former, where microbes take advantage of nutrients provided by the plant. In return, microbes may assist the plant, for example, by making nutrients available or by producing plant growth promoting compounds, or may cause harm to it, for example, by acting as plant pathogens. In general the microbes that inhabit the rhizosphere serve as an intermediary between the plant, which requires soluble inorganic nutrients, and the soil, which contains the necessary nutrients but mostly in complex and inaccessible forms. Rhizosphere microorganisms thus provide a critical link between plant and soil environments.
B. Rhizodeposition of Organic SubstancesCurrent Opinion Materials deposited by roots into the rhizosphere can be divided roughly into two main groups: first, water-soluble exudates such as sugars, amino acids, or-
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ganic acids, hormones and vitamins, and, second, water-insoluble materials such as cell walls, sloughed-off materials, and other root debris and mucilage such as lysates released when cells autolyse (3,4). In addition, carbon dioxide from root respiration often accounts fora large proportion of the carbon released from roots. Further, secretions such as polymeric carbohydrates and enzymes, depending on metabolicprocessesfortheirrelease,mayalsoberegardedasrootexudates. Some authors favor categorizing root exudates by their nature of release: however,since it isusuallyverydifficulttodistinguishexperimentallybetween “true” exudates and organic compounds from other sources such as secretions and lysates, many authorsuse the approach of Uren and Reisenauer (S) and Rovira (6), who consider exudates to be all organic substances released by healthy and intact roots to the environment. Meharg (7) has further suggested that categorizing organic carbon compounds according to the nature of their release tends to oversimplify the interpretationof carbon budgets in the rhizosphere, since this approachdoes not reflectsubstrateavailabilitywithintherhizosphere. It was suggested that perhaps a better categorization is to classify organic carbon coma microbial pounds lost from plant roots in terms of their subsequent utilization as substrate (i.e., as low-molecular-weight compounds readily assimilated by the microbial biomass; as polymeric and more complex compounds such as polysaccharides, polypeptides, nucleic acids, and pigments, etc., requiring extracellular enzymic activity to break them down before they can be assimilated; or as structural carbon sources such as cell wall materials, requiring saprophytic degradation before carbon becomes generally available to the rest of the soil biomass). This approach would appear to be more relevantto the study of rhizosphere microbial population dynamics. Either way. microbial populations in the rhizosphere generally have access to a continuous flow of organic substrates derived from the root. Newman (8) found that these soluble and insoluble rhizodeposits ranged from 10-250 mg/g root produced for a numberof plant species. This significant rhizodeposition has been shown to enhance microbial growth in the rhizosphere (9,10), and due to this large availabilityof substrate in the rhizosphere, microbial biomass and activity are generally much higher in the rhizosphere than in the bulk soil. ConcentraIO“’ and 10” cells per tions of microbes in the rhizosphere can reach between gram of rhizosphere soil ( 1 l ) , and invertebrate numbers in the rhizosphere have been shown to be at least two ordersof magnitude greater thanin the surrounding soil (12). These increased population densities are largely supported by carbon inputs from roots.
C.CarbonRhizodeposition-CurrentEstimates The input of carbon into soil via rhizodeposition and the decay of roots has been quantified in several studies using either pulseor continuous “C02-labeling techniques (4,7.13- 1 S ) , and estimates of carbon rhizodeposition vary considerably.
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The proportion of net fixed C released from roots has been estimated to be as much as 50% in young plants ( 1 3) but less in plants grown to maturity in the field (16,17). Lynch and Whipps (18) estimated that as much as 40%of the plant's primary C production may be lost through rhizodeposition, depending on plant species, plant age, and environmental conditions. Although many studies have attempted to quantify the amountof rhizodeposition associated with various plant species, relatively little is known about the exudation process itself. While early studies of rhizodeposition assumed that once C compounds were lost from the root they were irretrievable. it has more recently been ascertained that this is an oversimplification of soil-root C fluxes. Studies by Jones and Darrah (19-21) found, for example,that the influx or resorption of soluble low-molecular-weight carbon compounds may play an important role in regulating the amount of C lost by the root. Good reviews of carbon flow in the rhizosphere and techniques used to quantify the dynamics of carbon flow include those of Meharg (7) and Grayston et al. (22).
D. NitrogenRhizodeposition-CurrentEstimates In addition to carbon rhizodeposition, also of considerable importance to nutrient cycling, is N rhizodeposition usually as NH,' (23). NO3- (24), amino acids (2527), cell lysates, sloughed roots, and other root-derived debris. Despite the fact that N deposition has a significant rolein N cycling and rhizosphere N dynamics, studies of N input from the root have been fewer, mainly due to methodological problems.JanzenandBruinsma(28)estimated that forwheat,up to SO% of the assimilated N was present below ground and approximately half of this was apparently released from the root into the rhizosphere soil. In barley, 32 and 7 1% of the below-ground N was present in rhizodeposits after 7 and 14 weeks plant growth. At maturity, the rhizodeposition of N amounted to 20% of the total plant N (29). It is also well known that substantial amounts of N may be released from roots of legumes (23,30,31). Jensen (29), for example, found that N deposition constituted IS and 48% of the below-ground N in pea when determined 7 or 14 weeks after planting. At maturity, the rhizodeposition of N amounted to 7% total plant N.
E. Coevolution of Plants and Rhizobacteria While rhizobacteria may derive obvious benefit from the significant quantities of root exudates released into the rhizosphere, the microbes of the rhizosphere, in turn, have a significant influence on the nutrient supply to the plant by competing for inorganic nutrients and by mediating the turnover and mineralization of organic compounds. Thus the deposition of organic materials stimulates microbial growth and activity in the rhizosphere, which subsequently controls the turn-
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over of C, N, and other nutrients (32-34). Rhizodeposition is also considered to be of importance for soil organic-matter dynamics in terms of nutrient mineralization and improvement of soil structure. These soil microbe-mediated processes of nutrient mineralization and N immobilization are strongly influenced by the covered presence of easily decomposable substrate in the rhizosphere-a topic in more detail in Chap. 6. While rhizodeposition strongly influences the size and activity of microbial populations at the soil-plant interface, the activity of these microbial populations in turn affects plant health and thus influences both the quality and quantity of to bacteria or a rhizodeposition. The potential for either an exudation response response by bacteria to exudation suggests a certain degree of coevolution between plants and rhizobacteria (35). Since the composition and extent of exudation is largely determined genetically (36,37) and may incur a substantial metabolic cost, exudation must providea selective advantage to plants. Indeed, Bolton et al. (37) have suggested that root exudation evolved in plants to stimulate an active rhizosphere. This is feasibleif one considers that there exists ahigh degree of selectivity for rhizobacteria according to host plant genotype and that certain microbial interactions in the rhizosphere have the abilityto improve plant growth and plant health. While componentsof the stimulated rhizosphere microbial community have the ability to be either beneficial or harmful to the plant, in terms of plant nutrition and plant health, there is most likely to be a balance between beneficial and detrimental organisms (38).
F. The Study of Soil-Plant-Microbe Interactions The increase in information concerning interactions at the soil-plant interface has resulted mainly from the development of new techniques to quantify microbial populations in soil, to collect and analyze root exudates, and to study microbial interactions at the root surface. The recent application of electron microscopy has further provided us with a greater understanding of the spatial distribution of microorganisms on root systems. The refinement of analytical techniques has permitted the elucidation of root exudate composition (see Chap.2). Radioactive labelling techniques havenot only permitted quantificationof root exudation,but have also facilitated the identification of the precise locations of exudation sites along the root. Rovira and Davey (39), for example, found that the region of meristematic cells behind the root tip is a site of major exudation of sugars and amino acids. Besides quantification of root exudation and microbial colonization, knowledge of the growth of microorganisms in the rhizosphere in relation to the supply of organic nutrients is still an ongoing research goal. Such knowledge is necessary for evaluating the significance of microbial processes affecting plants. Several authors have assessed microbial growthin the soil and correlated the energy input
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provided by the addition of organic matter with the sizeof the observed microbial biomass. Implicit in any such calculation is a factor representing the energy requirementformaintenance of theexistingpopulationgenerallyexpressedas maintenance coefficient ( m ) or specific maintenance ( a ) . Barber and Lynch (9) investigated microbial growth in the rhizosphere of barley plants grown in solution culture either under axenic conditions or in the presence of a mixed population of microorganisms. It was found that more biomass was produced than could be accounted for by the utilization of the carbohydrates released by the roots grown i n the absence of microorganisms, supporting the view that microbes stimulate the loss of soluble organic materials. It was suggested that the kinetics of growth i n the rhizosphere and soil approximate most closely to those i n "fed batch" culture. Pirt (40) had shown that a "quasi-steady state" could be achieved in such a system and that therefore the same equations could be used to describe microbial growth in the rhizosphere. Thus Overall rate of consumption = consumption for growth consumption for maintenance
+
where p is specific growth rate (h"), x is the biomass (g), Y is the observed growth yield (gdry wt substrate), Y(; isthe"true"growthyieldwhenno energy is used in maintenance (g dry wt g" substrate), and 171 is the maintenance coefficient (g substrate g" dry wt h"). Barber and Lynch (9) used this equation to recalculate data from previous studies on microbial growth in soil, using a constant maintenance coefficient(m). They found no case where energy input exceeded the requirement for maintenance. and suggested, therefore, that apart from zones immediately around recently incorporated plant and animal residues, appreciable and continuous activity in soils can be expected only in the rhizosphere. As an example of how this related to microbial processes inlportant to the plant, nitrogen fixation was considered i n light of these results. Postgate (41) had shown thatfree-livingN?-fixingbacteriaproducedonly 10-15 n1g N g sugar consumed and that, therefore, this process was of negligible significance in the bulk soil because of the limited availability of substrate. In the rhizosphere, on the other hand, Dobereiner (42), having shown that Azotohrrctrr pnspali could fornl an association with the roots of the tropical grass P u s p l u m r w f u t u m , proposed that N2-fixing processes could be of significance to the N economy of the plant. The data of Barber and Lynch (9) were used to estimate if such an association could ever be of significance to temperate cereals. Assuming the total exudation of 0.2 mg sugar 1ng-l plant dry weight were utilized in Nzfixation with the efficiency quoted by Postgate (41), only 2-3 pg N nlg" plant dry weight would be fixed, which at most is about 15% of the total N content of the plant. The
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actual amount fixedwould be less than this,since evenwhen seeds are inoculated, azotobacters account only for approximately 0.3% of the total rhizosphere community of the resulting plants (43). Thus it was argued that this process could cause an appreciable increase in the N supply to temperate cereals only if larger quantities of carbohydrates were exudedby roots into the soil than were observed in previous studies (44) orthat the efficiency of N2 fixation greatly exceeded that observed by Postgate (41). Another microbial process important to the plant is the mineralization of organic matter in soil. This process is highly dependent on the growth and activity of microbes in the rhizosphere or those associated with organic residues present i n the soil to make mineral nitrogen available for plant uptake. Brimecombe et al. (45) found that inoculation of pea seeds with two P.seuck,rnonns juorescer~,s strains led to increased uptake of nitrogen from I5N labeled organic residues incorporatedintosoil. In contrast,themineralization of organicresidues i n the rhizosphere of wheat inoculated with the same strains decreased (46). Further work in ourlaboratorysuggests thatplant-specificchanges i n root exudation patterns mediated by the introduced strains are responsible for the changes to soilsaprophyticactivities,which,inturn,aremediated by effects on thesoil microfauna (unpublished).
G. The influence of Plant and Microbial Factors It hasbeenfoundthatmany environmentalfactorsinfluencetheamountand composition o f root exudates and hence the activity of rhizosphere microbial populations. Microbial composition and species richness at the soil-plant interface are related either directly or indirectly to root exudates and thus vary according to the same environmental factors that influence exudation. In essence, the rhizosphere can be regarded as the interaction between soil, plants and microorganisms. Figure2 shows some of the factors associated with these interactions, which will be discussed during the courseof the chapter. Herewe mention briefly the influence of some plant and microbial factors on root exudation and rhizosphere nlicrobial populations, while soil factors are discussed later.
1. Plant Species Gross differences have been observed in the amounts of fixed carbon released by annuals and perennials (47),with annuals releasing much less C than perennials. This effect may i n part be due to perennials having to invest more of their assimilates to survive year round. Between more closely related plants, several studies have reported that both the quantity and quality of root exudates vary between plant species (39,48,49). In addition, it is also recognized that different cultivars of the same species mayvary in their root exudation patterns. For example, Cieslinski et al. (SO) quantified low-molecular-weight organic acids released
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Speciedcultivar GrowtNDevelupment Nutrition
/
RHIZOSPHERE
MICROORGANISMS GrowtMSurvival Interactions Nutrient Supply
\
Physical - Tmpcrature, p1 L 0, availability, water content. Structural - soil type, porosity. clay content. f d t y . .4giculturaladdition, Fertilizer herbicide.C% pesticide application
Figure 2 Factorsinfluencingrhizosphereinteractions.
from the roots of five cultivars of durum wheat and four cultivars of flax and found significant variation between cultivars. The quality of compounds released by plant roots appears to strongly influence the bacterial composition and activity in the rhizosphere, as shown by the preference of certain bacteria for exudates of different plant roots ( 5 1,52). Differences in bacterial activity between cultivars of the same plant species have been shown to be related to differences in exudation spectra (i.e., subtle differences in compounds released by roots of the different cultivars) (53). This suggests that it may be possible to manipulate the rhizosphere flora through genetic changes in the host plant. Of particular interest is whether different varieties, by exuding different compounds,can influence the rhizosphere flora in a way that would benefit the plant.
2.
Plant Age and Stage
of Development
Root exudation and microbial colonization haveboth been shown to change with plant age and stageof development. The quantityof both proteins (54) and carbohydrates ( 5 5 ) released by herbaceous plants has been shown to decrease with increasing plant age. Liljeroth and Biith (56) found bacterial abundance on the
Effect
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rhizoplane of several barley varieties significantly decreased with increasing plant age. Keith et al. (16) measured relative amounts of carbon translocated to the roots and rhizosphere during different developmental stages of wheat grown in the field. As the crop developed,it was found that proportionally lessof the total photosynthesized carbon was transported below ground, with a marked decrease after flowering. Microbial numbersin the rhizosphere had previouslybeen shown to increase over time, reaching a peak around the time of flowering and then decreasing (57). Such changes in microbial colonization could be due to changes in total amounts of carbon exuded per unit root produced or related to changes in the quality of exudates released.
3. PlantGrowth Prikryl and Vancura (58) found that the release of root exudates from wheat roots was positively correlated with root growth. The amounts of substances released by the roots were directly associated with root growth, and in plants where almost no root growth was observed, almost no root exudation occurred even in plants whose shoots were actively growing. This study confirmed results obtained by Prat and Retovsky (59), who found that live, intact, but nongrowing roots had lower exudation rates than the roots of plants in the period of rapid growth. A possible explanation for this may be provided by the results of Frenzl (60), who found that exudation depends considerablyon the physiological stateof the superficial root cells. It would appear therefore that root exudation is likely to be greatest from plants with actively growing root systems whose superficial root cells are in an active state. This may also explain why root exudation decreases with plant age, as metabolic activity of superficial root cells decreases with plant age.
4.
Presence of Microorganisms
The presence of microorganisms in the rhizosphere has been shown to increase root exudation (58,61-65). This stimulationof exudation has been shown to occur in the presence of free-living bacteria such as Azospirillurn spp. and Azotobtrcrer spp. (66,67) and in the presence of symbiotic organisms such as mycorrhizae (68,69). Increased root exudation has also been shown to be species-specific; for example Meharg and Killham (65) found that metabolites produced by Pseudo1 7 1 0 1 1 m trerwginosn stimulated a 12-fold increase in ’“C-labeled exudates by perennial ryegrass. However, under the same conditions, metabolites from an ArrhroDacter species had no effect on root exudation.
II. MICROBIAL INTERACTIONS IN THE RHIZOSPHERE During seed germinationand seedling growth, the developing plant interacts with a range of microorganisms present in the surrounding soil. Plant-microbe interac-
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tions may be considered beneficial, neutral, or harmful to the plant, depending on the specific microorganisms and plants involved and on the prevailing environmental conditions. Plantbeneficialmicrobialinteractions can be roughly divided into three categories. First, there are those microorganisms which, in association with the plant, are responsible for its nutrition (i.e., microorganisms that can increase the supply of mineral nutrients to the plant). This group includes dinitrogen-fixing bacteria such as those involved in the symbiotic relationships with leguminous plants (for example,Rhizobilrrr~and Btudyrhizobiutn species) (see Chap.IO), with monocots (for example, Azo.spirill~ltnbrasileme) or free-living nitrogen-fixing bacteria such as Klebsiella pneumoniae. In addition, there are a numberof microbial interactions that increase the supply of phosphorous (for example, mycorrhizae) (see Chap. 9) and other mineral nutrients to the plant. Second, there is a group of microorganisms that stimulate plant growth indirectly. by preventing the growth or activity of plant pathogens. Such organisms are often referred to as hiocontrol agents, and they have beenwell documented. A third group of plant-beneficial interactions involve those organisms that are responsible for direct growth promotion-for example, by the production of phytohormones. Plant growth-promoting rhizobacteria (PGPR) or plant-beneficial microorganisms and their use to increase plant productivity have been the subject of several reviews (70-75), and examples are discussed below. Neutral interactions are found extensively i n the rhizosphere of all crop plants. Saprophytic microorganisms are responsible for many vital soil processes, such as decomposition of organic residues in soil and associated soil nutrient mineralizationhrnover processes. While these organisms donot appear to benefit or harm the plant directly (hence the term neutml), their presence is obviously vital for soil nutrient dynamics and their absence would clearly influence plant health and productivity. Detrimental interactions within the rhizosphere include the presence and action of plant pathogens and “deleterious rhizobacteria.” Root exudates play a key rolein determining host-specific interactions with, and the composition of, their associated rhizobacterial populations. Root exudates can attract beneficial organisms such as mycorrhizal fungi and PGPR (13,76), buttheycan alsobeequallyattractive to pathogenicpopulations(77,78). As mentioned above, it is the balance between beneficial and detrimental microorganisms thatultimatelygoverns plantnutritionandplanthealth.Beforediscussing some of the specific interactions mentioned above, considerationis given to the microbial colonization of the rhizosphere.
A.
Colonization of theRhizosphere
Studies have shown that rhizobacteria are better adapted to the colonization of roots than bacteria isolated from nonrhizosphere soil (79); this is further evidence
ExudatesEffect of Root
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to support the theory of coevolution of plants and their associated rhizosphere microbial populations. Root colonization can be consideredto involve four stages. The initial stage of root colonization is the movementof microbes to the plant root surface. Bacterial movement can be passive, via soil water fluxes, or active, via specific induction of flagellar activity by plant-released compounds (chemotaxis). The second step in colonization is adsorptionto the root. This is a step required before anchoring and can be defined as nonspecific and based on electrostatic forces, whereas anchoring can be defined as the firm attachment of a bacterial cell to the root surface. Following adsorption and anchoring, specific and/or complex interactions between the bacterium and the host plant may ensue, which lead to induction of bacterial gene expression. Table 1 cites examples of root exudate components involved in these processes.
B. SpecificMicrobialInteractions 1. BeneficialInteractions C I. Dinitroger1Fixation. Species of Rhizobiutn and B~-n&rl~izobiunzhave long been known to induce the formation of root nodules on leguminous plants (see Chaps. 7 and 10). Once formed, a differentiated form of the bacterium, the bacteroid, converts atmospheric nitrogen into ammonia, which is then used as a nitrogen source by the plant. In return, the plant provides a carbon source to the bacteroid, probably in the form of dicarboxylic acid (74). Nodulation is hostspecific, and each (Bmdy)r.hizobiunz species can nodulate only a restricted number of legume species. Both nodulation and nitrogen fixation are complex processes, involving interactions betweena number of bacterial genes and their gene products and plant products. Specific compounds released by roots of young lein attractingthesesymbioticbacteria to theirroots(see gumesareinvolved Table 1). Followinginfection of thehostrootsystem,flavonoidcompounds released by the root hair zone of the plant are believed to be responsible for the induction of bacterial nodulation (nod) genes in Rhizobium species (91), which results in the biosynthesis of Nod factors. Nod factors are a group of biologically active oligosaccharide signals whose effects on the host legume are similar to the early developmental symptoms of the Rhizobium-legume symbiosis, including root hair deformation and nodule initiation (92). The role of organic signaling molecules between plants and microorganisms and the biochemistry of the association between rhizobia and their host plants are discussed more thoroughly in Chaps. 7 and IO, respectively. In addition to N,-fixing symbioses with legumes, associations between N2of monocotssuch as cerealsandforage fixing microorganismsandtheroots grasses have beenreported (93). However,the benefitofnitrogenfixation to nonlegumes was thought to be small, with studies showing that the contribution
Table 1 Root colonization” Process I . Movement of microbes to root surface A. Passive B. Active-induction of AageIlar activity (chemotaxis)
2. Adsorption 3. Anchoring A. Bacterial appendages (pili. fimbriae) 8. Agglutination C. Formation of cell aggregates 4. Gene expression A. Nodulation genes-production of nod factors
B. Virulence penes-production
of virulence factors
Exudate component
Microbial species
Luteolin/Phenolics Acetosyringone Benzoate/arornatics sugars Amino acids. nucleotides and sugars Serine Unidentified Unidentified Unidentified
Rfii,-cibiurn rnelilriti Agrohucterium tionefucieiis Arospirillimr spp. Agrobucreriurn tumefuciriis Pseudomorius laclz~mans
Lect i n s
Flavonoids
Reference
80 81
82 83 83
Pseuilomoncis aerugiriosa Pseudomnncis jhorescens Pseudonionas putida A;ospirilhcm briisilenw
83 83 83 84.85
Pseudomonos f7iioresrriis Pseudoinonas pufida Enterobiicter ciggloriieraris
86 8738
Rhirobiurn spp. Brudl\rliirobium spp. Agrobacteriirm fuinefuciens
YO,91.92
Table lists examples of root exudate components involved in chemotaxis. anchoring, and induction of gene expression.
89
90
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of N, fixation by Azospirillurn to plant growth was minimal, withyield increases ranging from 5-18% (85). It was also found that mutants unable to fix nitrogen ( N t f ) were capable of increasing plant growth, like a wild-type N2 fixer (94). It was suggested that the observed beneficial effects (increased yield, increasedwater and mineral uptake) of Azospirillum may derive from improvements to root development as a result of the action of phytohormones such as IAA, gibberellins, and cytokinin-like substances producedby the strain rather than from its nitrogenfixing abilities (85,94,95). However, more recentwork has initiated a reappraisal of the theory of N, fixation as a mechanismof plant growth promotionby Azospirillurn spp. (85). Mostimportantlyperhaps,resultsindicatethatgraminaceous plants are potentially capableof establishing associations with diazotrophic bacteria in which high ammonium-secreting Awspirillurn mutants provide the host with a source of nitrogen. 0. Mycorrhizrre. Thebiochemistryof theassociationbetweenmycorrhizae and the plant is extensively discussed in Chap. 9. Arbuscular mycorrhizal (AM) fungi interact symbiotically with approximately 80% of all plant species (96). Mycorrhizal symbioses are presentin most natural and agricultural ecosystems, where they are involved in manykeyprocesses-includingnutrient cycling, maintenance of soil structure. plant health, and enhancement of nitrogen fixation by rhizobia (97). Their primary effect on the plant is the improvement of plant growth by increasing the supply of mineral nutrients from the soil to roots. Arbuscular mycorrhizal fungi are known to influence phosphorus (P) uptake and plant growth in P-deficient soils (98,99). Several mechanisms have been proposed to account for the increases in uptake of phosphorus, one of the most important beingthe acidificationof the rhizosphere (100,I O l ) , which may explain how vesicular arbuscular mycorrhizal fungi increase the uptake of P as a result of plant utilization of ammonium (NHJ' -N). Higher P uptake as a result of plant utilization of NHJi occurs in bothneutral andalkalinesoilsand isdiscussed further on. More efficient utilization of ammonium by mycorrhizal than nonmycorrhizal plants has been shown by several authors ( 102,103) and may lead to increased H' secretion i n to the rhizosphere as a result (104,105). Rhizosphere pH has been reported to be altered by the form of nitrogen added both in thc presence and absence of mycorrhizae (102,106,107). It has also been reported that infection by ectotnycorrhizae significantly enhanced the capacity of plant roots to release H+ into a medium, which can increase the bioavailabilityo f compounds not readily soluble at higher pH (108). If a mycorrhizal plant induces a decrease in its rhizosphere pH, this effect may contribute to more P uptake by solubilizing calcium P and iron and aluminium phosphates, and thus increasing P availability to both the root and hyphae. I n general, the addition of nitrogen stimulatestheuptake of P by theplant,especiallywhen NH,+ is applied ( 109,l I O ) . Ortas et al.( 1 1 I ) found that application of ammonium led to increased
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plant dry weight and P content of sorghum as compared to plants treated with nitrate. These differences were enhancedby inoculation with mycorrhizae. It has also been shown that arbuscular mycorrhizal fungi can take up significant quantities of Cu, Zn, and Cd ( I 12), providing a mechanism bywhichplants avoid exposure to toxic quantities of these metals. The improved phosphate, nitrogen, or micronutrient uptake by plants as a result of mycorrhizal associations also has secondary effects on the uptake of other ions, such as potassium, sulfate, and nitrate. In addition, the increased uptake of phosphate indirectly stimulates nodulation and nitrogen fixation (74), since rhizobia require substantial amounts of this element for their activity. The biochemistry of the association between mycorrhizae and plants is covered i n depth in Chap. 9.
c. Biocontrol. Evidencesuggests that monoculture of cropseventually leads to decreased yields as a result of an increase i n plant pathogen numbers in the soil (74).Crop rotation has previouslybeen employed as a methodto alleviate this problem, by denying pathogens a suitable host for a period of time so that their numbers in soil decline. However, i t has also been noted that in some soil systems repeated monoculture eventually leads to a decrease in plant disease (74). In these cases the disease-conducive soils are converted to disease-suppressive soils. This phenomenon has led to the isolation of a wide range of microbial biocontrol agents from disease-suppressive soils ( 1 13). Mechanisms by which these organisms are known to antagonize plant pathogens are varied, and some of these are discussed below. In some instances, controlof a pathogen may result from a combination of two or more of these mechanisms. I. PRODUCTION OF ANTIBIorICS. Theproduction of secondarymetabolites with antimicrobial properties has long been recognized as an important factor in disease suppression (see Chap. 7). Metabolites with biocontrol properties have been isolated from a large number of rhizosphere microorganisms, including the fluorescent pseudomonads (Table 2). Further discussion is not given here since this is the subject of recent reviews (122,123).
11. PRODUCTIONOF SIDEROPHORES. Manyplantgrowth-promotingbacteria, especially pseudomonads, produce high-affinity Fe3'-binding siderophores under conditions of low iron concentration ( 1 24,125). This may result in severe iron limitation in the rhizosphere, which could limit the growth of other rhizosphere bacteria and fungi. Many authors have demonstrated the importance of siderophores in the inhibition of both fungal and bacterial pathogens (126-129). This topic is discussed in Chap. 8. 111. PRODUCTION OF VOLATILE COMPOUNDS. Volatilecompoundssuch as ammonia and hydrogen cyanide are produced by a number of rhizobacteria and are also believed to play a role in biocontrol. For example, Pseudomorfns jluorescens strain CHAO can produce levels of HCN that in vitro are toxic to
Effect of Root Exudates vi
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pathogenic fungi such as Tl~ieluviopsis hasicoh, and it is possible that this is the mechanism responsible for prevention of black root rot of tobacco in the field ( 1 30). I V . PARASITISM. Lysisbyhydrolyticenzymesexcreted by microorganisms is a well-known feature of mycoparasitism. Chitinase and p-1,3 glucanase (laminarase) are particularly important enzymes secreted by fungal mycoparasites capable of degrading the fungal cell wall components, chitin, and p- I ,3 glucan (131-134). Many rhizobacteria are classified as chitinolytic and, for example, Serruticl tncwsescrtzs, which excretes chitinase, was found to be an effective biocontrol agent against Sclerotium roljkii (135). Similarly, Aerornonus cclviue was found to reduce disease causedby Rhizoctonia solani, Fusariumo.vy.spor~c~n, and Sclrrotiurn roljkii (136). There is also evidence to support the role of p-1,3 glucanase in biocontrol of soil-borne plant pathogens (137).
v.
Elad and Chet ( 1 38), studying biocontrol of Pythiurn damping-off by rhizobacteria,suggestedthatcompetitionfor available carbon and nitrogen sources may account for observed disease reduction. They found competition for nutrients between germinating oospores of Pythi r m e ~ p h c ~ r ~ i d r ~ t ~ and ~ c rbacteria t ~ r t ~ ~(whichwasunique to isolatedbiocontrol strains) significantly correlated with suppression of the disease. It appeared that bacteria were competingwith germinating oospores for available C and N. and by eliminating these resources, the bacteria effectively reduced oospore germination. COMPETITIONFOR NUTRIENTS.
V I . COMPETITION FOR ECOLOGICAL NICHE. An alternative mechanism involved in biocontrol is that of niche exclusion. For PGPR to function successfully in the field, they must inevitably be able to establish themselves effectively on plant roots. Pseudornorlcls strains, for example, are able to establish on roots from inoculated seeds relatively easily. This is a major factor contributingto the success of these strains as biological control agents. However, root colonization is not the only criterion for defining a successful PGPR strain. Most diffusible root exudates are associated with the zone of root elongation, inducing colonization by PGPR in this zone. However, some studies have indicated establishment of populations along a greater. more undefined areaof the root surface, including root tips, zones of elongation, and zones of lateral root emergence. Since phytopathogenic bacteria occupy particular niches in the rhizosphere, it has been proposed that the deliberate application of a nonpathogenic mutant of the same species may prevent pathogen establishment through niche exclusion by the nonpathogenic mutant. An example of this was the deliberate release of a nonpathogenic Ice-. mutant of P.srudot~1onu.ssyringue to compete with plant pathogenic lce' P. syringae to prevent frost damage (139,140).The Ice' (pathogenic) strain of this organism causes frost damage to plants by making an ice
Effect of Root Exudates
111
nucleation protein that initiates ice crystallization at temperatures not normally favorable for ice formation. It was envisaged that deliberate release of the Icemutant of P. syrirrgrre from which theice nucleation protein gene had been deleted could prevent frost damage by colonizing niches previously occupied by the Ice' strain. In the field, it was found that frost damage was indeed decreased following the application of the genetically modified Icc- strain. v11. I N D U C E D DISEASE RESISTANCE. Anothermechanismresponsiblefor or the biological control of plant disease is induced systemic resistance (ISR) induced disease resistance. ISR protects the plant systemically following induction with an inducing agent to a single part of the plant. The action of ISR is based on plant defense mechanisms that are activated by inducing agents (141). ISR, once expressed, activates multiple potential defense mechanisms thatinclude increases in activity of chitinases, p- 1,3-glucanases, peroxidases, and other pathogensis-related proteins (142); accumulation of antimicrobial low-molecularweight substances such as phytoalexins (143), and the formation of protective biopolymerssuchaslignin,cellulose,andhydroxyproline-richglycoproteins (144-146). A single inducing agent can control a wide spectrum of pathogens. I n cucumber, for example, treatment of thefirstleafwitha necrosis-forming organism protects the plant against at least 13 pathogens, including fungi, bacteria, and viruses (147). Caruso and Kuc (148) showed systemic protection in cucumber and watermelon resulting from induction with Colleototrichrrm orhiculore against challenge inoculum of the same pathogen. Many other cases have been reported, but widespread application has not been accomplished, as classical ISR employs pathogenic organisms as inducing agents. More recent work, however, has demonstrated that some PGPR may act as inducing agents, leading to systemicprotectionagainstpathogens. In response toearlierobservations of PGPR-mediated ISR in the greenhouse, Wei et al. (149) carried out three 2-year fieldtrialstodeterminethecapacity of PGPR to inducesystemicresistance against cucumber diseases. Results indicated that PGPR-mediated ISR was operative under field conditions, with consistent effects against challenge-inoculated angular leaf spot and naturally occurring anthracnose. Furthermore, ISR induced early-season plant growth promotion and increased yield. In these studies, the pathogen and the resistance-inducing PGPR were applied at separate locations on the plant, excluding direct antibiosis and competition as mechanisms of disease suppression (149). d . Prodrction o f P h t Growth-Promoting Compounds. Alargenumber of rhizobacteria, such as strains of A z o s p i r i l h m . Azotohocter, P.~eurlor,lorlrl,s and Bucillus, have been shown to produce plant growth-promoting compounds such as indoleaceticacid(IAA),gibberellin, andcytokinin-likesubstances (93,95,150- 152). The presence of such compounds in the rhizosphere appears to stimulate plant growth directly.
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2.
DetrimentalMicrobialInteractions
cl. P l m f Parhogens. Interactions between microbial pathogens and plants are in general host-specific and consequently have been considered to be influenced by root exudate components. There are examples where amino acids, sugars,andotherexudatecomponentshave been shown todirectlystimulate pathogens (77,153). Stimulation of chlamydospore germination of Fuscrriurn solanifi phaseoli (154) and Thielviopsis hnsicola (155) was found to occur at the surface of bean seeds and roots but not in soil distant from the host. Phyrophora, PJIrhium,Aphanomyces, and other examples of pathogenic oomycetes have motile zoospores and consequently tend to accumulate around roots via chemotactic or electrostatic responses to root exudates (156). Germ tubes from spores of Phytophora cir~nnrnorni have also been shown to exhibit chemotrophisnl, becoming oriented toward the region of elongation of susceptible roots. Exudate-induced interactions unfavorable to propagule germination, growth, and colonization of roots by plant pathogens are often associatedwith the activity ofother miroorganisms in the rhizosphere. Sinceall these organisms occupy the same microhabitat, there is inevitably direct competition for nutrients and ecological niche. For example, Paulitz ( 1 57) found that hyphal growth from soil-produced sporangia of PyrhiLtnl ulrirnurn was stimulated by volatiles from germinating pea seeds, and this stimulation was reduced when seeds were treated with PseLc~lomonas ,puoresc'ens N 1R. It was thus suggested thatN 1 R may reduce damping-offby competing for and using volatile exudates from germinating pea seeds. In addition, plant pathogens may be antagonizedby compounds released by plant roots, plant residues, and other microorganisms present in the rhizosphere. Further, the survival and activity of specific plant pathogens may be affected by the action of antagonists present in the rhizosphere. To reiterate, it is the balance between plant pathogenic and antagonistic microorganisms that determines the effect on plant health.
h.DeleteriousRhizohacreria. Rhizobacteria thatinhibitplantgrowth without causing disease symptoms are frequently referred to as deleterious rhizobacteria (DRB), or minor pathogens. DRB caninhibit shoot or root growth without causing any other visual symptoms (158-160) and may be partly responsible forgrowthand yield reductions associated with continuous monoculture (160,161). DRB implicated in yield decline in a number of crops have been reviewed by Nehl et al. (35). Several mechanisms for growth inhibition by DRB have been proposed, the most likely being the production of phytotoxins such as cyanide (162,163) and other volatile and nonvolatile compoundsyetasunidentified (164,165). An alternative mechanism by which DRB inhibit plant growth may be through the production of phytohormones (160). IAA produced by DRB has been shown to inhibit root growth in sugarbeet (166) and blackcurrant (167). DRB may also compete with the plant and beneficial rhizobacteria for nutrients,
Effect
Root Exudates
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which may also contribute to decreased plant growth and yields. Further, DRB may indirectly reduce growth by inhibiting mycorrhizal development (168) or by counteracting the effects of nitrogen-fixing rhizobacteria (35). The pathogenicity of DRB to crop plants hasbeen shown to be host-specific (35) and thus is conceivably linked to root exudation. Alstrom (169) found that the pathogenicity of two isolates of Pseurlo~~~or~ns was determined by the major components of the broth culture in which they were applied to bean seedlings. Both isolates were pathogenic to bean seedlings when the broth contained sucrose and peptoneor sucrose and yeast extract. When the broth contained sucrose alone, one isolate was pathogenic and the other wasnot. Neither isolate was pathogenic when the broth contained yeast extract or peptone alone (169).
111.
MICROBIALCOMPOSITIONINTHE RHIZOSPHERE-METHODOLOGY
Root exudates are believedto have a major influenceon the diversity of microorganisms within the rhizosphere (78,170). Several approaches havebeen employed to measure the biodiversity of rhizosphere microbial communities. Most of the earlier studies relied on dilution plating procedures with selective culture media as a way to enumerate specific groups of microorganisms. Problems associated with such methods include thelow culturability of soil microorganisms ( 1 - 10%) (17 1 ) and the difficulty in adequately defining species of different microorganisms. Subsequently, techniquesof molecular biology and fatty acid analysis have increased the ease of identifying organisms without the requirement for isolation in pure culture. Community diversity in soil has been measured using a number of indices, some of which have been applied to the experimental study of rhizosphere microbial populations.
A.
MicroorganismsAssociatedwithGerminating and Young Roots
Seeds
Rhizospheremicroorganismsmayarisefromseed-bornepopulations thatsurvived seed storage and germinationor from soil-borne populations. The microbiological characteristics of the germinating seed are not often addressed but could be important for establishment of some saprophytic microbes intheendorhizosphere. It is thought that most recruitment of microorganisms for subsequent colonization of the plant root and rhizosphere soil takes place after growth stimulation of the microbes by the advancing root. The root tip may be described as a slowly advancing point source of substrate, which actsas a stimulus for resumed activity and growth by the dormant microbes in the bulk soil. Figure 3 shows
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l Noh-energylim t e d bacterla
Figure 3 Model of proposed interactions in the rhizosphere and in the bulk soil.
a model of proposed interactions within the rhizopshere in terms of microbial colonization and utilization of root exudates. Early microbial successions have been studied by Van Vuurde and Schippers (173, who found the invasion sequence of (in order of appearance) various rhizobacteria, coryneforms, true actinomycetes and microfungi associated with the onset of epidermal and cortical cell senescence in young roots (1-2 weeks old). Liljeroth et al. (174) washed 1- to 2-day-old root tips and 8- to 9-day-old root bases of wheat seedlings before maceration and plating onto 10% tryptone soya agar. Early colonizers were characterized in terms of carbon substrate utilizationandclusteredinto 1 1 groups of both gram-negativeandgram-positive bacteria. Utilization of simple sugars such as lactose, galactose, mannose, xylose, and mannitol and of organic acids such as citrate and succinate was found more often in populations from the root tips as compared to those isolated from root bases. The advancing root tip is considered to be an important site for exudation of simple sugars and organic acids, which may have stimulated the growth of rhizobacteria utilizing these compounds. Kleebergeretal. (175) found fluorescent P . s e u ~ i o ~ ~ ~ spp. o r ~ ato s bethe largest group of gram-negative bacteria and coryneforms the largest group of gram-positive bacteria to colonize the endorhizosphereof wheat and barley when surface-sterilized root segments were plated onto plate-count agar. They also suggested that the coryneforrns were primarily located in the external part of the
Effect of Root Exudates
endorhizosphere (i.e., the mucilage layer) segments with untreated roots.
B.
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by comparing surface-sterilized root
Microorganisms Associated with Mature Roots
Several studies have indicated that the species diversity of indigenous soil communities will influence the species composition of ectorhizosphere populations (176). On mature roots, seasonal successions may be observed as the soil microbial activity varies with temperature, water content, nutrition, and root exudation. Acero et al.( 177) foundthat the composition of alder (Alnlrs) rhizosphere populaBacillus spp. in autumn and winter tions alternated between one dominated by and one dominated by Pseudor~~or~as spp. in spring and summer. Lambert et al. (178,179) investigated the total cellular protein compositions in culturable bacteria as a way of comparing microbial diversity in crop rhizospheres. Such proteinprofiles,characterizingtheprotein composition in each clone of the culturable bacteria, were used to distinguish specific strain clusters among fast-growing rhizobacteriain the endorhizosphereof maize and sugarbeet in twoseparatestudies(178,179). I n thesugarbeetstudy, proteinprofiles in whole-cell digestsof organisms grownon 10% tryptone soya agar were compared after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( 179), and it was found that P . jluorescerz.s or Spkingomorzas paucirnohilis were predominant on therootsurface of young sugar beets until June, after which time X c ~ n r h o m o n ~mnltophila ~.s and Ph):llob~~ctrril~rrz spp. were found at increasing densities. During the plant growth season, microorganisms become more dependent on mobilization of organic matter present in the soil. Gram-positive microorganisms, including coryneforms and true actinomycetes become increasingly more abundant in the rhizosphere of maturing plants (174). Miller et al. ( I 80) used 10% tryptone soya agar to obtain total bacterial counts in rhizosphere soil of 1to 2-month-old wheat cultivars. Using two selective media for the fluorescent Pseudornorm spp., it was found that this group accounted for only a small percentage (- 1 %) of the total population. This maypossibly reflect that pseudomonads are abundant only among rhizobacteria during the initial growth phases of wheat. They also found that coryneforms constituted a significant proportion of the total population. On two different wheat cultivars, coryneforms accounted for between 4 and IS% of the total populations. Coryneform numbersin the bulk soil made up to 30% of the total bacterial population, suggesting that these soilborne coryneforms colonized therhizosphereasplantsaged. In asubsequent study, Miller et al. ( 1 8 1) estimated the relative abundance of true actinomycetes in the rhizosphere of 3-to 10-week old wheat cultivars. Isolation on selective chitin-oatmeal agar gave relatively stable numbers of actinomycetes over the 3to IO-week growth period. It was concluded that root exudation did not control
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the establishment of true actinomycetes in the rhizosphere soil (181). It is suggested that their high capacity for polymer degradation, resistanceto desiccation, and relative insensitivity to toxic compounds gives these organisms a selective advantage when root exudates become sparser in mature plants. De Leij et al. (182) developed a method for describing microbial community structure based on the quantification of bacterial colonies in six or seven growth classes on agar media. The idea of quantifying bacterial populations according to their growth rateis based on the ecological conceptof r- and K-strategy ( 1 83). Typical r-strategists do well in uncrowded, nutrient-rich environments, are inefficient in breaking down recalcitrant substrates, and are also thought to be well in crowded sensitive to toxins in the environment. K-strategists do relatively environments that have reached their carrying capacity, having more efficient cell metabolism than r-strategists, and are able to utilize recalcitrant substrates. It is also thought that they are less sensitive to toxins than r-strategists. Under field conditions, it was found that as wheat root matured, the microbial commutonity changed from one dominated by r-strategists to one distributed more ward K-strategy. De Leij et al. (184) used this method to assess the impact of fieldrelease of geneticallymodified P. Juorescens on indigenousmicrobial communities of wheat. The concept of r-K strategy distribution for risk-assessment purposes is thought to be useful ( 1 84), since r-strategists are characteristic of environments that undergo rapid changes, while K-strategists dominate in stable, non-perturbed environments(1 83,185). Populations of r-strategists can markedlyincreasewhenconditionsarefavorable,whereas-because of their poor competitiveabilities andlack of long-termsurvival mechanisms-their numbers decline rapidly when conditions deteriorate. In contrast, the population size of typical K-strategists is more buffered against perturbation because of their slow growth and ability to form long-term survival structures. Rapidly growing bacteriaandyeasts,whichmightallberegarded as typicalr-strategists,were found to be sensitive indicators of environmental change and responded with large population decreases in the presence of wild-type and recombinant P.j u o rescens SBW25 during early stages of wheat development ( 184).
IV. INFLUENCE OF SOIL FACTORS In addition to the interactions between plants and microorganisms, a third factor, the soil, also plays a role in determining root exudation and the activity and diversity of rhizosphere microbial populations.In this section, physical and structural aspects of the soil are discussed in relation to their effects on root exudation and microbial populations. Consideration is also given to the role of agricultural management practices on rhizosphere processes. In addition, the role of other biotic factors, such as microfaunal predation, is discussed in relation to nutrient cycling in the rhizosphere.
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PhysicalFactors
The effects of physical factors such as temperature, soil moisture content, pH and oxygen availability on microbial survival and activity in soil are well documented (for a review, see Ref. 90). It is also widely acknowledged thatthese same factors may also influence plant growth and can therefore be presumed to influence both root exudation and rhizosphere microbial populations.
1. Temperature Extremes of temperature influence both microbial and plant growth. Cell division and expansion are temperature-dependent processes that control the rate of both root growth (186) and microbial proliferation. Above and below the temperature range to which a plant is adapted, root growth may be inhibited or even irreversibly arrested ( 1 87). Similarly, above and below specific microbial adaptive temperatures, cells may become inactive or decline in numbers in soil. Many aspects of cell metabolism are adversely affected by temperatures that depart from the optima, including cell division, protein synthesis, respiration, and ion transport ( 1 88). These effects inevitably apply to both plant (189) and microbial cells. Root exudation is affected both qualitatively and quantitatively by temperature, with sudden increases or decreasesin temperature stimulating exudation (for a review, see Ref. 22). For example, Martin and Kemp (190) found that a reduction in temperature from I5 to 10°Cincreasedcarbon loss fromroots of 1 I different cultivars of wheat into the rhizosphere. Qualitative differences in exudation patternswereobservedwhen Zea t n n y seedlings,growninitiallyat19"C,were subsequently grown at 5°C. This led not only to greater exudation but also to the exudation of three new oligosaccharides and sucrose, compounds not previously observed atthehighertemperature (19 I ) . Similarly, Rovira (192) found that raising the temperature increased the amount of root exudate andtherelative proportions of amino acids exuded by Trifolium repens and Lycopersicotl escw letltutn.
Unfavorable temperatures, especially when suddenly imposed, have been shown to cause leakage of ions and metabolites from the root to the surrounding medium (188). In addition, sudden changes of temperature cause a lowering of conductivity to water of roots and inhibition of ion transport ( I 88). This can indirectly affect the carbon economy of the whole plant by leading to more negative leaf water potentials, partial stomatal closure, and hence a slowing of net CO, assimilation. Some roots, however, display considerable ability to acclimate to suboptimal temperatures. For example,in rye or barley acclimated by previous exposure to 8"C, net transport of K.', Ca?+, and H,POJ- into the xylem sap of detached roots was enhanced by factors of 2 to 3 as compared to controls maintained at 20°C (193,194), with a threefold increase in flux of water to the xylem. These changes almost completely compensated for the smallerroot systems that devel-
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oped at the lower temperature. In addition, low-temperature acclimated roots contained 9- 15 times more soluble carbohydrates than the controls at 20°C (195). The carbon efflux to the rhizosphere under such conditions, therefore, might be considerably enhanced if permeability to the carbohydrates was unaffected by temperature ( 188). Freezing injury is another factor to be taken into account in considering the influence of low temperatures on rhizosphere dynamics. Freezing injury occurs whenicecrystalspenetratethe plasma membrane and cause mechanical rupture.Thiseffect is irreversible,anduponthawing,leakage o f solutesand metabolites from the cytosolis concomitant with cell death. The releaseof carbon compounds to the microbial community in the surrounding soil and rhizosphere of surviving roots is therefore likely to be considerable. In general, low temperature tends to slow root extension rather than cell maturation; consequently, the endodermis frequently becomes suberized and thickened closer tothetip. This can lead to a block in apoplastic movement of water and solutes. The effects of suberization on the transport of carbohydrates in roots and leakage of carbon compounds from the rootsto the rhizosphere have as yet received little attention.
2. Water Availability Anothermajorfactoraffectingmicrobialactivity insoilistheavailability of water, which is variable, depending on factors such a s soil composition, rainfall, drainage, and plant cover. The primary importance of soil water is to provide the moisture necessary for the metabolic activities of both soil microorganisms and plants. Under conditions of water deficit, both plants and microbes may be harmed. When roots extend into a dry soil, for example, the apical zone may develop a water deficit and lose turgor, so that cell expansion rates are slowed ( 196,197).Osmotic adjustment takes place over a period of days during acclimation to water deficits that are gradually imposed, so that the roots and shoots can maintain low or negative water potentials and thereby maintain the flowof water from the rhizosphere to the root without the simultaneous loss of turgor ( 1 88). In general, roots seem less sensitive to water deficit than shoots; however, growing cells in the root tip zone readily lose water to a drier environment, and-for example, in maize-irreversible damage to cells occurs when water loss exceeds 70% (198). Desiccation causes changes to the physical and chemical propertiesof plant cell membranes. In mature roots of barley, for example, desiccation caused degeneration of the epidermis and outer cortex (199), prcsumably resulting i n the release of appreciable amounts of carbon compounds to the rhizosphere. In an earlier study, it was suggested that both C secreted as mucilage and C released by root tissue increased in zones of localized water stress, even where other parts of theroot system had adequate water supply (200). Soil water stress in tree species has been shown to both enhance and reduce root exudation (22). Effects of water stress on root exudation are more fully discussed in Chap. 3.
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3. OxygenDeficit Waterlogging can lead to the development of anaerobic conditions and oxygen deficit. In well-drained soils, air penetratesreadily and oxygen concentrations are usually relatively high. In waterlogged soils, however, the only oxygen present is that dissolved in the soil water, and this is rapidly consumed by microbes or plant roots, leading to the development of anaerobic conditions. Under these conditions, roots and soil aerobes are in direct competition for oxygen; indeed, microorganisms have been shown to account for up to 50% of the O2 consumption of a soil densely rooted with arable crops (201). When this oxygen has disappeared from soil pores, a combination of chemical and biological transformations take place, resulting in the release to the soil solution of a sequence of reduced substances: NO?-, then Mn2+,Fe?', and H 2 S as well as microbial end products such as acetic and butyric acid. Some of these can reach concentrations in the rhizosphere thatare damaging torootsorphytotoxic if accumulated in theleaves (202). The rate of production of these compounds depends on the rate of supply of substrates for microbial activity and on the chemistry of the soil, including the presence of alternative electron acceptors such as NO3-, Fe3+,Mn'+, and SO4?-.However, lack of oxygen around roots alone is sufficient to induce injury (203). Root cells quickly experience a decline in aerobic respiration if oxygen is not replenished with a concomitant decline in energy status (204,205). Other physiological changes associated with oxygen deficiency include the inhibition of ion uptakeandtransport toshoots(203) andlowconductivitytowater (206,207). In addition, leakage of solutes to the soil environment can occur as a result of decreased energy status within the roots. Oxygen availability appears to have both qualitative and quantitative effects on root exudation. Lack of oxygen has been shown to enhance root exudation (208). Further, it has been reported, for example, that Pisurn srrtivum exudes more amino nitrogen under low oxygen concentrations (209). It has also been found that, under anaerobic conditions, Zra m a y s exudes ethanol at the expense of plant sugar content (210), and ethanol is a chemoattractant to plant pathogens. Other plant species (for example, peanut) have been shown to release more sugars under anaerobic conditions (21 I ) . Decreasing oxygen concentrations tend to increasethepermeability of the cell membrane, owing to a reduction inactive transport, resulting in increased exudation (22).
4.
pH and Availability of Nutrient Ions
Both pH and the availability of nutrient ions in soil play important roles in rhizosphere dynamics and are often dependent on one another. Nutrient ions movein soil toward plant roots either by mass flow with the soil water or by diffusion. Mass flow is the result of bulk convective movements of the soil solution toward roots, whereas diffusion occurs in response to a concentration gradient for a particular ion, which results from its absorption by the root and depletion from the
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surrounding soil. For typical agricultural soils, mass flow supplies CB2+, Mg”, Cl -,SO,’-, and usually NO-(-, but diffusion predominates for K’ and H 2 P 0 4 because of low concentrations of these ions in free soil solution. Where mass flow brings in ions at a rate much faster than absorption by the root surface, ions accumulate i n the rhizosphere. For example, concentrationsof Na” in saline soil can increase to injurious levels in the rhizosphere. Pronounced changes in the pH of the rhizosphere by as much as one or two units often occur under agricultural conditions, and especially in neutral soils where buffering capacity is least (212), due to the presence of both acid and alkaline cations. Such changes occur often because of the major influence of the form of nitrogen added on the cation/anion balance of plants. With NH,’-based fertilizer there is a marked solubilization of phosphate in calcareous soil because of the net release of H by roots. In contrast, NO1- fertilizer leads to more alkaline conditions dueto an efflux of OH- and tends to release phosphate from the chemical forms in which it is held in an acid soil. Such differences in pH caused by nitrate or ammonium nutrition can bring about large changes in the microflora (213,214) and i n the nature of microbial substrates released by the roots (38). The rhizosphere of legumes fixing N 2 also becomes markedly more acidic. The response of root exudation to acidic conditions is important; for example, it was found that exudation from wheat was higher at pH 5.9 than at 6.4 (215). It was postulated that the external pH altered the ionic states of compounds released and that this, in turn, affected their readsorption. The fact that external pH influences the state of released compounds (probably by modifying their charge) and affects their readsorption supports the suggestion of Jones and Darrah (1 9-2 1 ) that exudation is a balance reflecting both release and readsorption. Meharg and Killham (216) found that the amount of carbon lost from Loliunz per-enne increased from 12.3 to 30.6% with increasing pH from 4.3 to 6.0. It was suggested here that an increase in the microbial biomass combined with plant nitrogen limitation could have caused the observed increase i n exudation with increasing pH. It has also been noted that there are often large differences in rhizosphere pH between plant species growing in the same soil. In addition, differences in pH of more than two units have been found at different points along the root of an individual plant (217). In general, dicots tend to favor the uptake of cations over anions, so that acidification of the rhizosphere is usually greater than for monocots. which show a cation/anion uptake ratio close to unity. Nutrient availability also plays a major role in exudation, with deficiencies in N, P, or K often increasing the rate of exudation (218). It is believed that nutrient deficiency may trigger the release of substances such as organic acids or nonproteinogenic amino acids (phytosiderophores), which may enhance the acquisition of thelimitingnutrient(219,220). An examplehere might bethe release of phenolic acids such as caffeic acid in response to iron deficiency, which results in an increase in uptake of the cation (221). +
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5. Lightintensity Since most of the assimilated carbon in a plant is derived from photosynthesis, it follows that changes in light intensity may modify root exudation of carbon. However, Rovira ( 192) found that exudation of nitrogenous compounds was also affected by light intensity. In Trifoliunz repews grown under three different light intensities, the q~~antities of the amino acids serine, glutamic acid and a-alanine released were considerably reduced at the lower light intensities.In L.vcopersicorl e.scdetztum, quantities of aspartic acid, glutamic acid, phenylalanine, and leucine were significantly lower at the lower light intensities, but increases in the amounts of serine and asparagine exuded were observed.
6.
CarbonDioxideConcentration
C O z concentrations have been rising steadily over the last 40 years and are expected to continue to rise, though the magnitudeof the increase is uncertain (222). This could be expected to have important consequences for photosynthesis and hence exudation. However, Whipps (13) reportedthatthe loss of assimilated carbon from ZL'Nmn\a roots was unaffected by atmospheric CO2 concentrations up to 1000 pl I". In contrast, Norby et al., (223) found that carbon allocation to roots and root exudation increased in Lirioderdrotz tulipifer[/ grown in the presence of elevated COz levels. In Pitlus echinrrrtr seedlings, there was increased exudation under elevated COz after 34 weeks but not after 41 weeks (224). Rillig et a l . (225) carried out Biolog microplate analysis of soil C substratc utilization in the rhizosphere of Gderrezici scrrofhrrw grown in elevated atmospheric carbon dioxide. Compared to ambient CO? levels, they found polymers weremoreslowlyoxidized by themicrobialcommunity,amidesshowed no change. and all other substrate groups were more rapidly utilized, although there was 110 difference in the number of viable bacteria. This change in microbial function in response to elevatedcarbondioxidewithoutanychanges in total numbers of viable bacteria could have important impacts on nutrient cycling and may be due to changes in rhizodeposition under elevated COz.
B. StructuralFactors Soil type and structure also influence the dynamicsof rhizosphere microbial populations.Whethernutrientsareavailableforbacteria in therhizosphereoften depends on the sites in the soil where nutrients are present. Organic compounds tightly bound to the soil matrix are often less available for bacteria (226), and those present in smaller pore spaces can be physically protected against mineralization. However, disturbance of the soil often causes these nutrients to become more available to soil microbes (227). Soil textural aspects also influence bacterial survival, possibly by affecting
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the level of protection against predation by protozoa (90). The presence of clay minerals such as montmorillonite or bentonite has been shown to substantially improve bacterial survival (90,228). This appears to occur because some of the pore spaces in soil aggregates serve as protective microhabitats for soil bacteria against predation by protozoa (90), and it can thus be suggested that the greater the amount of protective pore space in heavier textured soils, such as clay soils, the greater the amount of protection against predation (229).
C. AgriculturalFactors 1. Application of MineralFertilizers There has been previous mention of the form of nitrogen fertilization and nutrient availability on rhizosphere pH andon the catiodanion balances in different plant species that affect both plant and microbial growth. However, the issue of nitroon root exudation and rhizosphere microbial gen fertilization and its influence populations is complex. Nitrogen fertilization has been shown to have variable effects on the ecology of the rhizosphere and may not always lead to predictable effects on microbial growth and activity in the rhizosphere. For example, Kolb and Martin (230) foundthat the application of N fertilizer stimulated root exudation in agricultural plants; they concluded that this may indirectly affect microbial growth in the rhizosphere. However, Liljeroth et al. (231) found that in wheat cultivars, stimulation of microbial growth seemed to be due to increased utilization of' theroot exudates themselves rather than to increased exudation rates. They also observed that N application resulted in higher bacterial abundance on seminal roots of young barley plants. In other cases it is believed that N fertilizer may sometimes stimulate root growth at the expense of exudation, and cultivars may respond differentlyin terms of stimulated exudation or increased root growth (176). In nonfertilized soil, however, lower nutrient availability may limit microbial utilization of root-released carbon compounds. This is an area that clearly needs further investigation. especially under the current trend to move toward sustainable agriculture and decreasing chemical fertilizer inputs.
2.
Effect of CroppingSystem
Alternative agricultural practices-including crop rotations, recycling of crop residues, and increased use of cover crops and green manures-contribute to high soil organic matter levels and improved soil quality, thus reducing the need for chemical fertilizers and pesticides. Agricultural management practices may be expected to have an impact on microbial diversity and thus affect soil health, It hasbeen hypothesized crophealth,and yieldandultimatelysustainability. thata more diverse soil microbial community will result in greater yield stability (232).
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Several studies have investigated the effects of different cropping systems and agricultural management practices on microbial biomass, activities, and diversityinsoil (233-235); however, comparatively few studies have looked at the impact of management practices on rhizosphere microbial populations and root exudation. Swinnen (236) studied carbon fluxes in the rhizospheres of barley and wheat under field conditions with conventional and integrated management using “CO2 pulse labeling. Compared with conventional management, integrated management lead to reduced transfer of carbon to the roots, reduced root growth, and lower total rhizodeposition. A 15-year study in which a conventional corn-soybean rotation was compared with two low-input systems (animal manure or legumes as N sources) was carried out by Buyer and Kaufman (237). The effects of the three cropping systems on diversity of fast-growing aerobic culturable bacteria and fungi in the rhizospherewerestudied.Followingextractionandplatingontosolidculture media, approximately 6000 bacteria were identified using fatty acid methyl ester analysis. Over 18,000 fungi were identifiedusing microscopic examination of spores. However, total counts and diversity were not significantly different between the three different cropping systems.
D. Biotic Factors In relation to nutrient cycling in the rhizosphere, another important factorto consider is the role of the microfauna (protozoa, nematodes, and microarthropods), and specifically their interactions with bacterial populations. There is increasing evidence thattheinteractionsbetweenmicrofloraandmicrofauna,especially nematodes and protozoa, are responsible for a significantportion of the mineralization of nitrogen in soil (238-243). Extensive studies on the relationship between soil protozoa and bacteria have revealed that soil protozoa can feed on a wide rangeof bacteria and that indeed bacteria are the most important food source for free-living heterotrophic protozoa (244). Since predation by protozoa removes bacteria, it might have been expected that this would lead to a decrease in bacterial activity and consequentlyin the decompositionof organic matter and mineralization of nutrients. However, the opposite has been observed many times. A stimulatingeffect of protozoangrazing onbacterialmetabolismwasdemonstrated for ciliates in marine habitats and for flagellates in freshwater habitats. Hunt et al. (245) developed a simulationmodel for the effectof protozoan predation on bacteria in continuous culture. Results suggested that upon predation by protozoa, the growth rate of bacteria increased even though the bacterial biomass was reduced. Bacteria were thought to respond to a higher level of available carbon, nitrogen, and phosporus upon predation. Elliott et al. (246) presented data suggesting a significant role of soil protozoa at the soil-root interface by accelerating the mineralization of microbially
et
124
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al.
immobilized nutrients. In the presence of protozoa, more mineral N was found in soil. In addition, plant shoot nitrogen concentration was higher as compared to plants grown in soils without protozoa. They hypothesized that the effect of protozoa on the mineralization of N would be greatest under the most N-limiting conditions-i.e., without the addition of mineral N fertilizer. However, it was only in the case where mineralN was added that protozoa accelerated the mineralization of microbially immobilized N. Data have also been produced indicatingthat bacteria can mineralize nitrogen from soil organic matter and that this process too can be increased by the presence o f protozoa (238). In the presence of protozoa, more nitrogen was made available to plants, and it was suggested that bacteria utilized the nitrogen from soil organic matter when supplied with a suitable energy source-i.e., root exudates. The nitrogen immobilized wouldthen become available to plants when predators such as protozoa consumed these microorganisms and excreted excess ammonium (238). Kuikman and Van Veen (239) investigated the impact ofprotozoa o n the availability of this bacterially immobilized nitrogen to plants. They found that protozoa reduced bacterial numbers by a factor of 8 and increased plant uptake of nitrogen by 20% in wheat. Grazing by protozoa was found to stronglystimulatethemineralizationandturnover of bacterialN.Grazingby bactivorous nematodes may also enhance the rate of N-mineralization in the rhizosphere (241,242)by excretion of ammonium and other nitrogenous compounds (247) or, indirectly, by dissemination of microbial propagules through the soil (248-250) or stimulation of bacterial activity by release of growth-limiting nutrients and vitamins(25 l ) . Furthermore, the removal of nonactive cellsby microbial grazers may provide new surfaces for microbial colonization.
V.
APPLICATION
Despite the fact that rhizosphere exudation patterns are governed by a large variety of factors (plant, microbial, and soil factors). it is clear that there is ample scope to modify root exudation patterns either by using agricultural practices or through plant breeding programs. Both plant breeding programs and agricultural practices have concentrated on reducing the negative impact of plant pathogens directly.Examplesincludebreedingprogramsthatrenderplantsresistant to pathogen attack, rotation schemes that prevent excessive buildup of pathogens, or application of pesticides that protect susceptible plants against pathogenic organisms. In relation to plant nutrition, plant breeding programs have concentrated on the development of plant varieties that give inherited high yields provided thatsufficientnutrientsareavailable. The latterconditionsaresatisfiedusing chemical fertilizers that are directly available to plants. Even though these practices have resulted in a dramatic increase in food production during the past half
ExudatesEffect of Root
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century. it is questionable whether these approacheson their own are sustainable in the long term. Resistant plant varieties have limited usefulness, as plant resistance will be broken by the pathogen in the absence of measures that reduce pathogen populations. Similarly, excessive use of pesticides will inevitably lead to the development of resistance in the pathogen population. Furthermore, lack of “organic” inputs will lead to a reduction of saprophytic biological activity in soil and, consequently, to a decline in the physical and chemical soil quality. This, in turn leads to an increasing dependence on chemical inputs i n the form of fertilizers and pesticides. The realization that increased yield and reduced incidence of plant damage resulting from pathogens can also be achieved indirectly by programs aimed at encouraging beneficial organistns in soil and the rhizosphere opens new possibilities for plant breeding and soil management programs. Root exudates released by the plant create a “rhizosphere effect,” resulting in intense microbial activity i n the vicinity of the roots. The influence of this microbial activity on plant health and nutrition depends on the net biological effect of the interactions between the rhizospherepopulations,theplant,andthesoilenvironment.Withincreasing knowledge of which factors influence this exudation process and their influence on rhizosphere microbial populations, it has become possible to nlanipulate these processes in favor of organisms that benetit the plant directly by providing biological control activity, growth stimulation. inductionof resistance, or by mineralization of organic residues. Indirectly, theactivity of soil (micro)organisms will result in improved soil quality (greater aggregate stability, improved soil structure, and better water-holding capacity), all of which benefit plant growth. Examples of the successful stimulation of microbial populations antagonistic to pathogens such as Take-all, Fusarium wilt, and cereal cyst nematodesusing continuouscroppingregimes,greenmanures, and farmyard tnanureshave been well documented (252-254). However, manipulation of rhizosphere populations using plant-breeding programs is at present not seriously pursued. The latter approach,when integrated with appropriate soil management strategies, might open new possibilities to release the full potential of microorganisms that benefit plants in a variety of ways. A further application of the manipulation of microbial activity in the rhizosphere is their potential to remediate contaminated land. Bioremediation involves the use of microorganisms that break down contaminants. Radwan et al. (255) found that the soil associated with the roots of plants grown in soil heavily contaminated with oil in Kuwait was free of oil residues, presumably as a result of the ability of the resident rhizosphere microflora to degrade hydrocarbons. The use of plants as a meansto accumulate pollutants such a s heavy metals (256,257) to degrade hydrocarbons and pesticides (255)is already widely implemented and has proven to be successful. In some cases, there is no doubt that it is the plant itself that is responsible for the removal of the contaminants. However, in most
Brimecombe et ai.
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cases, it will be the interactions that take place between plantroots, the soil biota, and the soil environment that result in the desired effect. Again, insight in the interactive processes associated with pollutant degradation will open opportunities to decontaminate land more effectively.
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16. H. Keith, J. M. Oades, and J. K. Martin. Input of carbon to soil from wheat plants, Soil B i o l o g y c u ~ r Biodlertlistr:\. l l8:4.55 (1986). 17. B. Jensen, Rhizodeposition by “C pulse labelled spring barley grown in small field plots on sandy loam, Soil Biology t r r d Biochemi.sty 25: 1.553 (1993). d 18. J. M. Lynch and J. M. Whipps, Substrate flow in the rhizosphere, Plur~tt ~ Soil 129:1 (1990). 19. D. L. Jones and P. R. Darrah, Re-sorption of organic compounds by roots of ZLW rnrcy.~L. and its consequencesintherhizosphere: I. Re-sorptionof “C labelled glucose, mannose and citric acid, P l m t ctnd Soil 143:259 (1992). 20. D. L. Jones and P. R. Darrah, Influx and efflux of amino acids from Zen r~1ay.sL. roots and its implications in the rhizosphere, Plnut t r r l d Soil 163:1 (1994). Zea 21. D. L. Jones and P. R. Darrah. Re-sorption of organic compounds by roots of mays L. and its consequences in the rhizosphere:111. Characteristics of sugar influx and efflux, P larlt nr~dSei( 178:1 53 (1996). flowintrees,in 22. S. J. Grayston, D. Vaughan, and D. Jones, Rhizosphere carbon comparison with annual plants: the importance of root exudation and its impacton microbial activity and nutrient availability. Applied Soil Ecology 5:29 (1996). 23. L. S. Brophy and G. H. Heichel, Nitrogen release from root of alfalfa and soybean grown in sand culture, Plrlnt urld Soil 116:77 (1989). 24. J. P. Wacquant, N. Ouknider, and P. Jacquard, Evidence for a periodic excretlon of nitrogen by roots of grass-legume associations, Plant o r d Soil I16:.57 (1989). 2.5. A. D.Rovira.Plantrootexcretionsinrelation to therhizosphereeffect: I. The nature of root exudates from wats and peas, Plant m d Soil 7 178 (1956). 26. A. D. Boulter. J. J. Jeremy, and M. Wilding, Amino acids liberated into the culture medium by pea seedling roots, Plant ancl Soil 24: I2 1 (1966). 27. M. G. Hale, L. D.Moore,and G. J. Griffin,Rootexudatesandexudation. Irlterwtiorls betn.ecw Not~-Pntlloget~ic Soil Microorgtlrlisn~s m t l Pltrr1t.r (Y. R. Dommergues and S. V. Krupa, eds.), Elsevier, Amsterdam, 1978, p. 163. 28. H.H.Janzenand Y. Bruinsma,Methodologyforthequantification of rootand rhizosphere nitrogen dynamicsby exposure of shoots to ” N labelled ammonia, Soil Biology cmd Biochertistry 21: I89 ( 1 989). 29. E. S. Jensen, Rhizodeposition ofN by pea and barley andits effect on soil N dynanics, Soil Biology urd Biochemistry 28:65 (1996). 30. A. L.Virtanen. S. VonHausen,and T. Laine, Investigations on the root nodule bacteria of leguminous plants: XIX. Influence of various factors on the excretion of nitrogenous compounds from nodules, Jourrml of Agricultural Scierm> 27332 ( 1937). 31. P. W. Wilson and 0. Wyss, Mixed cropping and the excretion of nitrogen by leguminous plants, Soil Scierlcc. Society of America Proceedjr1,q.s l ]:289 ( 1937). 32. L. Klemedtsson,P.Berg, M. Clarholm,and J. Schnurer.MicrobialtransformaSoil Biology (uld Biochemistry 19:55 I tionsintherootenvironmentofbarley, ( 1 987). 33. R. Merckx, A. Dijkstra. A. den Hartog, and J. A. Van Veen, Production of rootderived material and associated microbial growth in soil at different nutrient levels. Biology nr d Fertility of Soils 5 : 126 ( 1987). L
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34. D. Robinson, B. Griffiths. K. Ritz, and R. Wheatley, Root-induced nitrogen mineralisation: a theoretical analysis, Plant c m / Soil l I 7 185 ( 1989). 35. D. B. Nehl. S. J. Allen, and J. F. Brown, Deleterious rhizosphere bacteria: an integrating perspective, Applied Soil Ecology 5:1 ( 1997). 36. R.W.HedgesandE.Messens.Geneticaspectsofrhizosphereinteractions, The Rkizospl~ere(J. M. Lynch, ed.), John Wiley, Chichester. 1990, p. 129. 37. H. J. Bolton. J. K. Fredrickson. and L. F. Elliott, Microbial ecology of the rhizosphere, Soil Microhid Ecology (F. B. J. Melting. ed.). Marcel Dekker, New York, 1993. p. 27. 38. G. D. BowenandA. D. Rovira. The rhizosphere: The hidden half ofthehidden half. Plctr~tRoots, The Hiddet~H d f (Y. Waisel, A. Eshel, and U. Katkafi. eds.), Marcel Dekker, New York, 1991. p. 641. 39. A. D. Rovira and C. B. Davey. Biology of the rhizosphere, The plcrtlt root c u d its etIllirotlttrcwt (E. W. Carson, ed.), University Press of West Virginia, Charlottesville, 1974, p. 153. 40. S. J. Pirt, Pritrciples of Microhc c m ( / Cc4 Cdtiwrtiotl, Blackwell. Oxford ( 1975). 41. J . R. Postgate, New advances and future potential in biological nitrogen lixation. J o 1 w t d (fApplied Bnctc~riology3 7 I85 ( 1974). 42. J. Dobereiner. J. M. Day, and P.J. Dart. Nitrogenase activity and oxygen sensitivity of the Puspcrlurtl tlottrflotl-~;otohcccler-p u p d i association, Jolrrtltrl of Gcwcwrl Microhiology I 7 l03 ( 1972). 43. J. J.Patel, Microorganisms in the rhizosphere of plants inoculated withA ; o t o b ~ w r chrooc~occltrll,Pltrtlt c l t l d Soil 31 :209 ( 1969). 44. D. A. Barber and J. K. Martin, The release of organic substances by cereal roots into the soil, New Phytologist 7669 (1976). J. M. Lynch. Effect of genetically 45. M.J. Brimecombe, F. A. A.M.DeLeij,and modified P.selrtiottlotrct.sprrorc~.scetl.sstrains on the uptake of nitrogen by pea from "N enriched organic residucs, Letters it1 App/iecl Mictnhiology 26: 1 S5 (1998). 46. M. J. Brimecombe, F. A. A. M.De Leij, and J. M.Lynch.Effect of introduced P.sc.rrrlotrlot,as fhorcscctIs strains on the uptake of nitrogenbywheatfromI5Nenriched organic rcsidues, World Jorrnltrl of Mic,rol)iology (rtul B i o t r c h o l o ~ y . v15: 417 (1999). 47. W. F. Harris, D. Santantonio. andD. McGinty. The dynanlic below-ground ecosysfrortr Ii~~osystcwr At~rrly.sis.(R. H. Waring, ed.). tem. Forests: Fresh Per.sIx~ctivc~.s I 19. Oregon State University Press, Corvallis, 1980, p. 48. V. Vancura and A. Hanzlikova, Root exudates of plants: IV. Differences i n chemical composition of seed and seedlings exudates, Plrrrrt m d Soil 36:27 I (1972). 49. E. A. Curl and B. Truelove, Tlrc Rhi:o,spherc~,Springer, New York, 1985. I ~ . molecular weight organic 50. G. Cieslinski. K. C. J. Van Recs. and P. M. H L I ~ ILow acids released from roots of durum whcat and flax into sterile nutrient solutions. Joltrrd of' P/mt Nulritiorl 20:753 (1997). J. W.Rouatt.Theinfluenceofsoilandroot 51. E. C. S. Chan.H.Katznelson.and extracts on the associative growth of selected soil bacteria. Cutmlicul Jourtr(r1 Micwhiology!' 9: 187 ( 1962). AutIucrl 52. A. D. Rovira, Interactions between plant roots and soil microorganisms. Rc)l*icw uptake by water-extractable humic substances: involvement of root plas~nam c m brane H’-ATPase. Plunf Soil 215: 155 (1999). L. P. Solomonson and M. J. Barber, Assimilatory nitrate reductase: functional properties and regulation. AIIIILI. Rev. P l m f Pkysiol. Mol. Biol. 4/:225 (1990). Y.-F. Tsay, J . L.Schroeder, K. A.Feidmann,andN.M.Crawford, A herbicide sensitive gene CHLl of Aruhidopsis encodes a nitrate-inducible nitrate transporter. Cell 72:705 (1993). S.Cesco, S. Astolti, R. Monte, R. Pinton, and Z. Varanini, Stimolazione dell’ assorbimento di nitrato in radici di mais da parte di una frazione umica solubilein acqua. Proceedirlgs ofXVl1 Cortvegr~oNuziorrde S.I.C.A., Portoferraio, Italy, 1999. p. 13. S. Nardi, G. Concheriand G. Dell’Agnola,Biologicalactivityofhumus. Hrrrrlic Srthsttrrrce.~irr Terresrriul Eco.sy.sterr~.s(A. Piccolo, ed.), Elsevier Sciences B. V., Amsterdam, 1996, p. 361. G. Concheri, S. Nardi, F. Reniero, and G. Dell’Agnola, The effects of humic sub-
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72. C. C. Young and L. F. Chen, Polyamines in humic acid and their effect on radical growth of lettuce seedlings. Plant Soil 195:143 (1997). 73. A. Muscolo, S. Cutnlpi, and S. Nardi, IAA detection in humic substances. Soil B i d . Biockern. 3O:l 19Y (1998). 74. P. J . Davies, Plmnt Hortwnes: Physiology, Biochetnistly c m / Molecltlctr Riolocqy, 2nd ed., Kluwer Academic Publishers, Dordrecht, 1995. 75. B. S. Rauthanand M. Schnitzer,Effects of asoilfulvicacid on thegrowthand nutrient content of cucumber (Cltcltruis sntivus) plants. P / m t Soil 63:491 (1981). 76. G. Cacco, E. Attinh, A. Gelsomino, and M. Sidari, Effect of nitrate and humic substances of different molecular size on kinetic parameters of nitrate uptake rate in wheat seedlings. J. P l m t Nlctr. Soil Sci. 163:313 (2000).
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Mineralization and Immobilization in the Rhizosphere Luigi Badalucco Universita di Palermo, Palermo, Italy
Peter J. Kuikman Alterra-Green
1.
World Research, Wageningen, The Netherlands
INTRODUCTION
In recent years, the term rl~izo,sphere has appeared more and more frequently as a key word on papers that cover all basic and applied sciences related to soilplant-microbe relationships, such as soil fertility and biotechnology, microbial ecology, and plant nutrition, physiology, and pathology. A literature search with rhizosphere and (carbon or nitrogen) as requests for only the last 4 years would generate morethan 500 responses. Any physical, chemical, and biological change occurring within the root sphere or even indirectly mediated by its excretions and organic debris is called a rl~izosphere effect. Despite the demonstration of this effect on the basis of either an increase in number (1.2) or changes i n the biodiversity (3) of microorganisms, it has been much more difficult to produce experimental evidence for the rhizosphere effect in terms of carbon and nutrient flows specific to the rhizosphere environment (see Chap. 12).
A.
Objectives
In this chapter we review our current understanding with respect to carbon (C) and nitrogen (N) dynamics, the chemistry and biology of the rhizosphere, and aspects of immobilization and mineralization specific to the rhizosphere. Carbon 159
160 Kuikman
and
Badalucco
and nitrogen mineralization and immobilization in the rhizosphere is discussed in the light of the most recent concepts and models dealing with soil-plant-microbe interrelationships. In this context, emphasis is given to the aspects and possible roles of enzyme activities in the rhizosphere environment. We further review our understanding of the spatial and temporal distribution of substrates and their metabolism in soil and discuss the consequencesof the heterogeneous nature and distribution of rhizodeposits for their transformation into refractory organic matter in soil. Attention is given to estimates of the input rate, in situ decomposition, and residence time of rhizodeposits. Further, the rhizosphere as an environment for microbial activityis defined and discussed. Data on the size of the rhizosphere in relation to root mass are presented as affected by nitrogen supply, atmospheric COz concentration, and plant species. Specific rhizosphere deposition (carbon per unit of root mass, rhizosphere soil, also, ratios of rhizodeposition in rhizosphere and bulk soil) in relation to nitrogen supply and atmospheric COz concentration are treated. We further debate the methodologies used and the need for a protocolwhat such a protocol would look like-and speculate on other aspects peculiarto the rhizosphere: how long the rhizosphere effect lasts and whether it is transient, whether the effectis plant-or species-specific, the consequencesof distinguishing rhizosphere and nonrhizosphere, and the consequences of selecting organisms for C and N transformations.
II. RHIZOSPHERE A.
Biological,Chemical,andPhysicalComplexity
The soil-root interfaceis constitutively very variable, as plant roots are extremely dynamic in space and time. Soil niches correspondingto root tip, mucilage, meristematic zone. elongation/differentiation zone, or root hairs are not comparable for most aspects (4). Particularly favorable conditions in soil can led to unusual branching patterns in root systems. Thus roots vary enormously in their morphology, longevity, activity, and influence on soil as a resultof physiological, environmental, and genetic differences (5). Moreover, movement of water, nutrients, and microbes is more convoluted around the roots than in the bulk soil. Water potential regime in the rhizosphere soil is generally lower than in the bulk soil, thus causing a net mass flow toward the roots and a nutrient gradient acrossthe rhizosphere environment (6). Concentrations of available C are usually low around the root despite the rhizodepositions due to generally rapid microbial assimilation and depletion of the resource (7). Selective root uptake of ions causes some ions to be depleted, whereas those not assimilated tend to accumulate if not leached. Soil pH changes depend also on the root cation:anion ratio uptake (8,9). Redox potential in rhizosphere soil is generally much more negative than in bulk soil
and
Mineralization
161
(IO), also due to the higher oxygen consumption during respirationof both roots andmicroorganisms.Therhizospheregenerallyexperienceshighermineral weathering rates than bulk soil ( 1 I , 12). For all of these reasons, it is very difficult to obtain a representative sample of rhizosphere soil; consequently thereliability of any finding regarding soil rhizosphere processes depends strictly on the reliability of the model system adopted for the study. On the other hand, to study the soil and the plant as separate and independent entities is “nonsense” at many levels: pedogenetic, ecological (in the field plants cannot growin the presenceof particularly prohibitive climatic or environmental conditions), and methodological (any model system for studying soilplant relationships not involvingboth single components mayresult strongly misleading from a holistic viewpoint). As the soil as a whole is itself a complex body consisting of organic and inorganic components, also the use of simplified artificial soil-plant systems-for instance, plants grown either hydroponically or on mineral or organic support-may generate partial information that is totally unreliable, since interactions between components might be even more important than the mere sum of single contributions. From an operational viewpoint and depending on the investigation’s scale of resolution, the detailed description of the spatial and temporal variation of soil to the enormous number microhabitats, especially at the soil-root interface due of microbial species and underlying processes, it would require a huge amountof soil samples with, in any case, actually insurmountable difficulties in integrating results. Thus, at the moment, the only ongoing approaches for studying mineralization-immobilization in the rhizosphere are process-oriented and basedon manageable microcosms or mesocosms simplified at various extents in comparison with field conditions. More precisely, the spatial-temporal discrimination of the rhizosphere soil is lost when pot experiments are used. Thelevel of completeness at an immediately higher, the microcosm level, usually permits study of the rhizosphere effect both temporally and spatially, despite the addition of some constrictions for root growth and/or soil physical properties. Mesocosm levelstudies usually allow the same completeness of field conditions butin thermodynamically closed systems-that is, controlling the exchange of energy and matter with the exterior.
B. RhizosphereCharacterizationTheoretical Considerations By definition, all carbon (except above-ground littering) enters the soil via the rhizosphere, which is a highly dynamic and complex environment both in time and in space. As discussed in the Chaps. 2 and 4, the root excretions as well as root debris consist of a wide array of chemical compounds, most of which can be utilized by soil microorganisms (1 3). These compounds can be arbitrarily di-
162 Kuikman
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vided into two groups with distinct physical behaviors-i.e., soluble and structural particulate organic matter(14). Moreover, these groups relate to three distinctive environments: (1) growing root tip (exudation of solubles diffusing away from the source into the soil, characterized by a gradient of substrate concentraof solubles and cell matetions), (2) aging root (cortical senescence with release rial, diffusion, and hot spot), and (3) dying roots (structural compounds that are nonsoluble particulate characterized by a locally high concentration of substrates) (15). As already noted by Campbell and Greaves (16), the rhizosphere lacks physically precise delimitations and its boundary is hard to demarcate. Dimensions may vary with plant species and cultivar, stage of development, and type of soil. Soil moisture may affect the measurable size of the rhizosphere as well: wetter soils may stick better to roots than drier soils (Fig. 1). This will change the volume of soil regarded as rhizosphere soil upon separation of rhizosphere from bulk soil and thus alter the measured concentration in rhizosphere and nonrhizosphere soil of a response variable in exudate concentration or microbial production.
response variable
\
L
rhizosphere
non-rhizosphere
Figure 1 Schematic representation of the dynamics of a response variable, e.g.,concentration of rhizodeposited C, in the rhizosphere (dashed line) and the measured concentraThe vertical arrow indicates tions in rhizosphereand nonrhizosphere samples (solid lines). the separation of rhizosphere and nonrhizosphere soil; the effect of soil moisture is indicated by horizontal arrows.
Mineralization and Immobilization
163
C. Rhizosphere Dimensions High levels of available substrates in soil microsites may be utilized at the site at which they are released, or they may move through the soil due to mass flow or diffusion. Bremer and Kuikman examined the movement and utilization of 14C-glucose applied on a filter paper in contact with soil in laboratory conditions (Fig. 2). At the high application rate, 45% of added glucose C was respired, while only 36% was respired at the lower rate. This confirmed previous results from Bremer and Kuikman (17), who had added I4C-labeled glucose to two soils at seven geometrically increasing rates (from36 to 2304 pg C g" soil). An average of 42% of added I4C was mineralized by day 3 at glucose ratesZ 288 pg C g" soil in both soils, but only 30% of added I4C was mineralized at the lowest rate of glucose addition. From the filters, considerable movement of glucose C occurred into the soil (Fig. 2): up to 3 or 4 mm at the lower application rate and up to 6 mm at the higher application rate. The smallest proportion mineralized may indicate low C availability, whereas the largest proportion mineralized may indicate high C availability. Thus the volume of the actual rhizosphere will depend on the rate of exudation and impact of microbial utilization of rhizodeposits. For many years, the growth enhancement of microbes in the rhizosphere was illustrated by comparing planted with nonplanted soils. Only recently, data from rhizosphereandnon-rhizospheresoilhavebeenpresented (18-21), showing much larger differences than in planted versus unplanted soils. Analysis of the 14C distribution among soil and plant componentsby Cheng et al. (22) suggests that most root exudates were utilized by the microbes in the rhizosphere once
5000
10-1
4000
Ell-2
mm mm 12-4 mm 14-6 mm 06-14 m m
3000 2000 1000
0 24
240
pg C per c m 2 Figure 2 Distribution of total I4C in soil 4 days after addition of glucose I4C at two different levels. Labeled glucose was applied on a filter paper at 24 and 240 vg Ckm2 and placed in contact with moist, sandy soil. The microcosms (diameter 7 cm, height 40 mm) were incubated at 25°C for 4 days and then sampled in layers.
leaving the roots; only 3 4 % of the exudates were recovered from the bulk soil. amah (23), among others, regarded a zone of 1-2 mm around roots as the zone in which microbial growth was in~uencedby root exudates.
ssen (24) studied the effect of an elevated atmospheric the dist~butionof carbon and rhizode~ositionfor three ration of their data showed that the rhizosphere volume was inby a higher nitrogen supply through an increase in root m sphere volume per unit root m showed the opposite: at low e volume was twice that at high The amount of rhi~odeposition of rhizosphere soil at high N was twice as high as that at portional to root density. An explanation for this phenomenon ce in root ~ o ~ h o l o g y - i . e . , low N plants have finer roots, capable of exploring more soil volume for limited n u ~ e n t s , the utilization efficiency of rhizodeposits. That is, use at low so that su~stratesmust move away over longer distances. The total rhizosphere volume was equal for all three species. it root mass increased in the order ~ o Z ~ i e ~~ ~eA ~ ~ e , ca ovina. This means that ~ o l has i ~more ~ root mass per unit phere soil. Again, differences in root mo~hology,with ~ o l i ~ ~ r roots than ~ e s t ~ ccould a , explain this. The a ~ o u nof t rhizod~posit osphere soil was higher in soil planted with ~ o Z but i less ~ ~than could be expected on the basis of the root density ( i h a n , unpublished results). The total rhizosphere volume was 15% higher at high CO,. This was caused by an increased mass of the root system, as rhizosphere volume per unit root mass
Characteristics of Rhizosphere and ~onrhizosphereSoil for Three Crass Species Grown in a Continuous I4C-CO2Labelled Atmosphere After 8 Weeksa
hizosphere soil (dry wt) hizosphe~esoil per gram root r h , ~ ~ ~ ~ rhizosphere h ~ r e / ~ soil l4CbUlk per gram bulk soil
Ratio of 350 to 700 ppm C 0 2
Ratio of low to high N supply
0.86 1.02 0.84 0.86 0.79
0.52 1.96 0.49 0.53 0.41
a ~ i z o d e ~ o s i t i ois n measured as I4C recovered f?om soil adhering to the roots or soil not adhering to the roots. Soil moisture content was constant in all treat~ents.
and
Mineralization
165
was equal for both COz treatments. Root morphology seems not affected by COz concentration. At high COz, more rhizodeposition per gram of rhizosphere soil was found. This could indicatean increased rhizodeposition at high COz (unpublished results). In another experiment with four grass species (Holcus lanatus, Anthoxmthurn odorcctum, Festuca ruhra. and Festuca ovina), these results were confirmed: at the lower N supply, larger rhizosphere volumes (ratioof low N to high N : 1.4) were recovered and less IJCIg of rhizosphere soil (ratio of low N to high N:0.6) was found. No differences between lower and higher N supply were found in the non-rhizosphere soil (Kuikman, unpublished results). Clearly, the concentration of exudates and rhizodeposits depends on soil nutritional status and on plant species; this may affect the microbial utilization and subsequent turnover of rhizodeposits in soil.
111.
CARBON DYNAMICS IN THE RHIZOSPHERE
A.
InputRates of Rhizodeposition
Input rates of organic C into the soil system are hard to quantify, particularly for natural ecosystems and to a lesser extent for agricultural ecosystems. Whereas quantity and quality of carbon inputs vialitter fall and plant residues after harvest might be directly measurable, inputs via roots and rhizodeposition are more difficult to assess. The quantification of gross root production, rhizodeposition, microbial assimilation, and the production of organic materials in soil has made increasing progress ever since stable ( I T ) and radioactive ("C) carbon isotopes have been used (see Chap. 12). Measurements of soil organic matter dynamics without these isotopes are difficult due to the large amount present as compared to the smaller rates of input. Several authors have applied in situ pulse labeling of plants (grasses and crops) with 1"C-C02 underfield conditions with the objective of quantifying the gross annual fluxes of carbon (net assimilation, shoot and root turnover, and decomposition) in production grasslands and so assess the net input of carbon (total input minus root respiration minus microbial respiration on the basis of rhizodeposition and soil organic matter) and carbon fixation in soil under ambient climatic conditions in the field. to determine the fateof Recently, pulse labeling has frequently been applied carbon in crops suchas barley and wheat and the losses from roots and subsequent microbial transformations. In general, the results indicate that 15-25% of the net '"C assimilation is transferred to the roots and that there are seasonal differences in the distribution of assimilated carbon. Meharg and Killham (25) measured the C distribution in perennial ryegrass (L. perenne). At 8 days after the pulse with
166 Kuikman
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I4C, 26% of the label was recovered in shoots, 7% in roots, and 1% in soil; the remainder was returned to the atmosphere CO2 as (25). In field studies, the annual below-ground C transfer in wheat and spring barley has been estimated to be 475-1765 kg C ha" year" and 583-1652 kg C ha" year", respectively (26). Swinnen (27) estimated that more carbon was transferred to the roots by wheat than by barley and gave a range of 1500-2300 kg C ha-' year-!. Total rhizodeposition constituted 29-50% of the carbon translocated below ground and was 1S-2.1 times the amountof C in roots left at crop harvest (28). Microbial respiration of rhizodeposits was higher under conventional than under integrated management. Of the annual root growth, about 50% had decayed by the end of the growing period (28-30). Several authors have shown, by pulse-labeling wheat and barley plants at four to five stages during growth, that the distribution of carbon changes during the growing season. Also, about 10% of total crop carbon first 24 h after labeling (31). fixationwasreleased within the soil during the Rattray et al. (18) used "C pulse labeling to determine the distribution of C between L. perenne and its associated rhizosphere microcosm. The translocation of I4C to below ground was already detectable 30 min after the pulse application and reached the maximal value withinh.3Then, the microbial biomass accounted for 74%of the total IJCrhizosphere pool and, after 24 h, about 30% of the assimilated I4C had been translocated below ground. Partitioning of recent assimilates changed with increasing COz concentration in the atmosphere. The proportion of 14C translocated below ground almost doubled from 18% at ambient CO2concentration (450 ppm) to 34% at 750 ppm CO2 concentration (18). B. Decomposition of Roots Wedin et al. (32) analyzed changes in the stable C isotope composition (I3C)of above- and below-ground litter of C, and C4 grasses and monitored the shortterm (4 years) accretion of soil organic matter in soils with a known isotopic composition. They concluded on the basis of the measured isotopic shifts that 14% of the total soil C was new C over the period of 4 years. This amount was estimated to equal 30% of net primary production (NPP) over these 4 years. Kuikman (33) gives similar estimates on the basis of a field trial where pulse labeling was applied to study annual carbon fluxes and decomposition of roots in a managed grassland. The total stockof C was approximately 30000 kg C/ha in the top 30 cm; annual input due to rhizodepositions was 2200 kg/ha; and between 550 and 710 kg C of this was stored in soil per year. In addition to this below-ground input, 1550 kg C wasgainedfromabove-groundlittering. Applying the same turnover rates to the litter C input as in soil, 375-500 kg C of this C was stored in soil per year, which equals 3.1-4. l % of the total C stock in soil and represents a rapid turnover of the soil organic matter pool.
Mineralization and Immobilization
167
C. Soil Respiration The CO2 evolved from soil with living roots can originate from three sources: ( I ) root respiration, (2) microbial respiration of root-derived materials (rhizomicrobial respiration), and (3) microbial respiration of soil C. Different sources of substrate are concurrently available and used. Due to many technical difficulties and the intimate association of root and microbial respiration, most research in this area has been done in gnotobiotic systems and in nutrient solutions or artificial soils (34). However, questions can be raised as to whether the results obtained are representative for the complex interactions at the soil-root interface in natural conditions. Few methodologies are available to distinguish root from microbial respiration and microbial respiration of rhizodeposits from soil organic matter, respectively, in soils. Three different approaches have been used in recent years, giving highly variable results. Swinnen (35) applied trace amounts of ‘“Clabeled “model” rhizodeposits to planted soils. This madeit possible to fractionate the overall soil respiration into root respiration and microbial respiration on the basis of root exudates. The proportionof root respiration in overall soil respiration of total C assimilated by plants was greater than 75%. Johansson (36) used a different approach and gave an estimate of 32% for microbial respiration over a 7-week period. Cheng et al. (37) saturated planted soils with unlabeled substrates to eliminate the microbial use of labeled substrates from a pulse-labeled plant and estimated that microbial respiration accounted for 60% of the overall respiration. The disadvantage of the latter method is the short time period for redistribution of the pulse labeling and the potential for roots to absorb and use addedunlabeledsubstrates; both mechanisms will underestimaterootrespiration. A recent advancementis the useof 13Cdifferences betweenC! and C 4plants or soils to separate root and microbial respiration in the rhizosphere (22,38,39). Rochette and Flanagan (40) monitored soil respiration during a growing season and separated root respiration and microbial-rhizosphere respiration from microbial soil organic matter respiration in a soil developed under C 3 plants and now planted to C, corn (Zen r m y . ~L.). The CO?respired by the rhizosphere was equivalent to 18-25% of crop net photosynthate and 24-35% of crop net CO? assimilation during most of the growing season. This methodology assumes that the organisms in the C3-planted soil are not different from organisms in a C,-planted soil and are able to utilize the C,-plant substrates. We conclude that recent advances in the use of tracers, albeit I‘C or I3C, offer a solution to some of the technological difficulties in quantifying and separating microbial from root respiration. Combinations of these tracers may even overcome the last of the problems in discriminating between all three sources of CO2 released from soil.
168 Kuikman
and
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D. Metabolization of Rhizodeposits Swinnen (35) added labeled “model” rhizodeposits to planted soils to measure their respiration and residue and compared a number of soil factors and different to represent uctual rhizodeposits. A substrates.Hediscussedtheirsuitability higher fraction of the soluble substrates (38% of the glucose, or 37% of root extracts) were respired than those of root residues (26%) during the 21 days of incubation in a climate room. Incubation in field soils reduced the respiration of substrates compared to the incubation in climate rooms (23-25% for solubles and 12% for root residues). Naumova and Kuikman (unpublished results) studied theinfluence of threelevels of glucoseaddition on microbialutilization of ground-root residues (solubles extracted with water) and nativesoil organic matter and their interactions during an incubation of 24 days (Table 2). The mineralization rate of C derived from roots was stimulated during days 0-3 by the low levelof glucose addition and during days 3-24 by the high level of glucose addition. The cumulative CO, evolution, the amounts of extractable C (2 pg C/g soil) and the microbial carbon (29 pg C/g soil) produced during the decomposition from added root material were not affected by additionof glucose. However, the addition of glucose increased the mineralization rate of native SOM by l 1 and 22% under low and high levels of glucose addition, respectively (Table 2). Virtually all residual “C in soil at the low addition rate of glucose was glucosederived microbial biomass C, whereas at the highest rate, glucose-derived microbial biomass represented about 50% of the residual “C in soil (Table 3). This indicates that a high level of soluble substrate stimulates the turnover of newly formed microbial biomass. On the contrary, a low level of substrate may be less well utilized by the microbial population awaiting further mineralization (17) or result in a lower turnover, i.e., inducedby bacterial predators (41). It is concluded that the addition of soluble substrates such as glucose accelerates the turnover
Table 2 Cumulative COI Evolution from Root Residues (SOM). and Glucose During 24 Days of Incubation
(RM), Soil Organic Matter
C source for COz Total
Treatment“ (pg C per g soil) ~
SOM RM
Glucose
RM
+ glucose)
~~
128 No glucose Glucose (45 pg C) Glucose (541 pg C) ~
(% of added
133 126
253 280 308
~~
In all treatments, root material was
added at a constdnt rate.
Source: Naumova and Kuikman (unpublished results).
30 14
31
383
53
169
Mineralization and Immobilization Table 3 Soil C and Microbial C (pg C per gram soil) Derived from Glucose Ratio
Glucose (45 pg C) Glucose (541 pg C)
cwd
Cnurruhld
Cm~'roh~rl-to-C\u~l
12.1 156.8
13.1 72.5
I .08 0.46
and Kuikman (unpublished results) Table 2 for more details.
Sorrrcw Naumova
See
of microbial biomass (or soil organic matter) only at relatively high addition rates. Soluble substrates are respired to a larger extentthan particulate substrates and we hypothesizethat solubles do not interact or interfere with plant particulate substrates during their decomposition in soil. The latter can be explained by assuming that fractions with a different physical behavior in soil are decomposed by spatially separated microbial populations. This may lead to different turnover rates of the (microbial) products that are being formed. Clearly differences between soluble and particulate substrates exist in the short term but may not last at longer incubations.
E. Production of Refractory Soil Organic Matter From Rhizodeposits So far, we have no answer to the issue whether the nature of the substrate influences its transformation into refractory products. The hypothesis is that decomposability of rhizodeposits is not simply a characteristic of biochemical quality of the substrate but also of their physical characteristics and the rate at which they are supplied to the soil. Most simulation modelsthat are used in soil organic matter studies will partition residues into two to three pools from easily decomposable to recalcitrant organic compounds (42,43). Each of these pools will have a distinct turnover rate and a distinct probability to produce refractory soil organic matter. The easily decomposable substrate may very well end up in refractory pools, whereas substrate that initially is decomposed slowly will, in the end, be decomposed to a larger extent and result in less refractory soil organic matter. Organic matter formed from glucose is metabolized at a fast rate,but the residue is only further mineralized at a low rate (< 150 pg/g soil) and less at a low addition rate of glucose than at higher addition rates of glucose (17). Sarensen et al. (44) hypothesized that finer plant residues (i.e., upon grinding) may have better contact between substrate and decomposer organisms within the soil matrix and would decompose less extensively than coarser (i.e., intact,
170 Kuikman
and
Badalucco
unground) plant residues. They tested this hypothesis by measuring decomposition and biomass formation in soils with ground and unground plant residues and with glucose. The results led them to conclude that glucose-derived C was retained more in soil than residue-retained C; this confirms data reported by Ladd et al. (45). Sorensen et al. (44) also suggested that grinding plant residues, upon addition to soil, favors,on their addition to soil, a more intimate contact between the plant residues and the soil matrix, thereby enhancing opportunities for the colonization by decomposer organisms that are more protected against predation and further decomposition and result in higher retention.
F. Advantages and Limitations of Applying C Isotopes The methodology to study the C economy and C fluxes of plant-soil interfaces is fraught with problems (46). Studies employing isotopes of C, particularly “C. seem to provide the only feasible way to obtain accurate figures for root-derived C. Pulse-labeling is relatively simple and cheap and there is 110 need for complicated apparatus in the field (35). This approach provides an estimate of gross C fluxes underecologicallyrealisticconditionsbuthasthedisadvantagethat C fluxes are derived by fitting data to a mathematical model rather than by direct measurement(35,47).Thedecomposition of roots wasmeasured by leaving pulse-labeled plants in the field. The dynamics of the remaining carbon was followed during 1.5 years following the addition of shoots and labeling of roots, respectively. This approach has the advantage that root decomposition was studied in situ (35,47). The major assumptionin pulse labeling is that the distributionof the carbon assimilated during labeling with “C-C02 is representative for the carbon assimilated during that period. Jensen (26) addressed some of these assumptions by measuring the distribution of photoassimilated C in spring barley plants at different times after the onsetof light and at different light intensities during assimilation. Higher proportions (15-20% more) of assimilates tended to be transported below ground at lower light intensities and after labeling early in the morning (40-60% more). These results were confirmed by Swinnen et al. (27,28).
G. ScientificRelevance Thus, there is evidence that the methodology of pulse-labeling plants under field conditions can very well be applied to study carbon dynamics i n both the short term (root and microbial respiration and root exudation) and longer term (turnover and decomposition of roots). The release of organic compounds from roots of growing plants may have substantial effectson vital soil ecosystem processes, such as organic matter dynamics, structure formation, and nutrient cycling. Yet, proper quantification of the release of organic and inorganic C compounds from
and
Mineralization
171
roots or seasonal dynamics in various ecosystems is still lacking, and the fundamental mechanisms involved in their metabolism in soil are not fully understood. It is important to realize that any changesin the proportional allocation of carbon that form coarse and fine roots or in the rate of exudation may affect overall C sequestration in terrestrial ecosystems. Changes in the carbon distribution pattern over exudation, fine roots, and coarse roots may strongly affect subsequent decomposition patterns; some materials may beprotectedwithinthesoilmatrix while others remain unprotected (45). Even though exudates consist of simple sugars that are generally qualified as easily decomposable, Martin and Merckx (47) showed that some of this carbon is recovered from very recalcitrant organic materials in soil shortly after their release from roots. Swinnen (48) showed that exudation can amount to 25% or more of the total carbon translocated below ground in annuals such as barley and wheat. Thus, the contribution of rhizodeposition to the recalcitrant organic pool can be substantial.
W. ENZYMEACTIVITIES IN THE RHIZOSPHERE A.
GeneralConcepts
All soil metabolic processes are driven by enzymes. Themain sources of enzymes to be the in soil are roots, animals, and microorganisms; the last are considered most important (49). Once enzymes are produced and excreted from microbial cells or from root cells, they face harsh conditions; most may be rapidly decomposed by organisms (SO), part may be adsorbed onto soil organomineral colloids and possibly protected against microbial degradation (SI),and a minor portion may stand active in soil solution (52).The fraction of extracellular enzyme activity of soil, which is not denaturated and/or inactivated through interactions with soil fabric ( 5 l ) , is called ncrt~rrclllysttrbilized or immobilized. Moreover, it has been hypothesized that immobilized enzymes have a peculiar behavior, for they might not require cofactors for their catalysis. Extracellular enzymes are essential for the initial degradation of high-moor humic molelecular-weight substrates like lignin, cellulose, pectins, chitin, cules, which cannot enter microbial cell envelopes to be processed (53). The presence of immobilized extracellular enzymes in soil has been hypothesized to be necessary for the liberation of low-molecular-weight monomers that subsequently may diffuse into microbes for signaling the (temporary) presence of particulate substrates in the rhizosphere environment. This process induces an aimed enzyme biosynthesis for a consequent excretion and save the microbial biomass the high metabolic expense of secreting continuously and blindly a wide array of differenthydrolyticenzymes (53). Theecologicalmeaning of thiscrucial mechanism could get over the simple nutritional requirements of roots if we consider recent hypotheses on the positive influence on plasma membrane H+-
172 Kuikman
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ATPase and specific modification of root cell membrane permeability directly mediated by low-molecular-weight (5 mm from surfaceof pine roots (bulk soil)of different morphology and functional type (coarse woody roots of >2 mm diameter; fine roots of /> 011 Plcltlt Gr[)n.Ih-Prot,/otitlR Rhi:ohc/cteritr (A. Ogoshi, K. Kobayashi, Y. Homma, F. Kodama, N. Kondo, and S. Akino, eds.), Sapporo, Japan, 1997, pp. 44-48. 59. .I. Versalovic. T. Koeuth. and J. R. Lupski, Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nlrcleic At,itl Rcs. 1936832 (1991). 60. P. Vinucsa, J. L. W. Rademakcr,F. J. de Bruijn, and D. Werner, Genotypic character-
222
61.
62.
63. 64. 65.
66.
67.
Werner
ization of Brrrd~~rlIizobiltrII strains nodulating endemic woody legume of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16s rRNA (16s rDNA) and 16s-23s rDNA intergenic spacers, repetitive extragenic palindromicPCR genomic fingerprinting and partial16s rDNA sequencing. Appl. Envirot~rt~. Microhiol. h4:2096 (1998). K. Kersters, W. Ludwig, M. Vancanneyt, P. de Vos, M. Gillis, and K.-H. Schleifer, Recent changes in the classification of Pseudomonads: an overview. Systenl. Appl. Microhiol. 19:465 ( 1996). D. Redccker,T. Batinic, 1. S. Feder, K. Kosch, U. Schulz, P. Vinuesa, and D. Werner, Biocontrol strain P.sercr1onzorl~r.spuore.sc~erl.s W34: specifc detection and quantifcation in the rhizosphere of Cucurnis scrtivus with a DNA probe and characterization by DNA fingerprinting. Z. N~rtrr$or.sck.54c 359 (1999). E. A. Bell, J. A. Lacey, and R. M. Polhill, Systematic significance of canavanine in the Papilionoideae (Faboideae). Biochern. S y s t . Ecol. 6:201 ( 1 978). D. Werner, Syndiosis of Pltcrlts and Microhrs, Chapman and Hall, London, 1992. S. U. Schenk, F. Lambein, and D. Werner, Broad antifungal activity of p-isoxyzolinonyl-alanine, a non-protein amino acid from roots of pea (Pisrrm s c r r i v u m L.) seedlings. Biol. Ferti/. Soils. /1:203 (1991). T. Kohno, D. Kohda,M.Haruki, S. Yokoyarna,and T. Miyazuwa,Non-protein amino acid furanomycin, unlike isoleucine in chemical structure, is changed to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein. J. Biol. Chenr. 265:693 1 (1990). P. M. West, Excretion of thiamin and biotin by the roots of higher plants. Nuture ( L o n d o n ) 144:1050 (1939).
68. A. D. Rovira and J. R. Harris, Plant root excretions in relation to the rhizosphere effect: V. The exudation of B-group vitamins. Plant Soil 14:199 (1961). 69. R. Schonwitz and H. Ziegler, Exudation of water-soluble vitamins and of somccarbohydrates by intact roots of maize seedlings ( Z ~ rfrcrys N L.) into a mineral nutrient solution. Z. PJarlw~phy.siol. 107:7 (1982). 70. H. A. Azaizeh. G. Neumann, and H. Marschner. Effects of thiamine and nitrogen of fertilizer form on the number of Nz-fixing and total bacteria in the rhizosphere nmze plants. Z. P’arzzeneruaehr. Boderrkurde 159: I83 ( 1996). 71. M. Simons. A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg, Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pserrdornorlas bacteria. Molec. Plorlt Micro/w b~rercrct.9600 ( 1996). 72. W. R. Streit, C. M. Joseph, and D. A. Phillips, Biotin and other watcr-soluble vitaInins are key growth factorsfor alfalfa rhizosphere colonizationby f?/zi:ohiftmmeliloti I02 I . Molec. P l m t Microbe It~terccct.5:330 ( 1996). i~lr:~, 73. D. A. Phillips and W. R. Streit, Hhirospherc~Sigrra1.s crnd E c o c l ~ ~ ~ ~AbschldJberich1 des Sonderforschungsbereichs 305, VerlagChcmie,Weinheim.2000. In press.
Function of Siderophores in the Plant Rhizosphere David Crowley University of California-Riverside, Riverside, California
1.
INTRODUCTION
Siderophores are iron chelating agents that are secreted by microorganisms and graminaceous plants in response to iron deficiency. Given the essential requirement foriron by almost all living organisms, these compounds are not only important for iron nutrition but are also speculated to functionin the ecology of microorganisms in the plant rhizosphere (1). Siderophores have been studied for their importance in plant disease suppression by mediating nutritional competition for iron (23) and contribute directlyto the rhizosphere competenceof root colonizing bacteria (4). In research on plant ecology, siderophores have been investigated in relation to calcicole and calcifuge plant species and as one of the factors that may explain the distribution of various plant species in different soils ( 5 ) . Most recently,withthegrowinginterest in using plant-microbe systems for phytoremediation of heavy metals, new research is examining the role of siderophores and phytosiderophores in facilitating heavy metal uptake and food chain transfer of metals (6,7). Despite the wealth of information on siderophores, there is still considerable debate as to how they function in the plant rhizosphere and the degree to which they accumulate in soils. Much of this debate has been due to inadequate methodology for detecting siderophores at microsite locations in the rhizosphere and the lack of analytical methods for in situ study of the interaction of sideroin the phoresandother iron mobilizingsubstances.Usingsimplifiedsystems laboratory, i t is possible to examine many different scenarios as to how siderophores might function. Yet, for the most part, thereis still almost no information
223
Crowley
224
on what siderophores actually occur in the rhizosphere, which compounds are the predominant ironsources,andhowthisvariesatdifferentrootlocations. With the advent of new microsite sampling techniques for in situ collection of siderophores and root exudates, this situationis now poised for rapid change. As highlighted in this chapter, new molecular techniques for the fingerprinting of microbial communities and identification of the predominant microbial species in the rhizosphere will also enable us to better evaluate the role of siderophores in microbial ecology. One of the current questions in research on the function of siderophores in the rhizosphere is the influence of plants on microbial iron nutrition. Because plants have the first access to iron that is mobilized at the root tips, it is likely the roots take up much of the readily available iron in the rhizosphere prior to microbial growth. Thus, plantiron demand combined withrhizodeposition of carbon may be the driving force behind competition for iron during primary colonization of the plant roots (1). The degree to which root-colonizing microorganisms compete for iron depends on where theyarelocated in therhizosphere, the chemistry of the soil, and the efficacy of plant iron stress responses such as rhizosphere acidification, release of reductants, and secretion of iron-mobilizing root exudates that increase the bioavailability of iron to microorganisms. A major focus of this chapter is the review of plant microbial interactions that regulate siderophore production and their role in mediating competition for iron in the plant rhizosphere. In understanding the factorsthat control siderophore production, it is important to consider thatiron-mobilizingsubstances in the rhizosphere include not only siderophores, which are produced by virtually all microorganisms, but also a milieu of other compounds that chelate iron. Understanding of howsiderophoresfunction in microbialcompetitionalsorequires consideration of theirchelationchemistryandkineticfactorsthatcontrolthe dissolution of iron from minerals and exchange of iron between different sideroof microbial phores. Other topics that are considered in detail include plant use siderophores, the role of siderophores i n disease biocontrol and rhizosphere conipetence of microorganisms, and the relevance of siderophores to nitrogen fixation.
11.
FACTORSCONTROLLINGIRONAVAILABILITY IN THE RHIZOSPHERE
Under aerated conditions at neutral to alkaline pH, inorganic iron is extremely insoluble (g), such that plants and microorganisms rely absolutely on iron uptake from organic matter complexes or iron that has been solubilized by siderophores and organic compounds contained in root exudate. Low-molecular-weight root exudates that dissolve iron include organic acids that are secreted by plant roots as a specific response to iron deficiency (9) or that are released constitutively at
Function of Siderophores
225
low levels in root exudates and lysates (IO). In the rhizosphere of graminaceous plants, organic acids are released initially, but as the plant becomes more ironstressed, are followedby increased production of highly efficient chelators called phytosiderophores that are secreted in localized zones behind the root tips ( I I ) . Further adding to the complexity of the soil solution, humic and fulvic acids as well as microbial produced chelators containedin compost can also mobilizeiron for uptake by plants and microorganisms ( 12,13). Comprehensive analysis of root exudate composition by ' H and 'jC multidimensional nuclear magnetic resonance (NMR) and by silylation GC-MS has proven to be particularly useful for identifying the constituents of root exudates and how they change in relation to iron deficiency (14). As shown for barley subjected to different levels of iron stress, many organic and amino acids can be identified, as well as several derivatives of mugineic acid phytosiderophores, the major one being 3-epihydroxymugineic acid (Table I ) . Quantification of all major components during changes i n the plant iron nutritional status revealed a sevenfold increase in total exudation under moderate iron deficiency, with 3-epihydroxymugineic acid representing approximately 22% of the exudate mixture. As iron deficiency increased, total quantities of exudate per gram of root remained unchanged, but the relative quantity of carbon allocated to phytosiderophore increased to approximately 50% of the total carbon released by the roots. Given the inherent complexity of root exudates and mixtures of other organic substances in the rhizosphere-as well as theoretical consideration of all
Table 1 EffectofIronStressonChemicalComposition of Root Exudates Collected from Barley Plants. as Determined by Combined NMR and GCMS Analysis (Fan et al.. 1997)
PFe Conlpound
16.5
17.0
17.5
18.o
lactate alanine succinate y-aminobutyrate fumarate malate glycine betaine acetate 3-epi-OH-MA
1.1
4. I 4.4
5.0 I .S
4.8 0.7
0 .I
I .0
2.5
0.2 0.4
0.I
0 0. I 0
-
Total
0.3
0.5 1.6 1.1
5
0.3 2.8
0.2
5.9 4.1
1.7 1.7
x. 1
16.2
0.3 0.2 0.2 2.7 2.3 17.4
29
32
37
0.2
Barley plants grown in chelator huffered hydroponic solutions with free activity of iron a t glven pFe values rmglng from 16.5 (iron sufficient) t o pFe 18 (severe iron deficlency).
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of the factors that govern the production, diffusion, degradation, and chelation chemistry of these compounds-soil chemists have increasingly turned to computer models to describe the chemistry of the soil solution. These models include chemical speciation models that describe the partitioning of metalionsinthe presence of calcium and other minerals at a given pH and redox (15), as well as conceptual and mathematical models of factors that influence siderophore production and accumulation in the rhizosphere (1,16). A current objective is the validation of these models using data obtained from microsite analysis of the rhizosphere soil solution or with gene reporter systems that are calibrated to measure thepotentialproductionandaccumulation of siderophores in therhizosphere (l7,18).
111.
PREDICTION OF METAL CHELATE BEHAVIOR AND FUNCTION IN SOILS
Bioavailability of iron and other metals is largely controlled by dissolution of solid phase minerals. Dissolution rates for these minerals are describedby kinetic processes that are dependent on mineral crystallinity, ionic strength, and composition of the soil solution and the pH and redox conditions that govern the system (19). The dissolution of metals by direct attack of mineral surfaces is another kinetically driven process that has been studiedby surface chemists using various models. Metal chelation occurs with compounds that coordinate metal ions by providing multiple negatively charged functional groups that completely coordinate the metal atom and displace all of the water associated with the hydration shell. Complexes are formed by lnolecules providing one or more ligands that bind metals but which do not completely encapsulate the metal in a single molecule. Metals that are held by chelates and conlplexes maintain a chemical equilibrium with various iron hydrolysis species, which can be predicted from chemical equilibrium equations (15). This continuous dissociationheassocation results in slow exchange of metals between chelators having different stability constants. Computer-based models have emphasized equilibrium situations. However, since rates of exchange may be slow, i t is likely that nonequilibrium is the more typical situation in the rhizosphere. Models that can predict ratesof exchange are critical for understanding competition for iron based on preferential utilization of different siderophore types. There is also a need for more information on synergistic effects of mixtures comprising siderophores, organic acids, and dissolved organic matter in mobilizing iron. Recently, exchange of metals between siderophores and phytosiderophores has been proposed as a primary mechanism for plant use of microbial siderophores (20,21). Conversely, it has also been shown that microbial siderophores
Function of Siderophores
227
may strip iron from phytosiderophores (22). For this reason, there has been some debate as to how plants and microorganisms interactin their mutual problem of acquiring iron and the mechanisms that are used for iron acquisition from different iron sources. The partitioning of metals between different types of siderophores and other iron complexes depends not only on the stability constants but also on the concentrations of each chelator as well as the abilities of different chelators to attack the surface of iron minerals and undergo exchange. The major driving factor in competition for iron is the accumulationof excess siderophore caused by nonequilibrium with the solid phase. This process is describedin Fig. 1. In the presence of deferrated siderophores, the solution concentrations of the inorganic iron hydrolysis species are depleted to very low concentrations below equilibrium levels maintained by the mineral phase. Consequently, the mineral undergoes further dissolution to reestablish equilibrium concentrations. Nonequilibrium causedby slow dissolutionof metals from the solid phase results in accumulation of free ligands or complexes with weakly bound ions that can decrease soluble inorganic metal ion concentrations to extremely low levels. With strong chelators such as microbial siderophores, even a small amount of excess demetal-
Reduced
I
Siderophore
(Anaerobic)
.L
I
I
5
I
I
6
L
I
7
I
I
8
I
I
9
Figure 1 Solubility diagram for iron as Fe(III) and Fe(I1) as controlled by pH and redox. Solid line at 10-6M depicts the critical quantity of chelated iron required to support the growth of most plants and microorganisms. Under most conditions in nature, bioavailable iron is present only in the chelated form. Siderophores can be detected in most soils at backgroundconcentrationsof lo-* to concentration.During active growth,siderophoreconcentrationsmayincrease to concentration at localizedsitesofmicrobial activity but willbe present primarily inthe deferrated form. Under these conditions, microorganisms compete for iron based on their siderophore type and transport specificity.
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lated chelate can suppress inorganic metal concentrations down to levels of only a few atoms per liter (8,15). Under these conditions, the only available iron is that provided by metal chelators and complexes. Plant and microbial competition for iron involves very complex interactions that are influenced by a number of factors (Fig.2). These include differences in the level of siderophore productionby all of the competing microorganisms, the chemical stabilities of various siderophores and other chelators with iron, their
.... B
i....:Fe
..
.... . .
:;F;
.*.S.
0
....
t
...
..../ ....
i Fe:
0 Fe b
Competitionfor Iron Chelation kinetics Stability constants Ligand exchange
Quantity produced Transport specificity Degradation rate
Figure 2 Conceptual model showing competition for iron between two microorganisms producing different siderophore types. Much of the siderophore present theinsoil solution during active growth is in the deferrated form. The deferrated chelate mobilizes iron by two mechanisms, including sequestration of dissolved iron presentat very low concentrations, which causes further dissolution of the solid phase, and, second, by direct attack of mineral surfaces. Once in solution, the chelated iron can donate iron to other chelators by ligand exchange or by releasing inorganic iron into the solution in accordance with chemical equilibria governing chelate stability. Competitionfor iron is based on the quantity of chelator produced,its efficacy in mobilizing iron,its stability with iron under given pH, redox conditions, and its rate of degradation by other microbes that use the chelator as a carbon source for growth. At the organism level, competition is further mediated by transport specificity and the kinetics of iron uptake from different chelators.
Function of Siderophores
229
resistance to degradation, and the ability of different chelators to solubilize iron by attacking mineral surfaces. Moreover, all of the different siderophores i n the soil solution may interact through ligand exchange. At the level of the individual microorganism, cornpetition is further mediated by the ability t o use different siderophore types and the transport kinetics for these compounds. To date there has been very little research on the interactions of complex mixtures of chelators. The bestdata so far are those generated with computer models for synthetic chelators in simple systems withatmost twochelators i n chemically defined media at a specific pH. Metal chelation chemistry can be described by computer models such a s GEOCHEM, which uses stability constants, ion activities, and total metal quantities to predict the speciation of the metal chelators. In general, these models are appealing and highly useful tools for the design of hydroponic media to examine iron stress responses and to measure uptake rates by plants and microorganisms. However, thefewempiricaldataavailableindicatethatthesituationismuch morecomplex in soils. In soils,differences in metaldissolutionratesseldom correspond t o the stability constantsof different chelators and complexing agents but are instead controlled by the differing abilities of organic acids and chelators to attackmineralsurfaces (8,19.23). Undertheseconditions,metaldissolution rates arc controlled by surface characteristics of the mineral that influence binding, dissolution, and release of organic ligand-metal complexes from the mineral surfaces. Thus, predictions frolu chemical equilibrium models as to which chelators are the most important for metal solubilization in soils can be misleading and lead to the wrong conclusions concerning which chelators are effective for plant and microbial iron nutrition. To get around this problem, there is an increasing emphasis on using empirical data based on soil extractions with different chelators. However, ;I problem that has arisen in this methodology is the introduction of artifacts causedby using soil slurries in shaking flasks as opposed to percolation of intact soil columns. The former technique has been criticized for exposing new mineral surfaces and by disruption of soil pore spaces (24). Soil pores, which contain most oi the soil water, are exposed to continuous redox fluctuations :IS they undergo wetting and drying and are likely to have different mineral types, crystallinities. and surface properties a s compared t o the internal matrix of soil peds that are exposed during slurry extractions. Upon formation of a metal chelate or complex, the next rate-limiting step in delivering iron to the cell is the diffusion of iron complexes through the soil in response to diffusion gradients. In the vicinity of plant roots, metal chelates and complexes may also move by bulk flow in the transpiration stream as water moves from thesoil into the plant. However, depending on their charge characteristics and hydrophobicity, metal chelators and complexes can become adsorbed to clay and organic matter, which may then decrease their mobility and bioavail-
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ability to plants and microorganisms (25,26). This can greatly complicate the estimation of siderophore and metal chelator quantities that are actually functional in metal transport, especially if metal chelate concentrations are determined by soil extraction. Generally, concentrations of a chelator are estimated in relation to soil water content at an appropriate value-e.g., 10% soil moisture-which may vary depending on the soil texture. Repeated extractions of soil with water or buffered salt solutions or the use of large extraction solution volumes can result in overestimation of effective concentrations for chelatorsthat are normally sorbed to soils. Alternatively, concentrations of ionic, charged chelators and metal complexes that accumulate at soil microsites may be underestimated when they are diluted and become bound to clay and organic matter during the extraction process. For example, when the model microbial siderophore desferrioxamine B is added to soil, on average only 3% can be recovered, even after repeated extraction (27). The recovery is dependent on the clay and organic matter content. This latter problem may account forthe difficulty in detection of siderophores in soil extracts as well as the general finding that siderophore concentrations averaged over the entire rhizosphere are present in only nanomolar concentrations (27,28). These concentrations are orders of magnitude below that which would be needed to be physiologicallyrelevantforironuptake by eitherplantsormicroorganisms, which for some time has been a paradox for researchers investigating the function of siderophores in plant nutrition and microbial ecology.
W.
IRON UPTAKE MECHANISMS FROM SIDEROPHORES AND PHYTOSIDEROPHORES
A.
PlantIronUptakeMechanisms
Metal chelates and complexes deliveriron to cells by diffusion through plant and microbial cell walls, where they are presented to highly selective metal ion or metal-chelate transport systems that are deployedin the cell membranes of plants, bacteria, and fungi (see Refs. I and 29 for reviews). Charge characteristics of the wall itself, along with the accumulationof various ions and organic materials in the wall matrix and the size of the molecule in relation to the wall matrix, can all potentially influence the diffusion ratesof metal chelators across the cell wall. The operation of reductase enzymes present in cell walls can also strip iron from the chelate, which then results in the precipitation of amorphous iron oxides or sorption of the metal ion to the wall matrix (30). In order to be taken up by the cells, these metal ions must then be remobilized or else reside in the wall until they are released by decomposition by saprophytic microorganisms. This metal deposition process may occur primarily with low-stability metal complexes, but it has also been shown to occur with synthetic chelates such as EDTA and phytosiderophores produced by grasses (3 l ) . In this case, localized secretionof phyto-
Function of Siderophores
231
siderophores and microbial siderophores can remobilize precipitated iron (32). Other naturally occurring compounds-such as humic and fulvic acid or heme compounds-can also function in ironnutritionbydonatingiron to transport proteins or reductases at the cell surface. Recently, it has been shown that ironcontaining low-molecular-weight humic molecules can be reducedby both intact roots and isolated plasma membrane vesicles (33). Iron reduction may occur by means of an NADH-dependent iron stress-regulated reductase (34) that releases Fe?' from Fe3+ specific chelates and complexes and by biosyntheticreducing agents such as riboflavin (35) and caffeic acid (36), which have been proposed to enhance mobilization of iron that has been precipitated in the cell wall. In plants and microorganisms that are well adapted to iron-limiting growth conditions, the responses to iron stress are quite diverse and highly elaborate. Iron-efficient dicotyledonous plants, termed strategy I (37,38), produce organic acids that are released into the soil in the vicinity of the growing root tips to solubilize iron. They also increase their root surface area, number of root tips, and increase the activity of a proton ATPase that acidifies the rhizosphere and helps to increase iron solubility. Certain plants such as lupine release extremely large quantities of citrate in response to phosphorus deficiency but concurrently take up greater quantitiesof metals as a resultof this response(39). As mentioned above, plants may also increasethe activity of a plasma membrane-bound reductase in the rhizodermalcells that reduceschelatedferriciron to ferrousiron. Under iron deficiency, the kinetics of metal transport by the carrier protein are altered to permit a much higher Vmax suggesting increased induction of carrier protein synthesis or an alteration in the gate channel that regulates iron transport (40,41). The use of microbial siderophores by dicotyledonous plants appears to involve uptake of the entire metallated chelate (42-44), or an indirect process in which the siderophore undergoes degradation to release iron (45). As demonstrated in initialstudiesexaminingthisquestion,therewasconcern thatiron uptake from microbial siderophores may be an artifact of microbial iron uptake in whichradiolabelediron is accumulated byroot-colonizingmicroorganisms (46). Consequently, evidence for direct uptake of iron from microbial siderophores has required the use of axenic plants. In experiments with cucumber, it was shown that the microbial siderophore ferrioxamineB could be used as an iron source at concentrations as lowa s 5 pM and that the siderophore itself entered the plant (42). iron uptake from microbial The possible role of a chelate reductase for siderophores has been examined for several plant species (30.47). With certain microbial siderophores such as rhizoferrin and rhodotorulic acid, the reductase may easily cleave iron from the siderophore to allow subsequent uptake by the ferrousirontransporter.However,withthehydroxamatesiderophore,ferrioxamine B, which is produced by actinomycetes and used by diverse bacteria and fungi, it has been shown that the iron stress-regulated reductase is not capable
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of cleaving iron a t rates that would be relevant for plant nutrition. This suggest that ferrioxamine B may be utilized by another mechanism involving uptake of the siderophore. In studies with sterile onion plants, the rate of iron uptake and B was identical to that translocation by plants supplied with sSFe-ferrioxamine for ferrated ('"C)-ferrioxamine D, which was shown to accumulatein low concentrations in the root and shoot tissues (43). In the case of graminaceous plants, phytosiderophores are secreted to mobilize iron and are reabsorbedby means of a highly specific transport protein. These compounds are secreted at the root tips in short pulses 2 to 4 h in duration shortly after the onset of daylight, with both their synthesis and release being regulated by diurnal light and temperature rhythms ( 1 1,48). Differences in phytosiderophore production have been correlated with iron efficiency (9,38,49), but several other factors also have been shown to contribute to the relative iron efficiency of various monocot grasses. These include variations in the minimum iron requirement of the plant tissues for different species and cultivars, differences in the ability to respond by early and rapid production of phytosiderophores prior to the onset of deficiency symptoms, and alterations in the relative growth rate to prevent deficiency from becoming severe (Cries and Crowley, unpublished data). Alterin specific root length and numbers of ations i n root: shoot ratio and increases root tips also occur during the onset of iron stress (Table 2; Wu and Crowley, unpublished data).In addition to these physiological differences, iron uptake rates from synthetic chelators, microbial siderophores, and phytosiderophores may also be enhanced to different degrees depending on the plant cultivar (29). I n addition to iron, phytosiderophores also function for the uptake of zinc
Table 2 Changes in PlantGrowthParameter in Responsc to Iron Stress for Barley Plants Grown i n Chelator Buffered Hydroponic Nutrient Solutions
4.8
pFe,'
Shoot: root ratio
Specific length cndmg Root root root
16.5
2.4
17.0 17.5
2.3
2.0
5.4 5.0
18.0
1.9 3.0
6.7 6.1
19.0
tipslcm 2.6 2.1 1.8 1 .S
.' Iron concentrations expressed a s negative log of Fe(llI) huflered by HEDTA. Plants grown at pFe 16.5 were green. whereas thosc grown at increasingly lower Iron concentrations bccame increaslngly chlorotic
Function of Siderophores
233
and possibly copper and other trace metals that are fortuitously mobilized under iron deficiency conditions. Specific release of phytosiderophores in response to zinc deficiency has been shown for certain wheat species (50,51) but was not observed in barley (52). In contrast to the iron stress response, in which phytosiderophore release is correlated with iron efficiency, differences in zinc uptake efficiency among wheat cultivars do not correspond to differencesin phytosiderophore release rates (51). The mechanisms by which grasses utilize iron from rhizoferrin, ferrioxamine B, ethylenedianlinetetraacetic acid (EDTA), and deoxymugineic acid were recently studied for barley and corn plants grown in nutrient solution (20). By it was possible to supplying iron to the plants during the morning or evening, examine the effect of concurrent phytosiderophore release on iron uptake rates from the different iron sources. Uptake and translocation rates from Fe chelates in corn, in which paralleled the diurnal rhythm of phytosiderophore release except similar uptake and translocation rates were observed both in the morning and in the evening. Later, it was shown that for corn there was a constant rate of phytosiderophore release duringthe 14-h light period rather than the normal diurnal pattern observed in other plants. The results strongly suggestedthat iron supplied by rhizoferrin is taken up by strategy I1 plants by an indirect mechanism that involves ligand exchange between the felrated microbial siderophore and phytosiderophores.Thishypothesiswasfurther verified by in vitroligandexchange experiments showing the exchange of iron betweenrhizoferrinand phytosiderophore.
B. MicrobialIronUptakeMechanisms In the rhizosphere, microorganisms utilize either organic acids or phytosiderophores to transport iron or produce their own low-molecular-weight metal chelators, called siderophores. There are a wide variety of siderophores in nature and some of them have now been identified and chemically purified (54). Presently, three general mechanisms are recognized for utilization of these compounds by microorganisms. These include a shuttle mechanism in which chelators deliver iron to a reductase on the cell surface, direct uptake of metallated siderophores with destructive hydrolysis of the chelator inside the cell, and direct uptake followed by reductive removal of iron and resecretion of the chelator (for reviews, see Refs. 29 and 54). Depending on the abilityof specific transport systemsto utilize the predominant metal chelates present in the soil solution, competition may occur between plants and microorganisms and between different types of microorganisms for Pseuclomoncrs sp., availableiron.Thishasbeenparticularlywellstudiedfor which produce highly unique iron chelators that are utilized in a strain specific manner but which also retain the ability to use more generic siderophores pro-
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duced by other microorganisms (54-56). In pure culture in iron-limiting media, pseudomonads can produce extremely large quantities of siderophores that are inhibitory to iron uptake by other bacteria and fungi and that also can cause iron deprivation to plants (57,58).
V.
PLANT-MICROBIALINTERACTIONS
Attempts to unravel complex interactions in iron competition or cooperation between plants and microorganisms have generally involved pure culture systems in agar or liquid media or the use of hydroponic culture systems with plants. These studies have revealed basic mechanisms by which various plants and microorganisms respond to iron deficiency and their ability to use different metal chelators at defined concentrations. However, there has frequently been a tendency to extrapolate too far from these studies and overlook the differences that occur between soil and hydroponic systems. Hydroponic studies on plant use of microbial siderophores and phytosiderophores immerse the entire root system in uniform concentrations of the chelate.In reality, phytosiderophores are produced behind theroottips (59), where they accumulate tovery high concentrations in localized zones of the rhizosphere (60). Similarly, microbial siderophores, if produced in significant quantities in rhizosphere, are most likely secreted at sites of high microbial activity behind the root apices and at the sites of lateral root emergence ( 1,6 l ) . One particular controversy derived from hydroponic studies was the observation that organic acids and phytosiderophores are subject to rapid degradation (62) and that they would thus be ineffective for mobilizing iron in soils. This concern originated from hydroponic studies in which siderophore-degrading bacteria increased to such high population densities that phytosiderophores could not even be collected from the culture medium or were rendered ineffective for mobilization of iron from solid-phase minerals (29,58). It has since been shown that degradation is a localized phenomenon in sites of high microbial activity, which can be ameliorated by pulsed release of phytosiderophore at the root tips ( 1,481. In a carefully conducted study that examined the effect of bacterial distribution in the rhizosphere on phytosiderophore degradation, maize plants were grown axenically or inoculated with a mixture of microorganisms in a limestone substrate supplemented with iron oxide. Those that were grown axenically grew well and released phytosiderophores; whereas inoculated plants showed severe symptoms of Fedeficiency,suggestingmicrobialdegradation of phytosiderophores in the apical root zones (63). When the watering pattern was manipulated with a wick system, the plants were enabled to secrete enough phytosiderophore in localized zones to facilitate iron uptake. Similar results were obtained in a silt
Function of Siderophores
235
loam soil where short-term periodic flooding resulted in a uniform distribution pattern of rhizospheremicroorganismsand ironchlorosis. In contrast,nonflooded plants had lower numbers of microorganisms in the apical root zones and were not affected by iron chlorosis. Differences in the utilization of microbial siderophores by dicotyledonous plants have been correlated with the iron-efficiency stress reaction (64). In an experiment with iron-efficient and inefficient varieties of hydroponically grown carnation, the microbial siderophore pseudobactin was shown to serve as an iron source for the more efficient cultivar but not for the inefficient cultivar. Interestingly, the synthetic chelate FeEDDHA was equally well used by both cultivars, suggesting a unique mechanism foruse of the microbial siderophore that occurred only in the iron-efficient cultivar. In later research on monocots by these investigators, plant use of pseudobactin, the synthetic chelator EDDHA. and phytosiderophore was compared using barley plants grown under axenic and nonsterile conditions (65). Resultsshowedthatphytosiderophorewasthepreferrediron source but that pseudobactin at 4 pM enhanced iron uptake by barley under nonsterile conditions i n which the phytosiderophore was subjectto rapid degradation. In comparison, research by other investigators examining short-term Fe uptake from s'JFe-pyoverdine as compared with S"FeEDTAby cucumber and 5'Fe-phytosiderophores by maize showed that ferrated pyoverdine was a relatively poor Fe source for either maize or cucumber. Iron uptake from pyoverdine by cucumber iron uptake from pyoverwas 40 times slower than from EDTA. For maize plants, dine was 665 times lower than from phytosiderophores (58).
VI.
SIDEROPHORE PRODUCTION IN THE RHIZOSPHERE
A.
Types of SiderophoresProduced
The production and accumulation of different types of siderophores in the rhizosphere is largely unknown except for a few specific compounds that have been estimated using bioassays or antibiotic detection procedures (66,67; also see Ref. 68 for review). Using chemical detection procedures, only one study has reported thedirectextraction of siderophores in soil.Thisparticularstudyexamined schizokinen (69), which was produced in a flooded soil under rice culture. After extracting the compound from soil with water, the siderophore was sorbed on to a cation exchange medium, purified and analyzed by high-performance liquid chromatography. More commonly used methods for detection of siderophores have involved bioassays. In general, these bioassays use siderophore auxotrophic strains that require siderophores in order to grow on agar media or to cause turbidity during growth that can be used to estimate siderophore concentrations i n a variation of the most-probable-number assay.A,-th,-ohacfer.fi~~vescer~s JG-9 has been one of the most widely usedauxotrophic strains andis used to detect hydrox-
236
Crowley
amate type siderophores. Since Arthrobacter responds to a variety of hydroxamatesiderophores, the bioassay is usuallycalibrated to desferrioxamine B. which is available commercially. Another assay has used Esch~richiocoli mutants to detect hydroxamate and catecholate siderophores (66). Presently, several surveys have shown that hydroxamate siderophores commonly occur i n soil at nanomolar concentrations (28,66,70), which can be increased by amendment with various carbon substrates used to simulate rhizosphere conditions at nutrient-rich sites (7 l ) . In early studies on the rhizosphere, it was presumed that pseudomonads and various gram-positiveor gram-variable bacteria such asBcrcillus and Arthrobncter were the predominant bacteria that grow on plant roots. There has also been some investigation of siderophore production by nitrogen-fixing bacteria, mycorrhizal fungi, and selected fungi such as R h i z o p s . However, to date most of our knowledge of rhizosphere communities and the siderophores that they produce has been obtained in studies that focus on culturable microorganisms, particularlyrootcolonizingpseudomonads.Earlywork by Vancura(72)and Kleeberger et al. (73) suggested that pseudomonads were the predominant bacteria in the rhizosphere, comprising over 50-70% of the culturable bacterial species. In contrast, a more recent comprehensive study with sugar beet (Betcl v d garis) revealed 102 species from 40 genera, which were identified from among 556 isolates (74). The IO most common genera were Bacillus ( 14%), Arthrobucter ( 12%). Pseudottronus (1 1 %), Aureobacteriutn (9%),Micrococclrs (6%), Xctt~thottzotms( 5 % ) , A1ccdigene.s (4%), FlrrvoOcrcteriut~r (3%), Agrobacterim (3%),and Microbucterium (30/0),which accounted for 70% of isolates. Whether this general community structure is the case for most plants remains a matter o f speculation and would be somewhat surprising.Some 775 genera of bacteria have been isolated from the environment, and recent studies using genetic methods suggest that there may beas many as 4000 speciesper gram of soil (75). Culturable microorganisms thus may represent only l -S% of the total bacteria that occur in nature. Other evidence suggesting that this formidable level of diversity may be real are recent data showing that 64% of the bacterial species identified from a 16s rDNA library of rhizosphere and bulk soils from the southwestern United States are completely unknown (76). Many of these bacteria appeared to belong to a major bacterial lineage that has never been identified before except for one single cultured representative, and almost none of the bacteria that were isolated were from genera that are typically isolated on agar media. Based on these data, it has become increasingly apparent that we do not even know what siderophores to look for yet, much less how they function in rhizosphere ecology and plant nutrition. Siderophore production by rhizosphere bacteria that are culturable on agar media has been estimated by plating out colonies on an indicator agar medium containing chrome azurol, a weak iron-chelating complex that changes colorupon
Function of Siderophores
237
deferration (77). Among 240 isolates that were screened, 80% failed to grow on the assay medium, highlighting the problem of medium selectivity in working with soil bacteria. Bacteria that grew on the assay medium produced siderophores in vitro at concentrations ranging from 100-230 PM, a concentration range that would be effective for providing plants with chelated iron, in the case that they can use the specific chelator by either an indirect or direct mechanism. A nluch more fundamental questionis whether these concentrations ever existin the rhizosphere or instead are concentrationsthat might occur only temporarily at specific microsites of highmicrobialactivity or under carbon-rich conditions inirondeficient media. Sincemost bacteria and fungi have siderophore transport systems that are designed for much lower concentrations, which are suppressed by ironchelate concentrations above 5 to I O FM, it is unlikely that ferrated siderophore concentrationsabovethesevalueswouldeveraccumulateintherhizosphere. Hence, if large concentrations of siderophore are produced at microsites, it is likely that they would be in the deferrated form. The potential ability of pseudomonads to produce very high concentrations of siderophores at localized sites of high microbial activity in the rhizosphere is particularly well shown by the use of root fingerprints on iron limiting agar media. As shown in Fig. 3, for barley roots pressed onto an agar plate containing an iron-deficientmediumselectivefor Psrudomonns sp., culturable cells of root colonizing pseudomonads are located primarily in the zone of elongation behind the root tips. After growth of the bacteria on the agar plate, the siderophore produced by the root-associated bacteria is visualized by exposure of the plate to ultraviolet light, which causes the iron-chelating fluorescent siderophore to give off a green color that can be photographed. The green halo associated with the pseudomonadcolonies ishighlylocalizedatthese samelocations attheroot tips and sites of lateral root emergence. These sites correspond to those that are visualized with autophotography using a bioluminescent strain of Pscudonwwc~s (Fig. 4A andB ) and to those labeled and visualized with autoradiography following exposure of roots to radiolabeled-iron chelates (Fig. 4C). Iron uptake by bacteria at sites of lateral root emergence has been further confirmedusinganothertechniqueemploying7-nitrobenz-2-oxa-1,3-diazoledesferrioxamine B, which is a derivitized siderophore that becomes fluorescent after it is deferrated (78). In this case, iron uptake from the siderophore ferroxamine B was associated primarily with microbially colonized roots,both but plant and iron uptake from this chelate occurred in the regions justbehind the root tips. Several studies have shown that ferrated pyoverdine-type siderophores can be used as iron sources for plants when added to soils (79,80). However, to date almost all attempts to supply iron to plants by inoculation of hydroponic solutions with siderophore-producing bacteria or by inoculating soils with pseudomonads have been unsuccessful (58,63,81). In experiments with cucumber, inoculation of a hydroponic medium with P. putidu or with soil microorganisms and amend-
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Figure 3 Root fingerprints of Pseudomonas sp. associated with barley seedlings showing the production of siderophore by actively growing bacteria located in the zone of elongation behind the root tips. Roots were pressed on to an iron-deficient minimal medium selective for Pseudomonas. After growth of the colonies, the production of siderophore was visualized by exposure of the agar plate to ultraviolet light, which causes the siderophore to fluoresce.
ment with autoclaved soil as an iron source failed to provide iron for plant growth (58). In related work, the extent to which plants benefit from root colonization by siderophore-producing bacteria was examined for iron-efficient and inefficient oat cultivars inoculated with six strains of bacteria that produced high concentrain a calcareous soil (81). Three species of Pseutions of siderophores and grown domonas colonized the roots of both cultivars in numbers greater than IO6 cells g" root, but had no significant effect on Fe acquisition by the plants. Instead, plants fertilized with 5 pM Fe were larger and supported greater numbers of rhizosphere bacteria per gram of root than plants that were not supplied with iron and inoculated with bacteria.
239
Function of Siderophores
A
C
f' B
i Figure 4 Sites of high microbial activity on plantroots as depicted by autophotography using a bioluminescent pseudomonad P. juorescens 2-79RL that produces light during active growth. (A) Corn root system inoculated with the lux-marked bacterium.(B) Autophotograph generated by placing film over the corn root system, which was then exposed to light produced bythe bioluminescent bacterium. The autophotograph shows that microbial activity is greatest in the zone of elongation at the root tips and at sites of lateral root emergence. C. Autoradiograph of a nonsterile, iron-stressed oat plant showing iron accuB. Dark areas caused byexposure of film mulation from s5Fe-siderophore desferrioxamine to the radioactive iron are located in the zones of elongation and at sites of lateral root emergence, corresponding to the areas of microbial growth in Fig. 4B.
B. MicrositeDifferences in SiderophoreProduction Differences in siderophore production in the rhizosphere are likely to occur depending on the root location, the plant growth stage, its mineral nutritional status, the presence or absence of mycorrhizae, and on the soil chemical properties (1). Since siderophores are produced onlyin response to iron stress, which is a function of the relative growth rate, it cannot be presumed that similar of siderotypes phores will occur at specific root locations or that siderophores will occur in all root zones. Microbial growth rates are controlled by carbon availability, which varies along the root axis, and it is generally believed that there is a successional
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development of microbial communities as roots elongate and mature (82,83). New root tips that are elongating and secrete root exudatesin the zone of elongation are sites of high microbial activity and are presumably colonized by rapidgrowing,opportunisticmicroorganismssuch a s Pseudornottas sp.Older root zones, on the other hand, are crowded environments, which are relatively oligotrophic once the roots become suberized. Nonelongating secondary root tips and the sites of lateral root emergence are still other microsites that may be presumed to have unique microbial communities. Thus, it maynot bepossibletodraw general conclusions regarding which siderophores are predominant in the rhizosphere and whether or not any particular siderophore type accumulates at a concentration that would be relevant to plant nutrition when it is only produced at amicrositelocation.Similarly,siderophoresmayhavedifferentfunctionsdepending on the root zone. For example, it can be speculated that pyoverdines may contribute to rhizosphere competence of pseudomonads at the,root tips but are irrelevant in the older, carbon-limited root zones where iron is either not limiting or is chelated with various more generic siderophores. Recent studies have further examined the iron stress response of pseudomonads using an iron-regulated, ice-nucleation gene reporter (innZ) for induction of the iron stress response ( 17,18,84). This particular reporter system was developed by Loper and Lindow (85) for study of microbial iron stress on plant surfaces but was later employed in soil assays. In initial studies, cells of Pseudomot1N.sjll[)re.scCrl.s and P. syringae that contained the pvd-inaZ fusion were shown to express iron-responsive ice-nucleation activity inthebeanrhizosphereand phyllosphere. Addition of iron to leaves or soil reduced the apparent transcription of the pvd-inai! reporter gene, as shown by a reduction in the number of ice nuclei produced. This reporter system has sincebeen used in a number of studies examining regulation of the iron-stress response in soils and in short-term experiments after inoculation of plant roots (16). Ice-nucleation activity expressed by rhizosphere populations of P. juorescem Pf5 was at a maximum within 12 to 24 h following inoculation of the bacterium onto bean roots and typically decreased gradually during the following 4 days. The finding that iron stress decreased 1 to 2 days after the bacterium was inoculated onto root surfaces, suggested that iron became more available to rhizosphere populations of Pf-5 once they were established i n the rhizosphere. Thisled to speculation that iron acquisition systemsof plants and other rhizosphere organisms may provide available sourcesof iron to established rhizosphere populations of P. ,Puorescen.s. In concurrent research involving a longer-term study, the P. , ~ ~ ~ o r e s c e t ~ s Pf-5 strainwasinoculatedintosoilusedtogrowlupine (Lupitms d h u s ) and barley (Hordeum vlrlgare L.) over a several-week period (17). It was shown that iron stress was greatest in the zone behind the root tips as compared to older root zones and in the bulk soil. Calibration of the ice-nucleation reporter activity
Function of Siderophores
241
to siderophore production in vitro allowed estimation of potential siderophore production on a per cell basis in vivo. By using the regression between ice-nucleation activity and pyoverdine production, and assuming a P. ,fluore.~ce~l.s population density of IOx CFU g" root, the maximum possible pyoverdine concentration was estimated to be 0.5 and 0.8 nmol g- for lupine and barley, respectively. The low ice-nucleation activity measured i n the rhizosphere suggested that nutritional competition for iron in the rhizosphere may not be a major factor influencing root colonization by P. Juowsccw.y Pf-S (pvd-inaZ). These data also suggested that overall concentrations of this siderophore in the rhizosphere would be too low to be of relevance to plant nutrition. In another experiment with rice and barley, it was further shown that the iron-stress response of P. prtidrr Pf-S could be shut down by production of phytosiderophoresafterinduction of theironstress response byplantroots (84). When barley and rice seedlings were treated with foliar applications of ferric citrate, the iron-stress response of the plant was suppressed such that phytosiderophoreproductiondecreased and causedincreasedironstress in therootcolonizing pseudomonads. This studyalso tested the ability of this pseudomonad to use phytosiderophore and ferric citrate as iron sources for growth in vitro and showed that both were effective i n supplying iron and in suppressing expression of the iron-regulated ice nucleation reporter. These data strongly suggestthat siderophore production by root-colonizing microorganisms is induced only at a level necessary to supplement that which is not provided by phytosiderophores and organic acids released during theplant iron stress response. Thus, the plant iron stress response may control iron availability to microorganisrns in the rhizosphere.
'
VII. A.
EFFECT OF PLANT IRON STRESS ON MICROBIAL COMMUNITY STRUCTURE Denaturing Gradient Gel Electrophoretic Fingerprints of Microbial Communities
Perhaps one of the most powerful genetic techniques that has been developed in the past few years for characterization of microbial communities is the use of 16s rDNA fingerprints (86). Based on the pioneering work of Carl Woese (87), ribosomal DNA sequences are nowused for taxonomic classification of all living organisms. Using the 16s rDNA data base, portions of this 1500-base-pair sequence can be analyzed to provide domain, family, genus, or species identitications of bacteria, including those that are nonculturable on agar media. Initially, the extension of this technique for analysis of microbial communities involved polymerase chain reaction (PCR) amplification of 16s rDNA from environmental
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samples, followed by construction of a clone library that carried all of the sequences contained in the community. Individual clones were then analyzed to determine the 16s- rDNA sequence they carried. The community structure was characterized randomly by analyzing a large number of isolates, after which the different groups or species were tabulated to generate a frequency plot. Shortly i n community finafter development of this technique, the next major advance gerprinting was the use of denaturing gradient gels to simultaneously separate all of the 16s-rDNA sequences contained in a sample into discrete bands on a polyacrylamide gel. The end productis a broad snapshot of the community structure, which is manifested in a “bar code” pattern (86), in which each DNA band represents a different group or species of bacteria. The power of this technique lies not onlyin its ability to provide information on total diversity and community structure but also in its utility for identification and monitoring of specific microbial populations in environmental samples (88-90). T o generateDGGEcommunityfingerprints, totalbacterialDNAisextracted from the sample, after which all of the 16s-rDNA sequences from the communityareamplifiedsimultaneously using PCR. Denaturinggradient gel electrophoresis (DGGE) is then used to separate out the assortedl6S rDNA sequences based on differences in their guanosine-cytosine (GC) content and thermal melting, points as the DNA migrates through the gradient gel. This results i n discrete bands for each species or group of bacteria having 16s rDNA sequences with similar melting points. The end result of this process is a unique fingerprint that reflects all of the bacterial species present and their relative predominance, the latter indicated by the DNA band intensity. Once the DNA patterns are generated, image analysis is used to compare fingerprints from samples that have been subjected to different experimental treatments.
B. Analysis of Fe Stress Effects on Microbial Community Structure on Barley Roots In recent studies, examining the effect of plant iron stress on microbial communities in the rhizosphere, experiments were conducted with barley plants grown in replicate root box microcosms in an iron limiting soil. Half of the plants were treated with foliar applied iron to alleviate plant iron stress and shut down phytosiderophore production at the root tips. Previously, this treatment had been shown to cause an increase in iron stress for cells of P. jmrescelzs located at the root tips (84), due to increased reliance on endogenous siderophore production. In the experiment reported here, 1 -cm root segments were harvested from different locationsonFe-stressedandnonstressedplantsandsubjectedtoPCR-DGGE usinguniversal primers for eubacteria. Conlparison of the DGGE profilesfor eubacterialcommunitiesassociatedwithsimilarlocations onreplicateplants showed that the rhizosphere community associated with specific locations was
C
A +Fe
-Fe
Oblique Factor Plot
Factor 1
B
+ Fe
- Fe
Figure 5 Genetic fingerprintsofmicrobialcommunitiesassociatedwiththe roots of iron-sufficient and iron-stressed barley grownin an iron-limiting soil medium. Fingerprints were generated using polymerase chain reaction (PCR) primers for 16s rDNA, which was then separated on a polyacrylamide gel using denaturing gradient gel electrophoresis (DGGE).Each band corresponds to an individual species or group of bacteria having similar 16s rDNA sequences. The advantage of this technique is that it provides a cultureindependent method for analyzing microbial community structure and species composition. (A) DGGE gel of eubacterial communities from different root zones. Visual analysis of the profiles show that communities associated with apical roots of stressed and nonstressed plants have a similar structure and species composition. stress Iron caused a major shift in the community structure associated with the root tips. (B) Line profiles generated by image analysis of root tips from four replicate iron-stressed and nonstressed plants. (open (C) Factor plot showing separation of iron stressed (closed circles) and nonstressed circles) roots as variables that differentiate the rmcrobial communities associated with the root tlps of barley.
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highly structured and reproducible for replicate root segments obtained from the same plant or from replicate plants grown in different microcosms. Differences in microbial communities associated with different root parts are shown in Fig. 5A. In general, microbial diversity (species richness and species evenness) was greatest on the older root parts, whereas the communitiesthat formed on the root tips were relatively simple in structure and less diverse. Statistical analysis of the microbial communities associated with the root tips of iron stressed and nonstressed plants revealed the formation of distinct communities i n response to plant iron nutritional status (Fig. 5B and C). Communities associated with the older root parts clustered similarly, whereas sites of lateral root emergence and nongrowing root tips differentiated to a lesser degree than the communities associated with the rapidly growing primary root tips (data not shown). Comparison of thedifferentbandingprofilesshowedthatcertainbands were consistently present at allroot locations. To determine the microbial species that comprised these predominant bands,the DNA bands were excised from each lane and used to construct eight separate clone libraries representing each of the four root locations from iron stressed and nonstressed plants. Two clones were then picked from each location and sequenced to obtain species identifications. The bacterial species associatedwith these bands were distinct for eachroot location (Table 3). Aut-eohactrriwn was identified at the sites of lateral root emergence: whereas H~/?horllic.robilrirl and B$c/obacfer.iw?~ were identified among the predominant bacteria at the root tip. Nitr'osococclrs and an unidentified eubacterium were identified on the older root parts. Neither H?.i?hortzicr'ohiurtlnor Blfic/obcrcter'i1m7 have been previously reported in the rhizosphere. The presence of Aur-c.oDac.fcJr'irrt?l at the sites of lateral root emergence, which is a cellulose de-
Table 3 MicroorganismsIdentitied by SequenceAnalysis of Prominent 16s rDNA Bands Isolated from Barley Roots
Iron
Function of Siderophores
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grader, is in accordance with the availability of cellulose provided by sloughing of the root cortex tissue and damage to root cortical cells at this location. The presence o f Ni/ro.sococc~r,s on the older root parts suggests the operation of nitritiers i n the oligotrophic environment of the mature rhizosphere. The importance of siderophores in the ecology of these bacteria is completely unknown.
VIII.
FUNCTION OF SIDEROPHORES IN RHIZOSPHERE COMPETENCE
One of the driving forces behind research on siderophores and their importance in rhizosphere colonization has been the finding that various bacteria can have a wide range of effects on plant growth. Certain bacteria, termed p h f g r o d prorttoting, are thought to produce hormones, vitamins, and growth factors that enhance plant growth, whereas other bacteria produce compounds that are deleterious to plants. The abilityto manipulate rhizosphere populations via inoculation with siderophore-producing bacteria or by using soil amendments t o alter iron availability has thus been a matter of practical concern. In a recent study (91), over 100 bacterial strains were tested for their plant growth-promoting and plantdeleterious effects. Thcse strains belonged to the genera Azotobetcrer, AmspirilI L ~ Bcrcillus, , E ~ l / c ) r o b e ~ t eand r , P.selrr1orllorzn.s and were all isolated from the rhizosphere of various crops. In vitro inoculation of maize seedlings with thesc strains resulted in a 50% decrease to a 70% increase in plant growth as compared to noninoculatedcontrols.Subsequentresearchexaminedtherelationship between siderophore production and cotupetitive fitness of these strains but was unable to demonstrate any relationship between the ability to use pseudobactintype siderophores and rhizosphere competence. Genetic studies have shown that the nucleotide sequence of the Psrurlorttotzas sp. strain M 1 14 pbua gene, encoding the outer membrane receptor for ferric pseudobactin M I 14, displays characteristics in common with other outer membrane proteins (92). This sequence also has strong homology with the TonB boxes of both E. coli and P.setlrlon1onrr.s receptors. More extensive honlohg' rles were found with the PupA receptor of P . p u t i h WCS358 and the FhuE and BtuB receptors of E. coli. Based on these findings, it was suggested that a PbuA-like receptormaybewidelydistributedamong P.seudorwrms rhizosphereisolates. Those areasof the PbuA receptor exhibiting the least homology between different pseudomonads may represent ferric siderophore-specific recognition sites for different types of pseudobactin. The specificity of certain pseudomonads for strain specific siderophores has been demonstrated for P . putider WCS358, whichcan be recovered efficiently pM pseudobactin 358 (93). Low population onamediumamendedwith300 densities of indigenous pseudomonads (less than or equal to IO' g" of soil or
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root) recovered on the pseudobactin 358 revealed that natural occurrence of the pispA gene, required for use of this siderophore was limited toaverysmall number of indigenous Pselrclomonc~sspp. that arevery closely related toP. puriclct WCS358. In a subsequent experiment (94), the potential of different Psr~udonwnus strains to utilize heterologous siderophores was compared with their competitiveness in the rhizosphere of radish. Interactions were examined for microbial populations of P . puticla WCS358 andP.,fluore.scerl.sWCS374 and between strain WCS358 and eight indigenous Pseudonzonas strains capable of utilizing pseudobactin 358. Duringfoursuccessive plant growthcyclesofradish,strain WCS358 significantly reduced rhizosphere population densities of the wild-type strain WCS374 by up to 30 times. In comparison, a derivative strain WCS374 (pMR), harboring the siderophore receptor PupA for ferric pseudobactin 358, was able to maintain its population density. While this suggest that the ability to use pseudobactin was directly involved in rhizosphere competence. parallel studies on interactions between strain WCS358 and the eight different indigenous P s e d o n w t m strains showed that population densities of these bacteria were all reduced by up to 20-fold by strain WCS358, despite the ability to use pseudobactin 358. The authors concluded that siderophore-mediated competition for iron is a major determinant in interactions between some strains of bacteria, but that other traits also contribute to the rhizosphere competence of fluorescent PseucloInOIlelS spp. The ability of pyoverdine-type siderophores produced by pseudomonads to suppress the growthof other bacterial genera has come under some challenge; and in fact, these compounds have been shown in certain cases to actually promote the growth of different bacteria. In a study conducted with mixed batch cultures, P s e u d o n ~ o n mcepacia stimulated the growth of Bacillus polwr.wcr i n low iron medium, and this effect could be replicated in pure culture by addition of pyoverdine produced by either P. JIuorescetIs or P. cepc.itr (95). In another study, two strains of BrerclyrhizoDiurnj q x ~ n i c w r n ,which normally utilize ferric also citrateandthehydroxamatesiderophoresferrichromeandrhodotorulate, were shown to use the pyoverdine-type siderophore pseudobactin St3 (96).
IX. ROLE OF SIDEROPHORES IN BIOCONTROL OF PLANT PATHOGENS Unraveling the role of siderophores in disease suppression has been complicated and is one of the best examples of the problems that may be encountered by extrapolatingbroadconclusionsfromsimpleexperimentalsystems.Earlyresearch frequently employed screening assays using coculture of pseudomonads and plant pathogens on agar plate media to demonstrate that siderophores may be responsible for suppressing the growthof various fungal pathogens under iron-
Function of Siderophores
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limiting conditions. More recently, it has been recognized that this is a narrow approach but that these assays may still have utility for ruling out whether or not siderophores have an effect on the pathogen (97). In these plate assays, the siderophore sequesters all of the available iron to the detriment of the pathogen. Competition for iron is due to the superior ability of Pseudortwrm SP. siderophores to chelate iron, which is based on their high stability constant for iron as compared to the siderophores produced by most fungal pathogens. Siderophores have also been added to soil in spent media to achieve biological control and in Some instances are as effective as adding the bacteria themselves (2). However. much larger quantities of siderophores are produced during growth in nutrientin soils, and the relative importance rich, iron-limiting media than are detected of siderophores i n disease suppression in comparison to antibiotic production has been controversial. An increase in the number of siderophore-producing organisms in the rhizosphere is associated with increased disease suppression and can be achieved by amending soils with compost (98). However. it now appears that while Inany disease-suppressivebacteriaproducelargequantities of siderophoreswhen screened in vitro, the function of siderophores in directly antagonizing pathogens in the rhizosphere is questionable. Suppression of fungal pathogens i n the rhizosphere by root-colonizing pseudomonads involves several mechanisms that may operate individually or collectively to antagonize the pathogen. These mechanisms involve nutritional competition, production of antibiotics (99), release of cyanide (100). and production of siderophores. Some pseudomonads also cause disease suppression by producing a systemic resistance response that is induced by the host plant after colonization by pseudomonads that produce pyoverdine and other salicylate-based siderophores ( 101). One of the most powerful methods for studying the function ofsiderophores for biological control of root disease-causing microorganisms has involved the generation of mutant strains that are defective in producing antibiotics or siderophores. The bacteria are then inoculated into soils to assay their efficacy in disease suppression (for review, see Ref. 102). Examples where siderophores cause direct antagonistic effects on pathogens are rare; this phenomenon appears to operate i n a strain-dependent fashion in very specific disease interactions. For example, the sole mechanism involved in the suppression of Fusnviwtz wilt of radish by P.sc~~rdorr~or~rrs strainWCS358 is siderophore-mediatedcompetitionforiron, whereas strain WCS374 suppresses this disease by induction of systemic resistance (103). Other examples where siderophores have been shown to have no with Etlter-ohtrcror discernablefunctionindiseasesuppressionincludestudies cloc~caeCT-501, which suppresses Pytkiurn damping-off of cucumber and other planthosts. Thisbiocontrolbacteriumproduces the hydroxamatesiderophore aerobactin and a catechol siderophore tentatively identified as enterobactin. After generatingmutantsdefective in eitheraerobactin or enterobactinsynthesisor
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double mutants defective in both of these siderophores, it was shown that neither of these siderophores contributed to the abilityof E. cloaccrc to suppress Pythiurrl damping-off of cotton or cucumber (104). Similarly, the antagonism of five bacterial isolates, including Acitletobrrcter sp., Bacillus polyrtlyser, Btrcillus .subtili.s, P. ceptrcia, and P. p u t i d e r ) against the pathogens Sclerotirlitr sclerotiorurrr, Sclerntirlitl rrlirlor, and Gac~url~nt~norllyces grtrrr~ir~is was shown to be due to antibiotics rather than siderophore production (104). Interactions between the induction of systemic acquired resistancein plants and the involvement of siderophores in direct antagonism of root pathogens have turned out to be subtle and difficult to discriminate. I n one recent study (IOS), theinfluence of iron availability on induction of systemic resistance i n radish (Rtrpkarlus strtiws L.) against F~r.scrriur11 wilt as mediated by P. J ~ I I O ~ ~ S ~was ~ C I I S examined. In the actual experiment involving the pathogen(Fu,scrrirrrl~o.q"spor~or~ sp. raphani) and a strain of P.sc~ucl~)r~~oncr,s, salicylic acid (SA) or a pseudobactin was applied at separate locations on the plant root. Strain WCS374 and its pseudobactin-minus TnS mutant gave greater disease control in the induced systemic resistance bioassay when iron availability in the radish nutrient solution was low than when it was high. Mutants of P. .fluore~ce~~.s strains WCS374 and WCS4 I7 lacking the 0-antigenic side chain of the lipopolysaccharide induced resistance at low but not at high iron concentrations. Interestingly, the purified pseudohactin of strain WCS374, but not the pseudobactins of strains WCS3S8 and WCS417, induced resistance to Fustrriurr~.It was subsequently shown by gas chromatography that strains WCS374 produced high concentrations of salicylic acid under conditions of low iron availability and that salicylic acid also induced systemic resistance to root disease. This led to the conclusion that salicylic acid produced by selected P. ,jhorescerIs strains as well as pseudobactin produced by WCS374 may both be involved in causing induced systemic resistance. Other research examining the effects of salicylic acid i n causing induced systemic resistance under iron-limiting conditions suggests that salicylic acid itself is a siderophore, since it chelates iron and is released in response to iron stress (106). The plant growth-promoting rhizobacterium P.seuclomo~~as crenrgitwsa 7NSK2 produces three siderophores when it is iron-limited. These include pyoverdine, the salicylate derivativeof pyoverdine called pyochelin. and salicylic acid. The role of pyoverdin and pyochelin in the suppression of Pythium splenclerls wasinvestigated by usingvarioussiderophore-deficientmutantsderived from P. creruginoscr 7NSK2 in a bioassaywith tomato (Lycopersicorl esc~derlturrl). Production of either pyoverdin or pyochelin proved to be necessary to achieve wild-type levels of protection against Pytkium-induced damping-off. Since pyoverdin and pyochelin are both siderophores, siderophore-mediated iron competition could explain the observed antagonism, but the authors could not exclude the possibility that the siderophores acted in an indirect way. The relative importance of antibiotic production versus production of siderophores for disease suppression has recently been studied by Mulya and CO-
Function of Siderophores
249
workers (107). I n experiments with P. jluorescem strain PfG32 isolated from the rhizosphere of the onion, it was shown that the bacterium actively suppressed the occurrence of bacterial wilt disease of tomato and produced both antibiotics and siderophore in pure culture. After isolating several mutants defective in antibiotic substances or siderophore production, the suppression of bacterial wilt by the different strains was compared. Mutants that did not produce antibiotics but produced siderophores were less effective for disease biocontrol than were strains that produced both substances or that produced antibiotic but no siderophores. This suggeststhat for this particular bacterium, the antibiotic was more important than the siderophore but that both substances contributed to the ability of the bacterium to cause disease suppression. in disease suppresWhether antibiotics or siderophores or both are involved sion, an obvious role of siderophores is the enhanced competitiveness that may be associated with the abilityto produce and use siderophores for iron acquisition under iron-limiting, high-carbon conditions in the rhizosphere. Generally, pseudomonad population densities of I O ' C ~ L Iper gram of soil are associated with disease suppressiveness. Disease suppression in soils providingnatural biocontrol of take-all decline of wheat is associated with high numbers of pseudomonads that produce the antibiotic 2,4-diacetylphloroglucinol(108). Below this density, disease suppression rapidly declines: whereas above this threshold density, there appears to be no additional benefit for further enhancing the disease-suppressive effects. The importanceof maintaining a critical threshold density for suppression of Fuscwiurn wilt of radish was also shown in soil inoculated P. putick/ (94). In this study, population densities of IO5 cfupergramofrootwererequired to provide biocontrol by either strain WCS3.58, which produces siderophores, and strain WCS374, which causes systemic resistance. Althoughit may be speculated thattheability to utilizeadditionalsiderophoresmayincreasethefitnessand population density of pseudomonads used for biocontrol, it appears that crossfeeding among pseudomonads does not necessarily result in enhanced numbers of pseudomonads in the rhizosphere ( 109). Transfer of a plasmid conferring the ability to utilize additional pseudobactins had no effect on increasing the population size of pseudonionad inoculants in soil.
X.
FUNCTION OF SIDEROPHORES IN NITROGEN FIXATION
Iron is required in large quantities for nitrogen-fixing bacteria. and the importance of siderophores in the iron stress response of these microorganisms hasbeen well established ( 1 IO, 1 I 1 ; for review. see Ref. 1 12). Iron is an essential component of the nitrogenase enzyme complex and is required for respiration, oxygen binding by leghemoglobin and for synthesis of ferredoxin, which delivers electrons to nitrogenase. Siderophores may alsobe important for temporary storageof iron
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i n the peribacteroid spaceof the symbiosomeof Rhizobium nodules( I 13). Siderophores found in the peribacteroid space have been tentatively identified as members of the pseudobactin family of siderophores and are different for various species of Rhizobium. The prototypical siderophore produced by Rhizobium is termed rhizobactin and is structurally characterized as a derivative of citrate in which the distal carboxyl groups of the citrate molecule are joined by an amide bond with two side chains ( 1 14). More recent studies have revealed that not all Bradwhizobiurn and Rhizobium strains produce siderophores and that the types of siderophores produced include both hydroxamate and catecholate-type siderophores ( l 15). In screening assays of 3 1 strains, it was shown that siderophore production was correlated with nitrogen-fixing efficiency. Other studies suggests that this may not always be the case (1 16,l 17). Differencesinsiderophoreproductionhavebeenobservedfor thefreeliving forms of the bacterial symbionts (1 18). However, it has not been determined whether the production of high concentrationsof siderophores corresponds with an increase in nitrogen-fixation efficiency or adaptation of Rhizobium strains to iron-limiting soils. In one study examining various siderophore mutants of R. meliloti, it was shown that the high-affinity iron-acquisition system expressed by the free-living form of this bacterium is not essential for nitrogen fixation, although it can affect the early events of nodulation ( 1 15). In contrast, results of another study suggest thattheability toproducesiderophoresisessentialfor nitrogen fixation to occur. In this particular case, mutants of R. rnelilofi strain 1021 that were defectivein rhizobactin synthesis produced nodules on alfalfa but fixed insignificant amounts of dinitrogen ( 1 17). Mutants of R. rneliloti constitutive for rhizobactin synthesis also produced nodules, but the levelsof dinitrogen fixed were low. These contrasting data suggest that there is a need for siderophore production during symbiosis but that the exact role of siderophores in nodule formation versus nitrogen fixation is still unclear. One explanation for the disagreement over the relative importance of siderophore production by Rhizohiunl may be plant-host-related effects that influence iron availability to the symbiont and the ability of free living Rhir.ohiurn to obtain iron from sources other than its own siderophore. Jadhav and Desai ( 1 18) showed that while cowpea RhizobiurnGNI (peanut isolate) produced siderophore under iron-starved conditions, other nutritional and environmental factors a h affectedsiderophoreproductionand ironassimilationbythismicroorganism. Maximuln siderophore production was obtainedwith maltose and urea as carbon and nitrogen sources, respectively. With citrate as a sole source of carbon, there was complete repressionof siderophore production without any effect on growth. The involvement of citrate in iron transport was confirmed by "Fe-citrate uptake studies. This suggests that plant species that produce large quantities of citrate in response to iron stress may provide iron for Rhiwbiutnin the rhizosphere and during nodulation. Positiveinteractionsbetweenplantgrowth-promotingrhizobacteriaand
Function of Siderophores
251
Rhizobium have been reported, buttodatethereislittle evidence that siderophores produced by pseudomonads are beneficial for promoting nodulation and nitrogen fixation. In experiments examining the role of siderophore productionon nodulated clover plants, siderophore-defective mutants were shown to stimulate growth of nodulated clover plants similarly to the siderophore-producing parent strain ( 1 19).
XI.
SUMMARY
The primary function of plant and microbial siderophores in the rhizosphere is for iron acquisition under iron-limiting conditions. Almost all microorganisms have been found to produce siderophores and can potentially compete with each other for iron, depending on their ability to utilize different siderophore types or based on the uptake kinetics of their siderophore transport systems. Similarly, plants and microorganisms can compete for iron under certain conditions, although the extent to which this influences plant ecology in nature can be questioned. Since the discovery of microbial siderophores by Lankford in the 1950s andphytosiderophores by Takagi in the 1970s, therehasbeenatremendous amount of research on the biochemical characterization of siderophores, the proteins that function for the uptake of iron, and the molecular regulation of the high-affinity iron transport systems used to synthesize and transport these compounds. In comparison, much less is known about the function of siderophores in microbial ecology. I t is becoming increasingly evident from studies on the rhizosphere that the plant’s iron stress response is the dominant factor in determining whether microorganisms are subjected to iron stress and thereby are induced to secrete siderophores ( 1 8,32,61,84). In response to iron deficiency, dicotyledonous plants release increased quantities of organic acids, acidify the rhizosphere, and secrete reductants, all of which serve to increase iron availability to the plant and the root-associated microflora. Grasses that produce phytosiderophores in response to iron deficiency can also increase iron availability to microorganisms that utilize these iron chelating substances. Many microorganisms have iron transport systems that function with organic acids such as citrate, and both organic acids and phytosiderophores have been shown to serve as effective iron sources for pseudomonads that colonize plant roots (84). For this reason, it can be assumed that siderophoresareproduced by microorganismsonly in quantitiesnecessaryto augment iron that is not provided by the plant’s iron stress response. Initial studies examining the function of siderophores in the rhizosphere have focused on practical problems related to agricultural biotechnology. Early research suggested the involvement of siderophores in plant disease suppression by certain root-colonizing pseudomonads (2). Since then, it has become apparent that siderophore-mediated interactions areonly part of the story of how microor-
252
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ganisms interact during the colonizationof plant roots (102). Many factors influencecompetition in soils,including thefitness of microorganisms in a given niche, the ability to use different carbon substrates, and the growth strategy of microorganisms thataredifferentiallyadaptedtocopiotrophic or oligotrophic growth conditions. Keeping i n mind Liebig’s law of the minimum, competition for iron will occur onlywhen all other growth factors havebeen optimized. In the rhizosphere, easily utilizable carbon substrates are provided as root exudates and lysates but are localized in specific root zones that comprise a relatively small fraction of thetotalrootsurfacearea. Thus, production of siderophores is mostlikely to occur in spot locations during intense competition for easily used carbon substrates. In the oligotrophic environment of the older root zones, which comprises most of the rhizosphere, carbon is available only in the formof much more recalcitrant substrates such as cellulose. On the older root parts, it is unlikely that competition for iron will ever occur, since sufficient iron Inay be provided by turnover of plant tissues, microbial cells, background siderophore concentrations, and organic matter iron complexes. The production and release of siderophores is very expensive in terms of carbon allocation for cells. In constructing a carbon budget for the oligotrophic zones o f the rhizosphere, it should be evident that any microorganism releasing large quantitiesof siderophore would be a t a disadvantage andwould simply be providing a labile carbon substrate for other bacteria and fungi. The extent to which siderophores shape the ecology of the rhizosphere is an interesting question that remains to be investigated. New tools in molecular ecology are beginning to provide insightinto microbial community structure and species composition in different root zones (86). Preliminary data presented in this chapter suggests that the plant’s iron stress response can alter the structure of the microbial community at the root tips but that there is little effect of plant iron nutritional status on the microbial community composition of the older root zones. An interesting question that remainsto be investigated is whether competition for iron that occurs during primary colonization of plants also has an effect on microbial community development i n older root zones as these communities mature and undergo succession into a crowded, oligotrophic community. Plants, like microorganisms, appear to have the ability to use a variety of iron sources for iron nutrition. Studies in hydroponic culture have suggested that siderophores can function similarly to synthetic chelates in providing iron, which is taken up by a variety of mechanisms (21,42-44,58,64.65,79). Depending on the chelator, these uptake mechanisms can include reductive release of it-on. degradative release of iron during decomposition of labile siderophores, uptake of the intact iron chelate, and ligand exchange with phytosiderophores and organic acids. However, the extent to which plants rely on microbial siderophores for iron nutrition is questionable, since siderophores probably never occur at high concentrationsthroughouttherhizosphere;andwhenthey do occur, theyare
Function of Siderophores
253
primarily in the deferrated form, which is inhibitory to plant iron uptake. Background levels of siderophores detected in soil extracts are generally very low (66-7 I ), being measured at nanomolar concentrations that may be of some use for oligotrophic microorganisms but are too low to be of physiological relevance for plant nutrition. Depending on their charge characteristics, siderophores can also be strongly adsorbed to clay and organic matter; at low concentrations, they may not diffuse readily through the soil matrix (28,29). The importance of siderophore production in the rhizosphere competence of bacteria and fungi will very likely depend on the growth strategyof the microorganism. Most studies on rhizosphere microorganisms have focused on pseudomonads, which are generally characterized as opportunistic bacteria that grow of newroottissues. rapidly on root exudates and thus are primary colonizers Certainly siderophorcs play a role in the rhizosphere competence of these bacteria, but since much of the root system is relatively oligotrophic, it would not be surprising to find that the predominant microorganisms that occupy most of the root surface are little influenced by siderophores. Nonetheless, the fact that most culturable bacteria and fungi maintain high-affinity iron transport systems suggests that there is selection pressure for maintenance of these systems for occasional growth under conditions that require the synthesis of siderophores for iron acquisition. Given the complexity of modeling soil solutions and predicting the chemistry of siderophores i n complex mixtures with different solid-phase minerals and constantly fluctuating ionic strength and redox conditions, it is unlikely that soil chemists will ever unravel how siderophores mediate competition for iron in the near future. The immediate need for scientists studying the plant rhizosphere will be to develop a conceptual understanding of how siderophores can potentially function in different root zones and under different soil conditions rather than jumping to broad conclusion from various scenarios devised i n the laboratory or from a computer model. One of the most important avenuesof research for future studies on siderophores will be the investigation of how these compounds influence heavy metal transport and bioavailability in soils and the possible role of siderophores in groundwater contamination and food-chain transfer. Understanding of these compounds may also have application i n the development of plant microbial systems for phytoremediationand the use of siderophores as iron fertilizers. For the future, the ability to manipulate siderophore production in the rhizosphere to improve plant trace metal nutrition and biocontrol of root disease will remain as a significant challenge.
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87. C. R. Woese, Bacterial evolution. Microbiol. Rev. 51:18 1 (1987). 88. S. Jensen, L. Ovreas, F. L. Daae, and V. Torsvik, Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16s rDNA fragments. FEMS Microbiol. Ecol. 26: 17 ( 1 998). 89. T. Vallaeys, E. Topp, G. Muyzer, V. Macheret, G. Laguerre, A. Rigaud, and G. Soulas, Evaluation of denaturing gradient gel electrophoresis in the detection of 16s rDNA sequence variation in rhizobia and methanotrophs. FEMS Mirwbiol. E d . 24:279 (1997). 90. J. R. Stephen, G. A. Kowalchuk, M. A. V. Bruns, A. E. McCaig, C. J. Phillips, T. M. Embley, and J. I. Prosser. Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing. Appl. Environ. Microbiol. 6429.58 (1998). 91. G. Forlani, R. Pastorelli, M. Branzoni, and F. Favilli, Root colonization efficiency, plant-growth-promoting activity and potentially related properties in plant-associated bacteria. J. Genet. Breed. 49:343 (1995). 92. J. Morris, D. F. Donnelly, E. O’Neill, F. McConnell, and F. O’Gara, F. Nucleotide sequence analysis and potential environmental distributionof a ferric pseudobactin receptor gene of Pseudonwnas sp. strain MI 14. Mol. Gen. Genet. 242:9 (1994). 93. J. M. Raaijmakers, W. Bitter, H. L. M. Punte, P. A. H. M. Bakker, P. J. Weisbeek, andB.Schippers,SiderophorereceptorPupAas a markertomonitorwild-type Pseudotnonns prrticla WCS358 in natural environments. Appl. Environ. Microbiol. 60: 1184 ( 1994). 94. J. M. Raaijmakers, M. Leeman,M. P. Van Oorschot, 1. Van Der Sluis,B. Schippers, and P. A. H. M. Bakker, Dose-response relationships in biological control of fusarium wilt of radish by Pseudornorus spp. Phytopathology 85:1075 (1995). 9s. L.Chiarini, S. Tabacchioni,and A. Bevivino,Interactionsbetweenrhizosphere microorganisms under iron limitation. Arch. Microhiol. 160:68 (1993). 96. 0. Plessner, T. Klapatch, and M. L. Guerinot. Siderophore utilization by Bru&rhiz o l ~ i l t mj(lponicunl. Appl. Environ. Microbiol. 59:1688 ( 1993). of crop 97. S . A. Omar and M. H. Abd-Alla. Biocontrol of fungal root rot diseases plants by the use of Rhizobia and Brrrdwhizoh. Folia Microbiol. 43:43 1 (1998). 98. M. A. De Brito Alvarez,S. Gagne, andH. Antoun, Effect of compost on rhizosphere microflora of the tomato and on the incidence of plant growth-promoting rhizobacteria. Appl. Environ. Microhiol. 61: 194 (1995). 99. R. J. Cook, L. S. Thomashow, D. M. Weller, D. Fujimoto, M. Mazzola. G. Bangera, andD. S. Kim, Molecular mechanisms of defense by rhizobacteria against root disease. Proc. Natl. Acad. Sci. U.S.A. 92:4197 ( 1 995). G. Defago, C. Keel,andD. 100. J. Laville,C.Blumer,C.VonSchroetter,V.Gaia, Hans, Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudornor~asfluorescells CHAO. J . Bacteriol. 180:3I87 ( 1 998). 101. M. Maurhofer, C. Hase, P. Meuwly, J. P. Metraus, and G. Defago, Induction of systemic resistance of tobacco necrosis virus by the root colonizing Pseudonlonns of the gacA gene and of pyoverdine production. jfluorescem strain CHAO: Influence Phytopcrthology 84: 139 ( I 994). 102. J. E. Loper, Contributionsof molecular biology towards understanding mechanisms
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Mycorrhizal Fungi: A Fungal Community at the Interface Between Soil and Roots Francis M. Martin INRA Center of Nancy, Champenoux, France
Silvia Perotto and Paola Bonfante University of Torino, Torino, Italy
1.
INTRODUCTION
The rhizosphere is a dynamic environment in which bacteria, viruses, fungi, and microfauna,includingarthropods and nematodes,develop,interact with each other, and take advantageof organic matter released by the root(1). A substantial consequence of this richness in comparison withthe bulk soil is intense microbial activity, with feedback effectson root development and the growthof the whole plant. The complexity ofthemicroorganismsresident intherhizospherehas of severalstudies.Plantgrowth-promotingrhizobacteria formedthesubject (PGPR), for example, are beneficial organisms whose genetic traits can easily be manipulated to some extent before their release in the field (2). Mycorrhizal symbiosis, a mutualistic plant-fungus association,is an essential feature of the biology and ecology of most terrestrial plants, sinceit influences their growth aswell as their water and nutrient absorption and protects them from root diseases ( 3 ) . Detailed investigation of the occurrence and role of mycorrhizal fungi in the rhizosphere began very recently (4). They reside in the rhizosphere as spores, hyphae, and propagules and occupy the rhizoplane during their interaction with the root ( 5 ) . The more specific terms rnvcorr.lzizo.sp1zer.eand rnycorrl~izoplatze (i.e., the surroundings and the surface of mycorrhizal roots) (6) are ac263
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companied by the h?phos~~here-namely, the region not directly influenced by the root where mycorrhizal hyphae and soil particles interact (7). The realization that mycorrhizal fungi are among the most significant rhizosphere populations has stimulated investigation of their interactions with other microorganisms and their beneficial effects on plant nutrition and health as well as soil stability (3). There is an extensive literature on the molecular, cellular, andphysiologicalaspects of plant-funguscommunicationinsidemycorrhizal roots (8-14), whereas the biology of mycorrhizal fungi in the rhizosphere has received less attention despite its paramount importance for fungus development and survival and for the establishmentof symbiosis and the mobilization of nutrients. This chapter offers a synoptic overviewof the literature on the interactions between mycorrhizal fungi and the rhizospheric environment. Their establishment of a bridge between the soil and theroot; their crucial role in the acquisition and assimilation of nutrients and water, metal detoxification, and stabilization of the soil: and their colonization of neighboring plant roots is concisely described. The major insights derived from cellular, biochemical, and molecular studies of their development at the soil-root interface is summarized and the gaps in our current knowledge are highlighted.
II. MYCORRHIZAL FUNGI ARE GENETICALLYDIVERSE Mycorrhizal fungi are a heterogeneous groupof soil fungi that colonize the roots of ab5ut 240,000 plant species in nearly all terrestrial ecosystems. The taxonomic position of the plant and fungal partners defines the type of mycorrhiza, each association being distinguished by specific anatomical and physiological features (Table l ) . Because mycorrhizal plants occur in a wide range of habitats and soil conditions. a high degreeof diversity should be expectedin the genetic and physiological abilities of the fungal endophytes (3). About 6000 species within the Zygo-, Asco-, and Basidiomycotina have been recorded as being mycorrhizal. Determination of theirinter-andintraspecificgeneticdiversityhasnowbeen rendered possible by the recent advent of molecular techniques. In most ecosystems. roots are exposed to several mycorrhizal fungal species, each represented by a large population whose individuals almost invariably display some genetic diversity (1.5,16). This diversity has significant ecological consequences. Individual populationsvary in their potential rangeof host species, ability to colonize different host genotypes, and promote plant growth as well as their adaptation to abiotic factors, such as soil pH and toxic levels of heavy metals, likely to affect both the establishment and progresso f a beneficial symbiosis and their dissemination in the ecosystem. The physiological status of the root systems is highly dependent on the creation and efficient functioningof a symbio-
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sis. This, in turn, has a clear impact on the rhizospheric environment and the involved microorganisms through the secretion of carbohydrates, amino acids, secondary metabolites, and various ions. The temporal and spatial distribution of fungal communities and populations, therefore, and the origin and maintenance of their genetic diversity critically determine how and why they come and go during the development of an ecosystem. An understanding of these issues would help in dissecting the interactions that take place in the soil-rhizosphere environment. As already stated, molecular techniques have thrown a powerful light on the population genetics of mycorrhizal fungi (17-19,24). Until recently, the markers identifying mycorrhizal fungi for population studies were solely nlorphological (20,21) or allozymes only for a restricted rangeof species. Many endophytes, (22.23) and could be used in fact, do not generate reproductive structures in culture, like the sterile mycelia that form ericoid mycorrhizae. In addition, many structures needed for morphological identification and species differentiation are lost during the symbiosis. The arbuscules produced by arbuscular mycorrhizal (AM) fungi, for example, are very similar from one species to another. A widevariety of techniques can now be employed to detect DNA sequence variation in above-ground and below-ground ecto- and endotnycorrhizal fungal populations ( 17,18,19,24). Polymerasechainreaction(PCR)amplification of targetedgenomicsequences followed by restriction fragment length polymorphism (RFLP), allelespecific hybridization, direct sequencing, or single-strand conformation polymorphisms are increasingly used to detect ectomycorrhizal (25-27), arbuscular ( 1 S), and ericoid (28) fungi in natural ecosystems. PCR primers based on highly conserved regions of nuclear and mitochondrial ribosomal DNA havebeen designed (18,26,27,29-3 1) to amplify two variable noncodingregions-namely, the internal transcribed spacers (ITS) and the intergenic spacers (IGS). IGS are highly polymorphic and therefore provide a useful tool for molecular ecology. Microsatellite-primed PCR, randomly amplified polymorphic DNA (RAPD), and repeated DNA probes are highly efficient approaches for identification of the genotypes of mycorrhizal fungi (32-35) and have been employed to determine the genetic structure of their populations and assess gene flux between introduced and indigenous strains ( 3 4 3 ) . TheseDNAmarkershavebeensuccessfullyemployed totrackspecific strain-associated loci in endo- and ectomycorrhizal populations from agricultural land, forest nurseries, plantations, and natural ecosystems. The simplest strategy (digesting PCR-amplified ITS with selected endonucleases) has identitied their symbionts in variousecosystems ( 1 8,36-38). Speciesdiscrimination by ITSRFLP matching can be improved by comparing data for the targeted DNA with those on sequence databases (37). These lnolecular techniques have allowed the monitoring of indigenous and
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introduced ectomycorrhizal strains in plantations and natural ecosystems. As a striking example, the persistence and dissemination of the beneficial ectomycorrhizal basidiomycete Lnccnria bicolor S238N on Douglas fir was evaluated on a nursery site and in plantations several years after outplanting (35,39). As many as eight genotypes of L. bicolor and the closely relatedL. lnccmta were identified in the plantations (35). An even higher number of Hebelotm cl.litldro.v/,orl~nl genotypes have been tracked in a coastal sand-dune forest ecosystem (34). DNA markers have also revealed considerable diversity in a single root system. Several typesof AM fungi were associated with a single HyIcinthus root (40). and two Pnrnelln species shared one type (41). Heather roots were interacting with a wide range of symbiont types, each colonizing a different part of the root system and creating a mosaicof populations. Moreover, roots 30 m apart (28). were sharing the same ericoid mycelium The genetic heterogeneity of AM fungi is becoming increasingly evident from the great variability in the sequences of their ribosomal genes or in the patterns of amplified fragments from the same spore and from spores of the same isolates, respectively ( 16,18,42,43),and has suggested a relationship between the presence of many nuclei in n spore and their variability.
111.
SIGNALING MOLECULES AT THE PLANT-FUNGUS INTERFACE
A.
Rhizospheric Signals
Signaling molecules produced early in the interaction between mycorrhizal fungi and their host plants elicit discrete responses in these partners as the initial step in the cascadeof events leadingto contact at the host surface and eventual symbiosis (14,44,45). An interplay of signals probably coordinates and organizes the responses of the symbiont cells and modulates their respective biochemical and molecular differentiation. Identification of the signaling molecules that regulate the information flow between mycorrhizal fungi and host roots is currently an active research area (14.45). Fungal spore germination, chemoattraction of the hyphae by the root cells, adhesion to root surface, root penetration, and development of symbiotic structures in the root probably depend on precisely tuned hostderivedsignalingmolecules,whereasmoleculessecreted by the hyphae (e.g., phytohormones)generatedrasticmorphologicalandmolecularchanges i n the host roots. Root flavonoids that rnay act as signals for the initiation and development of endomycorrhizalandectomycorrhizalsymbioseshave beenidentified(see Chap. 7). Metabolites of the phenylpropanoid pathways apparently act as signaling molecules i n endo- and ectomycorrhizal interactions (14). The role of flavonoids is still controversial, but a variety of flavanones, flavones, and isoflavones
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enhance spore germination and hyphal growthof AM fungi in vitro (46-49) and promote their colonization. It hasbeenreportedthatflavonolsalonestimulate hyphal growth in an elevated CO? atmosphere, whereas all the other derivatives areinhibitory (50). Symbiosis is accompanied by qualitativeandquantitative changes in many secondary host root metabolites (coumestrol, daidzein, formononetin, glyceollin) (S 1-53). Root flavonoid levels are increased by the simple presence of the fungus in the rhizosphere without invasion of the root tissues (53). Formononetin produced in the presence of endomycorrhizal hyphae may intervene in the competition between AM species by favoring root colonization on the part of the one for which it promotes spore germination and stimulates mycelial growth (53). It has been argued that flavonoids are not necessary plant signal compounds in AM symbioses (54). The effect of flavonoids on spore germination and hyphal growth of ectomycorrhizal fungi is poorly known. However, several metabolites released by the plant roots trigger events leading to their infection(44,SS). In the saprotrophic phase, spores of several ectomycorrhizal fungi respond to stimulation by abietic acid, the diterpene resin acid, in root exudates (56).
B. Chemodifferentiation at the Root Surface During their initial contact with the host surface, ectomycorrhizal and AM fungal hyphae undergo a morphogenetic switch and produce repented apical branchings that result in a labyrinthine growth form on this surface (Fig. 1 ) (57,58). A concentration gradient of signaling molecules diffusing through the rhizosphere probably drives the tip toward the host surface and induces this switch only at the high concentrations on the surface itself (44,4S). Hyperbranching ensures intimate contact with the surface and appears to be a prerequisite for subsequent penetrationandfurtherdifferentiationofthespecializedsymbioticstructures [e.g., AM appressorium (Figs. 1,2) (S7,58), ectomycorrhizal mantle]. Identitication of these signaling molecules remains a major challenge for mycorrhizal research. Flavonoids-such as quercetin, present in Euctrlyptus root exudatesinduce rapid and striking changes in Pisolithus tirlctorius hyphal morphology, leading to hyperbranching and an increased proportion of hydrophilic hyphae (Lagrange and Lapeyrie, unpublished results). As growing hyphae of filamentous fungi contain a tip-high Ca’+ gradient thought to be vital for establishing and maintaining apical organization, morphogenesis, and growth (59), it is tempting to speculate that the signaling molecules involved in this hyperbranching alter the homeostasis by regulating or interactingwithiontransportsystems(e.g.. Ca’+-dependentATPases)andtransductionpathways.Thiscontention is supported by the observation that reduced levels of calcineurin, a Ca’ ’ /calmodulinregulated serinelthreonine phosphoprotein phosphatase, cause growth arrest preceded by hyperbranching in Neurospora c r m w (60). This hyperbranching may
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i
J Figures 1 and 2 (1) Extraradical mycelium produced by Gigaspora margunfa, an arbuscular mycorrhizal fungus, in the presence of a root. A network of larger (arrows) and thinner hyphae are formed as well as groups of auxiliary cells (F). X 100. (2) When the fungusGigasporu margarita makes contact with the root surface, aspecialized swollen and branched structure called appressorium (AP) is formed. X200.
be caused by alterations in the synthesis and rigidity of the cell wall, resulting in turgor pressure. Cloning of cDNAs coding for calcineurin and the Ca2+-binding protein calmodulin in P. tinctorius (Voiblet and Martin, unpublished results)will enable this question to be tackled.
C. FungalPhytohormonesAreProduced in the Rhizosphere Comparisons of root morphology and branching following auxin applicationor fungal colonization indicate that fungal auxins play a key role in the formation of ectomycorrhizae(55,61). Further evidencein support of this view comes from the observation that overproduction of indole-3-acetic acid (IAA) by a fluoroindole-resistant mutant of Hebeloma cyZindrospomrn induces abnormal proliferation of the intercellular network of hyphae (62),while the stimulationof ethylene production byP. tinctorius during early ectomycorrhizal formation is presumably triggered by the productionof IAA (63). Auxins and ethylene would thus appear
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to be chemical signals that regulate fungal penetration and several anatomical features of an ectomycorrhiza. This morphogeneticrole may imply targetedsecretion of auxins and their conjugates and local regulationof their metabolism. The part played by other fungal indolic compounds, such as theP . tinctor-ius metabolite tryptophan betaine (hypaphorine) (64), should also be investigated, since it greatly reduces root hair elongation in Eucalyptus (65). Apart from these clear indications that fungal auxins and their derivatives modulate root morphogenesis during symbiosis development, however, their precise role in the control of the cascade of molecular events leading to the mature ectomycorrhiza is still uncertain.
IV.
MYCORRHIZAL FUNGIDEVELOP STRUCTURES OUTSIDE AND INSIDE THE ROOTS
Despite their impressive genetic diversity, all mycorrhizal fungi complete their life cycle in close association with the roots through the establishment of a symbiosis ensuring a continuous flow of nutrients. They are divided into two main categories (Table 1 ): endomycorrhizae (arbuscular, ericoid, and orchid mycorrhizae) and ectomycorrhizae.
A.
PresymbioticMycorrhizalStructures in the Rhizosphere
All mycorrhizal fungi develop presymbiotic structures in the rhizosphere, sometimes independently of the host plant-namely, propagules (spores of either sexual or asexual origin) and hyphae organized in various ways: isolated hyphae, highly structured bundles, rhizomorphs, and sclerotia. The morphology of these structures has been often studied i n i n vitro conditions (S,66). Glomalean fungi (Zygomycetes) producevery impressive spores: huge round bodies rich in storage compounds (e.g., lipids, proteins, and glycogen), often containing symbiotic bacteria (67) and bounded by a thick, layered wall whose organization and compositionoffer an ultrastructuralcluetotheirlong-termviability.Sporopolleninis often present as a thin layer. In Glomus w r - s $ m w , it occurs between thc primary wall, composed of parallel chitin fibrils, and the secondary wall, characterized by a helicoidal architecture. Its chemicophysical characteristics suggest that it acts as a barrier against soil microorganisms. AM fungal hyphae living in the soil display a thick melanized wall whose texture is very different from that of the thin wall of those living inside the root (S). The morphology of AM fungal structures developing outside and inside the root is conserved even in the rhizosphere of transgenic plants, where accumulation of antifungal plant defense products-suchaslytic enzymes (e.g., chi-
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tinase) or pathogenesis-related proteins-is achieved by genetic transformation (68). In ericoid or ectomycorrhizal fungi, the thickness of the hyphal cell wall is much the same in presymbiotic and symbiotic hyphae, although the fibrillar material on the surfaceof ericoid fungi outside the root disappears during penetration (69).
B. Ultrastructure of MycorrhizalSymbioses Endomycorrhizal hyphae adopt a varietyof colonization patterns in their penetration of the host root cells. Glomalean fungi are highly dependent on their host and cannot survive for long in its absence. Their hyphae form appressoriaon the epidermal cells, penetrate the cortical tissue, and eventually form highly branched structures called arbuscules (Figs. 3-6) (10). Colonization of the Ericales takes place via simple hyphal structures present during boththe presymbiotic and the intraradical symbiotic phase. Hyphae of ericoid fungi may form loops and bundles outside the host plant, ill-defined appressoria and coils inside the epidermal root cells (Fig. 7) (69). Similarly, basidiomycetous fungi produce specialized coils (Fig. 8) in the orchid cells during both the protocorm and the mature stages (70). During the symbiotic phase, ectomycorrhizal fungi form the mantle sheath that surrounds the root, and progress into the apoplastic space of the rhizodermic (Angiosperms) and cortical cells (Gymnosperms), producing the Hartig net, the name given to an intercellular hyphal network inside the root tissues (Fig. 9,lO).
C. Cell Wall Alterations at the Plant-Fungus Interface Early interactions between the cell walls of the plant and the fungus and changes in their composition are essential morphogenetic events in the constitution of afunctioningmycorrhiza.Cellularandmolecularapproacheshaveprovided new insights into the complex and ever-changing scenario of these interactions. Cytochemical and in situ immunologicaltechniqueshavedemonstrated that bothstructureandfunctionarelesscomplexwhenassessedatthecelllevel (10,l 1,71,72). Bonfante et al. (73) used monoclonal antibodies and enzyme-gold complexes to reveal pectins and cellulose at the interface between the fungal wall and the host plasma membrane in AM roots (Fig. 6), and additional wall components have been investigated with other molecular probes (74-76). These studies indicate that the interface is an apoplastic space of high molecular complexity where the boundaries of the partners are defined. The examinationof other endomycorrhizal systems has demonstrated that their interface is morphologically simat the interface ilar but different in composition. Cellulose and pectins are present
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Figure 6 Detailof the interface space (IN) between the fungalwall (U) of Glomus versiforme and the host membrane of leek (PL), as seen by electron microscopy. Xyloglucan molecules are revealed by usmg a specific antibody and colloidal gold granules. P, host cell; F, fungus. X30,OOO.
in orchids only when the endophyte is collapsing (77) and absent in ericoid mycorrhizae (69) (Benhamou and Bonfante, unpublished observations). It is clear, therefore, that invagination of the host plasma membrane following fungal penetration is a common biotrophic interaction, whereas the nature of the molecules forming the interface is closely dependent on the nutritional capabilitiesof the fungus and its relationship with the host plant. of events These intraradical colonization patterns are clearly a consequence taking place at the root surface. Inof the one many studiesof this subject. Gollotte
Figures 3 to 5 (3) When Gigaspora murgarita, an AM fungus (F), penetrates the cortical root cells, highly branched intracellular structures are produced-the arbuscules (A). X200. (4) Scanning electron micrographyof a cortical cellof Ginkgo bilobacolonized by a Glomus strain. The symbiont produces a conspicuous arbuscule. X 1500. ( 5 ) Transmission electron micrography of a cortical cell of Ginkgo bifoba colonized by a Glomus strain. A large intracellular hyphais seen surrounded by smaller branches (F). Each hypha is surrounded by the invaginated host membrane (PL) (arrows). X5800.
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et al. (76) showed that AM fungi fail to develop regular structuresin pea mutants. The first sign of this failure was the deposition of callose on the surface of the epidermal cells. In ectomycorrhizae too, early contacts between the partners are followed by to the formation of alternations in cell-wall ultrastructure and composition leading a new interface (78-81). Plant and fungal walls are always in direct contact, a s shown in a detailed analysis of Corylu.7 avellnntr and Tuber-magnatum by Balestrini et al. (82), whose map of the symbiont cell wall components also showsthat the fungus causes subtle changesin the host walls. They suggest that a cementing material embedding the mantle hyphae and “obscuring” the plant-fungal contacts partly corresponds to an unregulated cell fungal wall component. Identification of the cell wall and surface proteins involved in fungal attachment and penetration is crucial to an understanding of how hyphae establish early interactionswiththeirhost (83). The protein composition of cellwalls of P. rirlctorirrs is strikingly altered by the symbiotic interaction (81). Many 31- and 32kDa, symbiosis-regulated acidic polypeptides (SRAPs), are foundin this cellular compartment (84). The central part of the SRAP sequence contains the RGD motif, a cell adhesion sequence found in several extracellular matrix adhesins, e.g., vitronectins and fibronectins. These ligands could interact with integrins, which mediate cell adhesion and signal transduction in mammal, yeast, and plant cells. Mycorrhizal development induces the accumulation of SRAP transcripts and the corresponding proteins thus gather in the cell wall when the ectomycorrhizal sheath is aggregating around the colonized roots and the infecting hyphae penetrate between the epidermal cells. Upregulation of the synthesis of cell wall proteins in ectomycorrhizae is not limited to SRAPs. Transcripts of three hydrophobins, another classof fungal secreted cell-wall proteins. similarly accumulate several-fold in P. tirlctor-ius hyphae colonizing the Euccr1yptu.s root surface (85). Changes in cell-wall protein composition may regulate the molecular architecture of protein networks i n a manner that allows new developmental outcomes for both fungal cell adhesion and root colonization. Further investigation of the structure and regulation of SRAP wall proteins will provide a more complete picture of their role in developing ectolnycorrhizal tissues. Incompatibility between ectomycorrhizal hyphae and the host roots detected during the initial con-
Figures 7 and 8 ( 7 ) Hair root of Ctrll~tr~tr ~ wl g c r r - iscolonized by an ericoid mycorrhizal strain. The ascomycetous fungus is a dark sterile mycelium and produces ;u1 intercellular coil, which is surrounded by the host membrane. X 15,000. ( 8 ) Detail of ;m orchid root cell colonized byan orchid symbiont. The basidiomycetous fungus (F) has ;I thick wall and is surrounded by the host membrane (H). X21.000.
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Figures 9 and 10 Fxtomycorrhizal root of Quercus suber and Pisolithus tincton'us. The fungus produces a well-developed mantle and a Hartig net involving the outer root layers.
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tacts is generally expressed in the form of polyphenol accumulation in host tissue and thickening of host cell walls abutting the incompatible isolate (86,87). Thesc complex interactions are evidently the result of early events in the mycorrhizoplane.
V.
THE ROLE OF MYCORRHIZAL FUNGI IN NUTRIENT CYCLING AT THE SOIL-ROOT INTERFACE
Symbiotic roots provide a niche for mycorrhizal fungi.T o bring about a symbiosis, the host plant must trade the fungus's demand for carbon for respiration and growth, which is met primarily by glycolytic and anaplerotic processes rcquiring carbon sources, against its provision of extra nitrogen, phosphate, and minerals (88-90). Hyphae prospecting the soil absorb nutrients by active metabolism and transport ions and assimilated metabolites to the host root via their strands and rhizomorphs. This mechanism is crucial for the absorption of nutrients that are poorly mobile, such as inorganic phosphate (Pi) and K + , or bound to soil particulates (NH,'). Since ions rapidly absorbed by nonmycorrhizal plant roots become scarce i n the rhizosphere, a zoneof deficiency forms andthe roots' absorption rate mainly depends upon their diffusion raterather than its own activity. Mycorrhizal hyphae counteract this deficiency, since nutrients translocate through the fungal cells to any sink, such as the root cells, more quickly than they diffuse in the soil (3). This faster translocation rate is sufficient to explain the enhanced absorption rates of symbiotic roots. In exchange, the fungus receives their carbon compounds. These two-way flows of nutrients and other metabolitestake place when physiologically active cells of both partners are in intimate contact (91).
A.
NitrogenAcquisitioninMycorrhizalFungi
The morphological and physiological dissimilarities between mycorrhizal symbioses probably determine their success and their distinct patternsin different ecosystems (92). Nitrogen (N) available to both AMandectomycorrhizalplants should not be regarded as a single pool open to free competition. Specialization of its acquisition and utilization in a given habitat is an important feature of plant and microbial community structure, while the fact that the ability to exploit its sources (and those of other limited nutrients) is not the same in all species may result in niche differentiation (93). If habitat specialization is a reflection ofdifferences between mycorrhizal types, ectomycorrhizal and AM species could cooccur because they exploit different niches in the same ecosystem. In the forest ecosystems of Eurasia and North America, where ectomycorrhizal associations are dominant, there are often wide variations in the environmental concentration of N and its forms, and its limited availability to plants is
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due to N microbial transformation in soil (94). The leaf litter produced by most tree species is relatively slow to decompose and thus forms a distinct layer of acidic,organicallyenrichedmaterial.Acidity,ahigh C:N ratioandseasons marked by low temperatures and surface drying are major obstacles to nitrification and ammonification. Mineralization of N is so slow in manyforeststhat available N becomes the main growth limiting factor (92.94,95). Most trees have therefore elaborated mycorrhizal associations (Fig. 1 1 ) and a wide range of alternative trophic adaptations (e.g., NI-fixing symbioses, cluster roots) to be able to compete for the limited resources of specific nutrients (96). The beneficial effect of AM colonization on N acquisition has been overlooked (3). Its significance in plant N uptake in both agricultural and natural ecosystem, however, is now becoming increasingly clear (92,97,98).
1.
Utilization of Organic N
The low temperatures and low soil pH that usually prevail at higher altitudes and latitudes (e.g., heathlands) restrainnitrification and (to a lesser extent) ammonification (92). Studies o f N relations in temperate and boreal ecosystems have dem-
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Figure 11 The different steps of nitrogen nv%holism in thc extraradical hyphae. cctomycorrhizal roots, and roots of the host plant. I . absorption: 2. assimilation; 3. storage; 4. translocation; A. extranlatricalhyphae; B. ectornycorrhizalsheath; C, Hartig net; D. root cortical cells; AA amino acids.
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onstrated the importance of its organic forms forplant nutrition (92). Some ectomycorrhizal and ericoid fungi use complex organic N, such as proteins, and their host plants have access to peptides and proteins. Soluble amino acids are also a substantial source for a l l types of mycorrhizal associations in these ecosystems (89.92). The extensive extramatrical mycelium of the ectomycorrhizal fungi is ideally placed for nutrient acquisition in the top I O cm of soil, where most of the in local pools is sequestered in organic form (92). A major contributing factor N acquisition by ectomycorrhizal trees is the continuous growthof this mycelium it absorbs and transports for into the patchily distributed soil resources, which storage by the colonized roots. Degradation of organic N residues gives rise to free amino acids and (through microbial processes) to inorganic N forms-i.e., NHJ', NOi-.Ectomycorrhizal fungi contribute to N nutrition of the host by co11into forms more readily utilized by either version of litter and complex soil N the fungus or the host, and by absorption. assimilation. and translocation of inorganic N compounds from the soil to its roots. Most ectomycorrhizal fungi readily assimilate amino acids, suchas glutamine, glutamate, and alanine,which predominate in soil solution, and peptides released by protein degradation (89,92). This hostwithorganic N ability is retained i n the symbiosis state and supplies the (92,99). Absorption of soil proteins requires their enzymatic degradation to peptidesandthenaminoacids. Bothericoidandectotnycorrhizalfungisecrete ;I wide range of proteinases when grown on animal proteins (casein, gelatin, albumin) and protein fractions from beech forest litter a s the substrate (89,99). Marked inter- and intraspecific variations in the ability to use protein N probably express genetic differences between fungal strains and i n the availability of host-derived carbon compounds. Competition forprotein N between saprotrophic and ectomycorrhizal fungi in forest soils is presumably very tight, but symbiotic funk' T I arc favored by the host's continuous provision of the carbon compounds needed to capture this N form through the synthesis of' proteolytic enzymes.
2. Uptake of NOs- and NH4+ AM and ectomycorrhizal fungal mycelia are extremely active scavengers of inorganicforms of N. suchaseither NHJ' and NO3- (98,100).Experiments performed on Pisolithus demonstrate that the fungus has access to N H 4 ' and Ca'. ions trapped in between the vermiculite 2: I layer ( 101). As vermiculite samples were separated from the mycelium by cellophane films, soluble fungal exudates may be considered responsible for phyllosilicate weathering. In this experiment. NH4' ions. usually considered as retrograded, were mobilized fromthe interlayer spaces and replaced by Mg", AI3', Ca", and Na' ions (101). NH.,' absorbed by mycelia,orderivedfrom NO1- reduction.israpidly assimilated into glutamate and glutamine, which are then used to synthesize other
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amino acids, such as alanine and gamma-aminobutyrate, within the foraging hyphae. Next, assimilated N is either incorporated into mycelial proteinsor translocated to the host, glutamine being regarded asthe main translocation form (102). NH,' and NO3- are apparently assimilated a long way from the mycorrhizal roots where the hyphal web is permeating the rhizosphere and various soil horizons. In natural ecosystems, therefore, primary NH,' assimilation is carried out by the occur (89). In fungus, then conversion to glutamine and its transfer to the host addition. both AM and ectomycorrhizal fungi mediate N transfer between plants through mycelial links (103). In grassland and forest ecosystems, where plants in betweenaregrown in verycloseassociation,thesewebsmaybecrucial plant N cycling.
3. The Pathways of N Assimilation in Mycorrhiza Primary NH,' assimilation takes place via the NADP-dependent glutamate dehydrogenase (NADP-GDH)/glutamine synthetase (GS) or the GS/glutamate synthase (GOGAT) pathways. Studies of ''NH,+assimilationprovidestrongevidence that NH4' is metabolized via the GS/GOGAT pathwayin AM associations (97,98). Investigation of the enzymes involved, however, has revealed considerable differences between one association and another (89,102). In conifer ectomycorrhizas, the NADP-GDH activityof the fungus is generally very high, irrespective of species involved, and NH,' is assimilated through the NADP-GDH/GS pathway, whereas in most hardwood ectomycorrhizas the GS/GOGAT pathway predominates.
B. Utilization of SoilCarbonCompounds by Mycorrhizal Fungi As described above (Sec. 1V.B) mycorrhizae differ in their morphology. Some have abundant external hyphae with high metabolic activity, others are smooth ectomycorrhizae with little or no exploratory mycelium. These features reflect differences in the need for carbon and the way in which it is shared between the symbionts. Carbon metabolism provides the mycelia and host cells of all types of mycorrhizae with energy and reducing power, and the skeletons required for the synthesis of various metabolites (e.g., amino acids). Pathways of hexose metabolism have been investigated in symbiotic and in free-living ectomycorrhizal fungi in axeniccultures (90). Carbon is acquired by thefungus via: ( I ) host photosynthesis and translocation, (2) carbon dioxide fixation in hyphal and root cells, and (3) assimilation following the degradation of soil carbon polymers. This section focuses on assimilation, which takes place in both the rhizosphere and the hyphosphere.
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Ectomycorrhizal and ericoid fungi typically inhabit organic soil horizons containing high levels of lignins, soluble phenolic acids and polyphenolics derived from plant litter. Most ectomycorrhizal fungi so far investigated have limited phenol-degrading activities, whereas those of ericoid fungi are well developed (104). A few ectomycorrhizal fungi (e.g., Hebelornn crLr.srulinjforrnr)have well-developed ligninolytic abilities (105), and their use of lignin as a carbon source reduces the amount of C needed from the host plant. The ecological significance of this ligninolysis, however, is unknown. Ericoid fungi, on the other hand, produce an array of hydrolytic enzymes during their extraradical phase and can thus exploit both simple and complex organic matter in the soil (106-108). Hvrtfenosc.vl~/zl,hus ericme, the strain that has been best characterized is ableto grow on a variety of complex organic substrates (see Ref. 106) and seemswell equipped to degrade mostof the polymeric components of plant and fungal cell walls included in the organic matter. Polysaccharides such as carboxymethyl cellulose, tylose, laminarin (109), and xylans (108) are utilized through the secretion of hydrolytic enzymes. Chitin, the structural polysaccharide of the fungal wall. is degraded. as well as proteins and even lignin (106). Ericoid mycorrhizal isolates in the Myxotrichaceae and Gymnoascaceae, telomorphs of Oidioderlclrotz, have also been described as cellulosolytic ( I IO). Pectin is another important component of the plant cell-wall debris and polygalacturonase, an enzyme involved in its degradation, is produced by a wide range of ericoid fungi ( 1 1 1). Several polygalacturonase isoforms are produced by ericoid isolates ( I 1 l ) , depending on the species involved. These biochemical data may be of great ecological significance together with the observation of multiple root occupancy by genetic analysis of ericoid fungi (28). Ericaceous plants may thus enhance their exploitation of complex soil substrates by widening their metabolic capabilities through an association with several fungi endowed with different functional enzymes.
C.
Utilization of Phosphorus by Mycorrhizal Fungi
1.
Ectomycorrhizal Fungi
The growth of ectomycorrhizal trees is frequently improved by their increased phosphorus (P) accumulation (3), and this, in turn, is related to the intensity of the mycorrhizal infection. Ectomycorrhizal fungi solubilize insoluble forms of AI and Ca phosphates as well as inositol hexaphosphates, though a wide interstrain variability has been recorded ( I 12). These complex P forms are digested by the secretion of extracellular acid and alkaline phosphomono- and phosphodi-esterases. Pi in soil solutions is easily taken up by ectomycorrhizal hyphae and then translocated to thehostroots.Itsabsorption and efflux are probably regulated
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by intracellular Pi and inorganic polyphosphates (Polyp) pools. Excess intracellular Pi is stored as Polyp by most ectomycorrhizal fungi. Most of these Polyp are oligophosphates with an average chain length of I O phosphate residues ( 1 13). Nuclear magnetic resonance (NMR) comparisons of Polyp in vivo with those in model solutions suggest that they are low molecular weight-soluble aggregates ( I 13). Polyp are the only macromolecular anions in the fungal vacuole ( 1 13), and their roles in basic amino acid and cation retention and osmoregulation have been demonstrated both i n vivo and i n vitro.
2.
Endomycorrhizal Fungi
Orthophosphate uptake is greatly enhanced duringan AM association ( 1 14- 1 16). There are many possible explanations for this increased efficiency ( 3 ) . An AM fungal mycelium explores the soil more efficiently than root the itself and spreads beyond the phosphate depletion zone (7). It takes phosphate from the soil and ( I 15) by using sources of P that might not be available transfers it to the plant root to roots ( 3 ) . The kinetics of P uptake into hyphae may differ from that of roots, leading to a more effective absorption. A gene coding for a high-affinity phosphate transporter isolated from the AM fungus G. vers~ortne( 1 17) is specifically expressed in the mycelium, which suggeststhat the transporter is a strong candidate for this initial phosphate uptake. Absorption of phosphate from soil has to be followed by translocation along the fungal structures and by transfer from fungus to plant across the symbiotic interface ( 3 ) . A satisfying picture of the molecular mechanisms which operate along the soil/fungus/plant pathway is not yet available. Cytochemical staining for phosphatase and H’ -ATPase activities indicates that the arbuscule-plant membrane interface is the most likely site for the fungus-plant phosphate exchange (3,91), but no plant transporters have as yet been identified on the periarbuscular membranes. Two transporter genes have been cloned in Medicqo truncatulrr and characterized for their affinity in a yeast expression to be influenced by heavy metal ions. Their expression in M. truncut d c r roots is induced by phosphate starvation and downregulated during mycorrhizal association, even before the formation of arbuscules (1 18), suggesting that these transporters are needed for plant nutrition, but are not involvedin the transfer of phosphate from the fungus. Polyp granules accumulate in ericoid fungi in response to high external P concentrations ( 1 19). Ericoid mycorrhizae, however, mainly form on acid soils whose total P is mostly organic. Their fungi produce an array of extracellular phosphatases that decompose different sources of phosphomonoesters, such as phytin (120) and phosphodiesters, such as nucleic acids and phospholipids(1 07). Isoforms of a wall-bound acidic phosphatase are found in ericoid strains grown in pure culture (121) and in association with the plant roots( 1 22). These enzymes are activated by low and inhibited by high P concentrations. Their activity is
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enhanced in the mycelium next to the root, but absent in both the more distant hyphae and in the fungal coils inside the plant cells (122).
VI.
BACTERIA AND MYCORRHIZAL FUNGI IN THE RHIZOSPHERE
Understanding of the interactions between the microorganisms found routinely in the rhizosphere is an essential prelude to determinationof the nature of the soilplant interface. The interactionof mycorrhizal hyphae with other microorganisms either directly, or indirectly by system ( 1 18). Associations between mycorrhizal fungi and soil bacteria, particularly plant growth-promoting rhizobacteria (PGPR) such as rhizobia, pseudomonads and Azospirill~rm,have profound effects on plant health through beneficial synergisms. PGPR interact with both mycorrhizal fungi and the plant root in a wide variety of ways. Many experiments have demonstrated that bacteria stimulate mycorrhiza formation. Garbaye (1 39) has elegantly shown how bacterial strains increase a root’s ability to establish an ectomycorrhizal symbiosis. He has also proposed a new bacterial category to describe this effect: the mycorrhization helper bacteria (MHB). Similar effects have been reported for AM fungi, where several rhizosphere bacteria appear to stimulate the growth and development of endomycorrhizal fungi or increase root mycorrhization (4,140). Several mechanisms have been proposed to explain these effects, for example the production of vitamins, amino acids, phytohormones and/or cell-wall hydrolytic enzymes. Some of these effects may directly influence the germination and growth rate of fungal structures,whereasothersmay actonroot developmentandsusceptibility to infection. Better use of mycorrhizal fungi as biocontrol and biofertilizer agents requires a more mechanistic understanding of the biological balance between them and rhizosphere bacteria. Since bacteria form biofilms around the hyphaeof ectoand endomycorrhizal fungi (141,142), Perotto and Bonfante (143) have suggested that their attachment to the root and fungal surface is a crucial moment of the interaction. Investigation of the mechanisms by which this attachment takes place has shown that several molecular components-including adhesive proteins, flagella,andextracellularpolysaccharides-areinvolved(144).Severalbacteria described as good root colonizers also adhere to hyphae, indicating that similar attachment mechanisms are adopted. For example, PGPR isolated as strong root colonizers, such as strain WCS 365 of Pseuclomonas jhot-escens, form a coat around the hyphae of AM fungi, as do strains of Rhizobiutn legumirzoscrrun~ (141). The significance of this bacterial attachment to a solid surface is not clear.It may be a way to avoid dispersion by percolating soil water. In addition, according to Boddey et al. (145), rhizobacteria interacting with mycorrhizal fungi may use
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hyphae as a vehicle for their distribution and/or t o enhance root colonization. In view of the widespread distribution of mycorrhizal fungi and the ability of AM fungi to colonize most plants. this strategy would offer these bacteria a good chance of finding new niches.
VII.
DO MYCORRHIZAL FUNGI PROTECT PLANTS FROM HEAVY METALS?
Recent reviews have focused on the role of mycorrhizal fungi in the uptake of heavy metals from polluted soils and their transfer to the plant (123). Several experimental data provide clear evidence that both ectomycorrhizal and ericoid fungi protect their host against these metals ( 123- 125). The position with regard to the AM fungi is less clear ( I 23). This ability to grow i n polluted soils and withstand high heavy metal concentrations rests on complex mechanisms involving both avoidance through exclusion o f metal ions from the cytoplasm and tolerance of high internal metal concentrations (126), thisbeing often dependent on theinduction of specific genes and proteins ( 1 26.127). Increasing concentrations of toxic aluminum (AI) ;ire being reported in the acidic soils of temperate forests. Ectotnycorrhizal fungi on tree roots modify the compartmentation of absorbed AI and protect their host against its toxic effects (125,128,132,133), even if contrasting results have been reported (134). AI has been detected in P-rich granules in the vacuoles (129) and i n cells walls (128) by energy-dispersivex-raymicroanalysisandelectron-energy-lossimaging (128,129,135,136), suggesting that Polyp sequester heavy metal ions and AI is part of a fungus’s detoxification mechanism. In aqueoussolutions, AI3’ forms an octahedral hexahydrate, A1(H20),,?‘ a t acid pH. Carboxylate and phosphate groups are the strongest AI3’ ligands, and AI3’ frequently fornu solublecomplexes with phosphategroups i n biological systems. ”AI NMR has shown that mixed solvation complexesof AI-Polyp occur in the vacuoles of. fungal cells ( I 13). A comparison of the NMR spectra of mixed solvation Al”-Polyp complexes observed in vitro with those obtained in intact mycelium indicated that AI3+ forms at least three complexes with Pi and Polyp, that there is more than one complex present under any set of conditions, and that the equilibria between these complexes is markedly affected by AI concentration i n the medium ( 1 13). Other cellular ligands (e.g.. organic acids) may occur in the fungal cell, but AI absorbed by rapidly growing L. bicolor is efficiently and prominently sequestered in the Polyp complexes, indicating that its AI tolerance is mainly due to sequestration. The AI-Polyp complexes observedby NMR likely correspond to the P-rich granules containing AI detected by microscopy (129). Better understanding of these mechanisms and those that regulate transfer to the plant is needed so that they can be exploited in land reclamation and soil
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bioremediation. Metal-specific tolerance mechanisms have been demonstrated for ectomycorrhizal fungi ( I 13,128,129), but no conclusive data are available for ericoid and AM fungi. However, mycorrhizal 0. rt7~iu.sisolates from soils contaminated with zinc and cadmium (K. Turnau, Krakow, Poland) grew better in vitro in the presenceof these metals than same-species isolates from non-polluted soils (130,13l ) . It was found that specific extracellular proteins are produced in the presence of metal ions, some of which are involved in oxidative stress response. Secretion and activityof extracellular enzymes involvedin ericoid fungal nutrition. such as polygalacturonase, were also found modifying host physiology and thepattern of rootexudation.wasdemonstratedlongago (137), andhas beenextensivelyinvestigated(138-140). In someisolatesfrom pollutedsoil, polygalacturonaseactivity rose in the presence of heavymetals,althoughthe regulatory mechanisms are not known. The results of the few experiments in this field show that the composition of the rhizosphere bacterial community is influenced by mycorrhizal fungi. Andrade et al. (146) foundthat the composition (but not the size) of bacterial communities in the rhizosphere and hyphosphere of Sorghwn in the presence and absence of mycorrhizal fungi variedbothwithin and among AM fungal treatments, suggesting that fungal taxa may have a qualitative effect on bacterial diversity. I n addition. a Brrrkoltleritr cepc~ciastrain was always present i n the rhizosphere (146). This result is of particular interest, since Blrrkoklc~ritrisolates have been described as endosymbionts living in the cytoplasm of spores of Gigtrsporc~ nwgaritrr, an AM fungus (67). Bacteria-like organisms (BLOs) in the cytoplasm of AM fungi were first observed ultrastructurally in the early 1970s (147), but confirmation of their prokaryotic nature was impeded by their refusal to grow on cell-free media. A combined morphological and molecular approach has now shown that the cytoplasm of G. rt~trrgwi~tr spores harbors a homogeneous population of Burkholcleria bacteria identified from the sequence of the 16s ribosomal RNA gene (67). PCR assays with oligonucleotides specific for this sequence have revealed these bacteria in all stagesof the fungal life cycle (spores and symbiotic mycelia). I n addition, isolates of different origin from the three AM fungal families(Glomaceae,Gigasporaceae,andAcaulosporaceae)displaybacteria when observed by confocal microscopy using a fluorescent dye specitic for bacterial staining. However, PCR amplification performed with universal eubacterial primers and primers specifically designed for the endobacteria of G. mrr,ycrrittr on DNA preparations from AM spores demonstrated that the endobacteria were aways present, whereas the Brrrkholckrir~were limited to the Gigaspornceae. It may be supposed that these intracellular bacteria are a general feature of AM spores and not merely sporadic components (148). Associations between endosymbiotic bacteria and the Homoptera, Blattaria, andColeopteraarecommon.One of thebestknownisthatbetween Brrchtzerlr and the aphids ( 149,150).Both partners are obligate and mutualistic symbionts, and the aphids cannot survive without the bacteria (150). Blrchnerrr, in fact,
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possesses genes involved in DNA metabolism, protein secretion, carbohydrate degradation, and ATP generation as well as amino acid synthesis, and one of its functions is the biosynthesis of the essential amino acids tryptophan, cysteine, and methionine for the aphid (150). The functional significance of an AM fungus’s endosymbiotic bacteria is not clear. Using the Buchrzem-aphid system as an analogy, we are investigating this issue. The finding that a genomic library developed from G. rnrrrgLzrifa spores is also representative of the Burkolderia genome (151) will help to establish the features of this rhizospheric bacterial population, which has so far remained hidden inside its fungal hosts.
VIII. CONCLUSIONS Mycorrhizae are usually described as resulting from a symbiotic interaction between plant and fungal genomes. There is increasing evidence that many of the complex events described inside theroot (morphogenetic changes, regulated development, nutritional exchanges) are the consequence of extraradical events, some of which have been highlighted here. These include development of fungal structures outside the roots, role of mycorrhizal fungi in the nutrient cycling, and plant protection from heavy metals. While there have been substantial advances in recent years in our understanding of developmentalprocessesleading t o the formation of mycorrhizal symbiosis, many questions concerning the morphogenesis of fungal structures in the rhizosphere remain unanswered. A major theme in future studies in this research area will be the molecular signaling involved in host recognition and the coordinated development of specialized symbiotic structures. The signals identitied might beanalogous to those that regulate formation of other symbiotic tissues andmaywell offerinsights into theprocessesthatregulateorganogenesis in fungi and plants. Mycorrhizal fungi are a crucial component of the rhizosphere, where they work at the soil-plant interface and are also free from the influence of the root by interacting with a number of other organisms to create a living underground web (103). The other topic of great interest will therefore be the interaction between mycorrhizal fungi and rhizosphere microbes. A good knowledge of these of “biofertilizers” multiple interactions will offer new tools for the development to be used in the frame of a sustainable agriculture. Last, it will be important to explore the potential of mycorrhizal fungi as bioremediation agents in polluted soils. There is a strong research need in this field. Molecular approaches will be surely crucial to clarify at least some of these problems. An improved knowledge will provide new stimuli for the better dissection of the role and functioning of mycorrhizal fungi in the rhizosphere.
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ACKNOWLEDGMENTS The investigations carried out in Francis Martin’s laboratory were supported by grants of the Bureau des Resources Genetiques, the Ministkre de I’Environment, a EU contract (PL 931742), and the Groupement de Recherches et d’Etude des Genomes. Research in Paola Bonfante’s laboratory was supportedby the Consiglio Nazionale delle Ricerche (P.F. Biotechnology-Subproject 2), by the Ministero della Ricerca Scientifica (MURST) and by the following European projects: ECC Biotechnology Project (IMPACT Project, Contract No. BI02-CT93-0053); and the ECC AIR Project (MYCOMED, Contract No. AIR2-CT94-1149).
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127. G. M. Gadd, Interactions of fungi with toxic metals. Neb47 Phytol. 124:25 (1993). 128. H. Vare, Aluminum polyphosphate in the ectomycorrhizal fungus Slrilllts wrriegutits (Fr.) 0. Kuntze as revealed by energy dispersive spectrometry. New Phytol. I16663 (1 990). 129. I. Kottke and F. Martin. Demonstration of aluminium in polyphosphateof L ~ ~ ~ l r i c l urtret/lystec.l(Bolt. ex Hooker) Murr. by means of electron energyloss spectroscopy. J. Microsco/>y I74:225 ( 1994). 130. E. Martino, K. Turnau, M. Girlanda, P. Bonfante, S. Perotto, Ericold mycorrhiza fungi from heavy metal polluted soil: their identification and growth in the presence of zinc ions. Mycol. Res. 104:338-344 (2000). 131. E. Martino, J. D.Coisson, I. Lacourt, F. Favaron,P.Bonfante,and S. Perotto, in Influence of heavy metals on production and activity of pectinolytic enzymes ericoid mycorrhizal fungi. Mycol Res. In press. 132. M. J. Hodson and D. A. Wilkins, Localization of aluminium in the roots of Norway spruce (Picerr uhies (L.) Karst.) inoculated with Pusillus irndutus Fr. Nebt' Phytol. 118:273 (1991). AI toxicityin 133. J . R. Cummingand L. H.Weinstein.Nitrogensourceseffectson nonmycorrhizal and mycorrhizal pitch pine(Pinusrigidn) seedlings: I. Growth and nutrition. Cm. J. Bot. 68:2644 (1990). 134. G. Jentschke, D. L. Godbold, and A. Hiittermann. Culture of mycorrhizal tree seedPhysiol. lingsundercontrolledconditions:effectsofnitrogenandaluminium. Plurlt. 8/:408 (1991). 135. H. J. Denny and D. A. Wilkins, Zinc tolerance in B e t l h spp: IV. The mechanism of ectomycorrhizal amelioration of zinc toxicity. New Phytol. /06:545 (1987). 136. I. Kottke, Electron energy loss spectroscopy and imaging techniques for subcellular localization of elements in mycorrhizas. Methods Microbiol. 23:369 (1991). 137. G. D. Bowen andC. Thcodorou, Interactions between bacterial and ectomycorrhizal fungi. Soil Biol. Biocher,~./ I : 119 (1979). 138. R. G. Linderman, Vesicular-arbuscular mycorrhizae and soil microbial interactions, Mycorrhizuc irl Sustainuhle Agriculture (G. J. Bethlenfalvay, andR. G. Linderman, eds.), 1992.pp.45-70. 139. J. Garbaye, Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytol. 128: 197( 1994). 140. J. M.Barea.Mycorrhiza-bacteriainteractionsonplantgrowthpromotion. Plant Grobct/~Prurrloting Rhi~obocteritr(A. Ogoshi,K.Kobayashi, Y. Homma, F. Kodama, N. Kondo, and S. Akino, eds.), OECD Press, Paris, 1997, pp. 1.50-158. 141. V.Bianciotto. D. Minerdi, S. Perotto,andP.Bonfante,Cellularinteractionsbetween arbuscular mycorrhizal fungi and rhizosphrere bacteria. Protoplnstrlu 193: 123 (1996). 142. E. L. Nurmiaho-Lassila, S. Timonen, K. Haahtela, R. Sen, Bacterial colonization patternsofintact Pirnrs syhvstris mycorrhizospheresindrypineforestsoil:an electron microscopy study. Curl. J. Microbiol. 43:1017 (1997). 143. S. PerottoandP.Bonfante,Bacterialassociationswithmycorrhizalfungi:close and distant friends in the rhizosphere. Tretlds Microbiol. 5:496 (1997). 144. T. F. C. Chin-a-woeng, W. Depriester, A. J . Vanderbij, and B. J . J. Lugtenberg, Description of the colonization of a gnotobiotic tomato rhizosphereby P.yplt&),tlo-
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JIuor(wws biocontrol strain WCS365, using scanning electron microscopy. Mol. Plant-Microbe Interclct. 10:79 ( 1 997). 145. R. M. Boddey, S. Urquiaga, V. Reis, and J. Doberreiner, Biological nitrogen fixat 137:1 I 1 (1991). tion associated with sugar cane. P l ~ Soil 146. G. Andrade, K. L.Mihara,R. G. Linderman,and G. J. Bethlenfalvay,Bacteria IKIS
from rhizosphere and hyphosphere soils of different arbuscular-mycorrhizal fungi. P l e ~ r ~Soil t 19271 (1997). 147. S. Scannerini and P. Bonfante, Bacteria and bacteria like objects i n endomycorrhizal fungi (Glomaceae),Syrt~biosi.~ us So~trcc c!fE\dutiortury Itvtovcliioti: Specicrtiotl w t l Morl.'l~o~etle.si,s (L. Margulis and R. Fester, eds.), The MIT Press: Cambridge,
MA, USA.1991,pp.273-287. S. Longato, D. Minerdi, M. Harrison, P. Bonfante. 148.M.vanBuuren,L.Lanfranco, Construction and characterization of genomiclibraries of two endomycorrhizal fungi: C1otm.s versifortnc and Gigrl.spor(l rtlargcrritn. Mycol. Res. (1999). 149. B. M. Unterman. P. Baumann, and D. L. McLean, Pea aphid symbiont relationships established by analysis of 16s rRNAs. J. Brrcreriol. 171:2970 (1989). 150. P.Baumann,L.Baumann,C.-Y.Lai,andD.Rouhbakhsh.Gcnetics.physiology and evolutionary relationships of the genus Rwhtwrn: intracellular symbionts of aphids, A t m r . Rev. Microbiol. 4955 ( 1995).
10 Functional Ecology of the Rhizobium-Legume Symbiosis Andrea Squartini Universita degli Studi di Padova, Padova, Italy
1.
EVOLUTIONARYRETROSPECTIVE OF BIOLOGICAL AUTOTROPHIES
Nitrogen fixation is today envisaged as a nutritional advantage. One may say that it slowly became one, and that everything was ultimately due to the advent of photosynthesis. A debate on “who came first” between rubisco and nitrogenase sees the first as a favorite. Atomic evidence from fossils ( I ) seems to indicate that, after a rich phase of microbial development occurred between 3.9 and 3.5 billion years ago, the massive sedimentation of carbon-containing compounds could have made carbon availability a major limiting factor for life. Selective pressure would therefore have favored the onset of carbon-fixing mechanisms using CO1 as a source. These would have been initially chemoautotrophic or photoautotrophic and not yet leading to oxygen development. However, given thenecessityofmaintainingthe C/N ratiobelow IO, as required in bacterial living matter, the capabilities of fixing carbon in unlimited fashion would have, in turn, rendered availability of nitrogen the new limiting factor. Considering the cell requirement of reduced forms and the abundance of gaseous NZ, a strong selection would have at that stage privileged the appearance of nitrogen-reducing reactions. Such potentialities were presumably already present among microbial communities, and thence they could have been recruited. It has been postulated that nitrogenase, presently the key enzyme for nitrogen autotrophy. could have been initially just a system of cyanide detoxification (2). In today’s world the capability of converting molecular nitrogen, amounting to a wealthy 78% of the surface atmosphere but too inert and oxidized to be easily drawn into biochemis297
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try, is the privilegeof a guild of prokaryotes to whom all remaining living beings, as consumers, owe a great deal of the nitrogen that shapes their proteins and nucleic acids. What, however, is the link bridging nitrogen fixation and symbiosis?Onceagainphotosynthesis may haveimposeditsearly influenceonthe events. The picture, a distant one, has to be seen in proper perspective. In a still aquatic habitat, under a reducing oxygen-free atmosphere, the reductions required for biosyntheses did not have to face the high redox slope imposed by today’s oxidizing conditions. Therefore, the first nitrogen-fixing systems did not require the elaborate devices aimed at protecting nitrogenase from oxygen, which are so evident in modern symbioses. The key to the change that has led to the present status is to be searched for around 2.5 billion years ago and consists i n the onset of oxygenic photosynthesis. Initially run by cyanobacteria, it was then joined by eukaryotic algae, and finally, during the last S00 million years, by higher plants. An oxygen-rich atmosphere, besides providing anew and versatile terminal electron acceptor that promoted aerobic life, has enabled the buildup of the ozone layer, at first catalyzed by UV radiation and eventually providing a shield from the same. Such protection allowed the nonlethal exploration and colonization of a highly irradiated environment. as the emerged land is. The possibility of living under high photosynthetic intensity brought plants to the massive development that characterizes their recent history: a history full of interaction with microbes.
II. SIGNIFICANCE OF THE EXCLUSIVE INVOLVEMENT OF DICOTYLEDON ANGIOSPERMS AND REASONS FOR THE TYPICAL OCCURRENCE OF SYMBIOSES ON LAND The adventureo f higher plants onto tcrrestrial environments is dominated by new limitingfactors,namelywaterandnitrogen in the ammonium form. The new atmosphere,withoutmethaneandammonia, andstronglyoxidizing,demands 225 Kcal for reducing of a mole of N2 to ammonium. This energy of activation it can be obtained could be sporadically dischargedby events such as lightning. or at pressures of 100 Mpa and temperaturesof 3SO°C, as in the expensive industrial production of nitrogen fertilizers; as an alternative, i t can be achieved simply at atmospheric pressure androom temperature within the cells of those microorganisms endowed withthe enzyme nitrogenase. Some of them live free in communities or rhizopheres of various plant species; others. typically cyanobacteria, can associate with more complex organisms such as algae, fetns, and gymnosperms. Only a few, belonging to the Rhizobiaceae family of gram-negative bacteria and to the Frankiaceae family of actinomycetes, establish profound interactions with higher plants and are eventually hosted inside newly formed structures of plants, the nodules, devoted to the fixation of nitrogen, which is passed to the plant. The
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plant species involved in symbioses with bacteria of the Rhizobiaceae are a majority of the Leguminosae and a genus, Par-crsporliu. belonging to the Ulmaceae. The actinomycetes of the Frankiaceae instead interact with at least 137 species, grouped in12 genera withinthefamiliesBetulaceae, Casuarinaceae. Coriariaceae, Eleagnaceae, Myricaceae, Rhamnaceae, and Rosaceae. Such a surprisingly wide plant host range, however, could be more an appearance than a reality, as recent botanical taxonomy has reached the conclusion that actinorhizal families form a more coherent group than has previously been thought. Therefore. the to a relatively capability of interacting with Frankiaceae seems now to be due common basis in the numerous actinorhizal plant species (3). The first angiosperms date back 200 (4) or 300 ( 5 ) million years. The two classes, mono- and dicotyledonous, diverged at an apparently early stage; nevertheless, monocots do not include clearly documented casesof true nitrogen-fixing symbioses. The dicotyledonous family that par excellence shows a tendency to form symbioses, the Leguminosae. is divided into three subfamilies.In the oldest, Cesalpinoideae (including about 2000 species), only 23% of the species form nodules, while the more modern Mimosoideae (3000 species)and Papilionoideae (13,000 species) are nodulated to the extent of 90% and 97%, respectively (6). It is interesting to note that the Cesalpinoideae originated in hot and humid climates, in many cases periodically submerged;they generally are not able to adapt to different habitats. But Bryan et al. (6) have described the presence of bacteria inside the roots of many Cesalpinoideae and reported measurable valuesof nitrogenase activity. Therefore,it appears that even the supposedly recalcitrant legume subfamily could be extensively involved in deep microbial interactions.Its difference from the cases of the two more modern subfamilies, which today have also conquered drier environments, would be indeed the lack of the nodule. Is there a correlation between nodule and dry environment? Nitrogenase activity requires a reducing environment, and the more we move away from the water and from anoxic conditions, the morethe reaction requires insulating expedients and barriers. In this sense, to attain effectiveness in a dry environment, one would need a system with relatively insulating walls, local oxygen scavenging systems, and vascular connections for fast exchanges: in essence, the nodule. A submerged environment presents instead a more favorable redox potential,but the exchange of the gases is limited by their low water solubility. It is possibly for this reason thatthefirst nodules formed by Leguminosae, in their tropical humid sites of origin, could have been on the stem and not on the root. The phenomenon is still observable in plants such as Seshar~iaand Aesck.vtzornet~e,typical of frequently flooded environments, and nodulated by the genera Azorhizobium and PI1oto&izobiutrl. Thesc rhizobia possess ancestral features ( 7 ) , such as the photosynthetic activity displayed by the latter, and suggest indications of the possible initial site of legume nodulation, a phenomenon that would then have moved underground upon legumes gaining access to drier habitats. Carbon autotrophy would not have
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been so crucial. as the plant would pass organic compounds to the bacterium, while protection from oxygen and from desiccation would have been of primary importance,thusimposingasubterraneanrelocation of thenitrogen-fixing chamber.
111.
THE ROOT NODULE, A LEAFLESS STEM BORNE ON ROOT
A
Nodules formedon leguminous plants differ anatomicallyfrom all other nitrogenfixing nodules, including both those made by actinomycetes on other angiosperms and those made by rhizobia on Pe/rcr.spouier (Ulmaceae). The causeis the position of the vascular tissue, a peripheral sheath like that found in a plant stem and not a central bundle as seen in the root. On the contrary, actynorhizal and P~rc~sponic/ nodules have central vessels. This feature calls attention to the old question “Why legumes?” When we look for common features in the Leguminosae family, the stem-like anatomy of root nodules is an important clue. It is not determined by the rhizobium as confirmed by the case of the nonlegume Parclsponier but rather by the legume itself. Cell divisions leading to legume nodules begin in the cortical cylinder, while those of other plants start off from the pericycle. as in the case of lateral root formation. Moreover, legumes appear particularly prone to events of spontaneous nodule formation evenin the absence of bacteria (8) or following treatment with hormone-like compounds (9). This plant family therefore appears exceptionally apt to the genesis of receptive pockets in hypogeous sites. But the entry permit to such chambers is not so easily offered.
IV. THECOMPROMISEOFNITROGEN-FIXING SYMBIOSES: A NECESSARY TOLL OR A SUPERFLUOUS GOOD? Any of today’s bacterial symbionts can represent the domesticated version of yesterday’s commensal or pathogen. It can be hypothesized that originally the invasion of plant tissues, useful to achieve shelter from predation, drying, and a rich trophic resource, did not involve passing combined nitrogen totheplant. Prokaryotic microorganisms, as well as animals, maintain in their cells a C / N ratio below 10. When the carbon flux is high, the nitrogen demand will be correspondingly high. Plants and fungi can instead sustain themselves with a much higher C/N ratio, up to 40 and beyond. Plants feature large quantities of structural polymers with no or little nitrogen, as cellulose, lignin, cutin, or suberin, providing them with the solidity, stance, and resistance required for the colonization oflands. In proportion to carbon,plants canfunctionwithlessnitrogenthan
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bacteria o r animals. While in parasitized plants the carbon loss is uncontrolled and allows unregulated growth of the invader, in plants having became capable of controlling the situation the carbon flux can be modulated to limit the microbe growth within precise limits beyond which just the carbon for maintenance would be released. Needing to keep a low C / N ratio. when receiving less carbon the bacteria would be induced to give away some of the unused nitrogen. This compromise would finally satisfy the mutualism postulates. in present legumes, It must also be pointed out that, although stabilized symbiotic nitrogen fixation does not necessarily represent their main source of nitrogen nutrition but rather an integration of it. The genus Acacia. with its over IS00 species ( I O ) , is one of the largest of the family, and typically represents legumes that, starting fromthe original humid tropics. have then radiated to colonizedrierlands. The rapid expansion of Accrcia speciesfollowstheclimate changes that occurred at the beginning of the tertiary and renlarkably expresses thosephenotypicadaptations thataridand semiaridclimatesrequire. In such areas and, in a broader sense, in terrestrial environments, water availability may be the most severc limitation, and ;l deep primary root with extensive branching represents the main strategy for provision of water. By the same token, suchroot apparatus enables many legumes to access mineral nitrogen forms present in the soil. Actrcirr receives symbiotically only SO% of the nitrogen required for optimal growth and needs to extract the rest as ammonium from soil ( I I ) . Therefore, even today, evolved legumes show their reluctance to rely completely on symbiotic nitrogen nutrition, and they preset their maximum nodule numberto defined values even when ammonium deficiency limits their development to 50%.
V.
WHY HAVE CEREALS NOT YET DEVELOPED TIGHT NITROGEN-FIXING SYMBIOSES?
Despite the fact that monocotyledons evolved from dicotyledons shortly afier their appearance (4), and have therefore coexisted with their parent class plants for most of the angiosperms’ history, no instances of nitrogen-fixing symbioses comparable to legume or actinorhizal nodules have yetbeen observed in this class. Andthe efforts of biotechnology, aimed at the goal of cereal crops independent from nitrogen fertilization, have so far not yielded satisfactory results. Monocot ecology, it seems, hasnot urged modifications analogous to the root nodule. For which reasons? For one thing, from a structural point of view, monocots show a reductionof the predominant role of the primary root, which is substituted by a crown of fasciculate lateral roots. In general. secondary growth is limited, and as a consequence a secondary structure such as the nodule, branching from an axis, would not fit in the program. Also the hierarchical architectureof dicots, implying the primary/secondary concept, is missing, replaced by a fasciculate
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same-order anatomy. This idea is repeated in the vascular tissues, which consist of several single bundles within the parenchyma instead of a stele in the center of a cortical cylinder. Eventhe parallel nerves of leaves reflect the nonhierarchical structure of the transport system. In light of what was previously mentioned on the possible semipathogenic origin of root nodules, which would have become symbiotic due to a fine modulation of the carbon released to the bacteria, tight control of the fluxes directed to each compartment is an important premise. In legumes, nodule number and position are under systemic control. And that, as in any system, implies a structure with ramifications of progressive order. In an equal-rank architecture, such as the fasciculate apparati of monocots, it would be harder to modulate quantities destined to aspecificsite. While in ramified systems with successive orders of decreasing thickness, the flow delivery can be automatically determined by the structure itself. or, more Second, there may bereasonsforwhichthenitrogendemand. specifically, the demandof a large supplyof it in a defined period, is less pressing for gramineous plants than for legumes. The latter, producing seedswith predominantly proteinaceous reserve material,need an abundant and continuous nitrogen intakeduringthephase of seedmaturation.Graminaceaeinstead,featuring mostly amylaceous reserve in their seeds, would not share such need in the same temporal context and would have one less reasontoinvestresources in highintensity nitrogen factories as well as in dealing with a prokaryotic intruder. But. in spite of that, would not gramineous plants benefit from extra nitrogen after of nitrogen all? It is clearly appreciated how cereal crop productivity is a function fertilization practices. The agricultural ecosystem, however, represents an unnatural condition. The potential zonal vegetation (and its associated microbiota) is substituted on an average annual basis with a new monophytic cover, the agricultural crop, constituting an artificially imposed situation. The distinct pedologic soil horizons are homogenized by soil tillage, aggregate structure is disrupted, and as a consequence organic matter is exposed to rapid consumption by action of r-selected aerobic bacteria. Fertilization and pesticide application further contribute to an already pronounced perturbation. On the contrary, when we analyze prairie systems dominated by gramineous species, we can record an abundant surface microflora where photosynthetic and free-living nitrogen-fixing cyanobacteria, periodically active after rainy periods, greatly contribute to maintain the ecosystem fertility (12). The renowned Broadbalk experiment in England has shown how wheat that has been cultivated for Over 140 yearswithoutfertilizationcanpersistentlygiveaconstant yield which, although obviously far from the records obtained in intensive farming, is superior to the average world yield for wheat (13). The phenomenon is mostly ascribed to cyanobacterial nitrogen fixation. The prairies, which are biomes where Graminaceae are well represented, constitute an environment where the significance of rhizosphere for microorganisms is different from that of other landscapes. Generally in any terrestrial ecosystem it is possible to distinguish a bulk
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soil from a rhizosphere soil rich in C substrates. I n a prairie the rhizosphere is a shallow subsoil protile where fasciculated roots are extremely dense, giving rise to a sort of rhizosphere continuum i n which plant-microbe interactions are spatially quite homogeneous and do not strongly show that gradient called the rhizosphere effect, typically surrounding the tap-rooted systemsof less gregarious plants. In this sense, in natural environments, the plant needs can be fulfilled by less strict, but more homogeneously extended plant-microbe interactions, such as those that cereals undertake with free nitrogen fixers (e.g., azospirilli or cyanobacteria). and that often involve not only nitrogen exchange but also hormones and other signals. From a broader perspective, the question is: Would it really be an advantage for every plant species t o have a nitrogen-fixing symbiosis? Let's judge legumes. It is interesting to note that despite their nutritional autonomy for carbon L~IIL~ nitrogen, which should make then1 potential dominators of several habitats, i n practice they never get to be the main component of the vegetation, nor has it been possible to encounter pure stands of Leguminosae in nature (14). Despite an initial advantage of legumes for colonization of poor soil, their growth and the subsequent death of rootsandnitrogen-richtissuesprogressivelyenriches the N content in soil, rendering no longer advantageous the possess of a direct atmosphericNz-exploitingsystem. In suchconditionsthenonsymbioticplants are the group that benefits from the fact that a legume has preceded them, and they can easily compete and displace it from the site. While a limiting environment promoted pioneers, a gentle environment is ground for fierce competition. Therefore, the dynamicsof colonization by legumes involvesa continuous escape toward nitrogen-poor zones,and leaves behind enriched patches that will be filled by nitrogen-hungry plants. such as Graminaceae, giving rise to mixing prairie stands. The growth of those weedy, non-N-fixing plants will in turn impoverish the site, which will be prone to a future return of the legumes. This itinerant, wandering characteristic of symbiotically nitrogen-fixing species is also linked to a certain rapidity of expression of their growth potential. Many herbaceous legumes tend to generate the maximum of green matter in a shorter time than that taken by the gramineous plant sharing the same habitat. This scheme is repeated also in a longertimerange at theforestscale,wherewoodylegumes such as black locust (Robirlitr p.~elrclocrcacin),introduced in Europe from North America, show an aggressive pioneering behavior and an apparent supremacy in degraded woodlots. Within a few years, however, this expansion extinguishes its vigor and yields space to more moderate. non-nitrogen-fixing species. For a comprehensive survey on plant ecology see also refs. 15 and 16. I n conclusion, there are no absolute reasons in nature that would indicate a convenience for every species to embark o n [he risks and costs of a profound morphophysiological remodeling, at either the root or stem level, and to face the odds o f hosting and feeding invasive bacterial populations. In many instances
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the mere opportunism in exploiting environments inhabited by free-living fixers or ameliorated by symbiotic plants can warrant equal or even greater success.
VI.
ECOSYSTEM-DRIVENRHlZOBlALTRANSGRESSIONS INTO GRAMINEOUS PLANTS
Given the fact that legumes and cereals may share the same environment, could not rhizobia be tempted to enter the cereal roots? As a matter of fact, they did. For over seven centuries rice has been cultivated and rotated with berseem clover ( T t i f o / i mrr/r.r~~nrlritzur~~) in the Nile delta inEgypt. It is known how many cereals host an abundant microbial community both at the rhizosphere level and in more internallocationssuchasthecorticalcylinder,theendodermis,andeventhe vascular system (17,18,19). These microorganisms initially referred to as endorhizospheric have later been called endophytes or internal root colonizers (20). In light of these facts an analysis of the endophytes has been made of Egyptian rice in search of rhizobia1 clover symbionts that could have adapted to colonize riceinternally(21).Differentstrains of Rhizobilrm /eSlrmit?o.sclr.lrrl?bv. tr(fi)/ii could be recovered from the inside of surface-sterilized rice roots. The bacteria, present at a density of up to 1 Oh cells per gram of root fresh weight, were capable of effectively nodulating clover and of colonizing rice endorhizosphere when reinoculated. Some of the rhizobium strains can promote an appreciable increment of the rice yield, by stimulating root and stem growth, when inoculated on plants cultivated either in growth chambers or in the field. The mechanism does not appearlinked to nitrogenfixationbutpossibly to hormoneproductionor other causes positively affecting rice physiology (21). The main point is that an organism such as rhizobium, besides being able to live saprophytically in the soil or to enter in symbiosis with legumes, could access a third niche of a trophicprotectivenature,exchanging benefits with a cerealplant. Thephenomenon shows that a microorganism can adapt to alternate symbioses, and that even in this case a monocotyledon, notwithstanding its coexistence with a nitrogen fixer, has not yet felt the needto subject the basisof a rewarding symbiotic relationship to the toll of nitrogen.
VII.
LIVING IN THE SOIL AS A BACTERIUM
What is the life of a prokaryote like? What kind of a life does it experience in the soil? In the soil, not yet in the proximity of a root, or waiting for a root that never passes by? The answers to these questions have been gathered from a detailed body of studies on soil microbial ecology (22,26). The physical environ-
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ment, structured through centuries of pedogenesis by the influence of three factors, weather, bedrock, and time, is the resultof a process in which a mass having a small exposed surface per volume unit, slowly becomes a mass with an enormous exposed surface per volume unit. While a gram of rock might have had less than 1 cm? of exposed surface, a gramof mature soil, with its inner labyrinth of clay sheets cementedby organic matter, can easily measure 500 m’. Soil moisture and temperature fluctuate in various ways, but the main factor that really limits life in soil is the scarcity of nutrients, the lack of easily usable organic in soil tends to be extremely resources. As a consequence, microbial life intensity low. Microbes which, in a laboratory flask of rich medium, would accomplish 15 generations overnight may be leading a lagging existence in the soil that lets them afford, energywise, not more than two or three generations per year. Which species do we find there? Taxonomy manuals are not exhaustive as only a few thousand taxa have been described, presumably a small fraction of the far higher number of species that we can estimate to exist. But our lack of knowledge is not due to the factthat unknown species live in remote virgin forests or inaccessible places. On the contrary, unlike plants and animals, the microbes living on the different continents are basically the same, but the great handicap in assessing 99% of soil bacterial species microbial biodiversity is the simple fact that over are in the state of viable but not culturable cells. They are recalcitrant to culture in laboratory media, thus eluding characterization by the microbiologist, at least in a classical sense. Experiments of soil DNA reannealing have led Torsvik et al. (27)to estimate that thousandsof different bacterial species, mostly nonculturable, share a single gram of soil. Methods such as 16s rRNA amplification and denaturing gradient gel electrophoresis, reviewed in ref. 26, are helping the modern microbiologist to conduct these studies. What about quantity? How many bacterial cells do we encounter in a detined volume of soil? Microbial biomass can be measured eventhough the single species are not culturable (28). In average agricultural situations, about 2 mg of bacterial cells are found in a gram of soil, corresponding to a billion cells. An anthill-like environment packed with life? Not if we consider the hundreds of square meters of exposed surface, and suppose to evenly position cells of the an individual space of a square size of a micron or two. They would result having millimeter, a picture comparable to a man every square kilometer (22,23). This is a nutritional desert, harshly carbon-limited and beaten by predators such as protozoa and nematodes, a mineral solitude that has led many species to retine the strategy of quiescent survival, disregarding temporary nutrient surplus, maintaining an extremely slow but constant life rhythm, viable but not culturable. Other species choose instead to bet on timing, on the rapid exploitation of nutrient abundance, as for example when a full river of nutrients is passing by, under the form of the growing root of a higher plant. Indeed in the proximity of roots, in the rhizosphere, the underground landscape rcuiiccrlly changes.
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We must also keep in mind that many of our views on underground life apply, for practical interest, to the cultivated agroecosystem. The difference between poor mineral soil and root oasis and the subsequent ecology is once again a result of the repeatedly interrupted establishment of relationships between soil and vegetation. In more stable environments one might even find a reverse scenario. In a pasture, for example, the nonrhizosphere soil could be richerin organic matter than the soil around harvested crop roots, resulting in high microbiological activity (29). In such conditions fast r-strategist species can be abundant also in zones not proximal to the plants. By the same token, once we look at the closest plantinterface,therhizoplane,theconditionsoccuring in undisturbedsystems make it possible to reach a certain degree of stability and the support of k-strategist bacteria. An analysis of the colony growth kinetics from different microsites supports this view (29). The concept can be generalized, up to the point of seeing a plant rhizosphere as the stimulatory growth-driving belt, while therhizoplanewouldbethemoreexclusiveandstable,k-selectingboundary where interactions are refined, specific, and mutual.
VIII.
ENTERING A LEGUME,THEMOLECULAR COURTSHIP
Rhizobia represent a small fraction. from100 to 100,000 units, among the billions of bacterial cells inhabiting a gram of soil. Nevertheless, if the passing root belongs to the right plant, they will end up hosted by i t and fed in an exclusive and privileged matter. What is so special about rhizobia? Do they have peculiarities indicating their linkto plants? One clueis the findingof digalactosyl diacylglycer01s in Brndyrhizobiutn membranes (30). These typical plant glycolipids are rarely found in prokaryotes and suggest an ancient inheritance of genes from the plant kingdom. Rhizobia and legumes recognize each other due to specific a molecular mutual talk. This molecular dialogue is now partially known due to extensive research of the rhizobiologists (see Chapter 7). Growing roots of legumes, such as thoseof many other plants, release flavonoids. These compounds have a particular meaning for rhizobia, acting specific as gene inducerson a groupof transcriptional units, the notlulation genes. Flavonoid activity is concentration dependent. 10"' At IO"' M. far from the root surface they act as chemoattractants, while at M, at closer range, they induce gene expression (31). Flavonoids play a role in host specificity since the bacterial response is mediated by their interaction with a regulatory protein, NodD, that in turn will activate the different ~ z o operons. d Spaink et al. (32) have shown how mutations in the nodD sequence may lead t o a flavonoid-independent transcriptional activation by its product that extends the host plant's range.
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It has been postulated that the occurrence of difference types of flavonoids could be an evolutionarymove to avoidinterferencefromcompetingspecies (33). Activation of the proper genes brings about the synthesis of the key molecular signals, which travel from bacterium to plant and affect the behavior of the host plant so as to impose the revision of the root morphogenetic program, triggering meristem activities that will lead to the formation of a nodule to host the visitors. A real neoplasia is apparently set up by the specific action of a chitinbased compound with a fatty acid tail, a chitolipooligosaccharide.In the bacterial world chitin is not a common product, belonging rather to fungi cell walls or to arthropod exoskeletons, where it is found however in longer chains. The nod genes, exclusively present in rhizobia are therefore sending anunusualsignal that orders plants to build up the rooms where bacteria will transfer themselves. A strict host specificity can exist between rhizobium type and legume species, as shown typically by herbaceous species of the temperate zone. For example, Rhi:ohium leglrtnirlosarunz bv. viciae nodulates genera including Vicicl, Pisurn, h t h y r u s , and Lem because it sends off a signal in which the first of the four or five chitin residues is acetylated and the fatty acid tail is I8 atoms of carbon long with four unsaturations in precise positions (34). The nucleotide sequence of the nod genes in different rhizobia is responsible for the structure pecularities of each species’ signals. Interestingly, supplementinga plant with the purified compound, even in the absence of rhizobia, will make it form nodules; obviously empty and nonfixing but structurally complete (35). There are casesof unrelated rhizobia that produce identical r~odfactors but nodulate different hosts, e.g.. Pllmeo1rl.s and Lotlrs. In this case again it is the specific flavonoid-mediated activation that discriminates the response. Another main specificity custom is represented by plant surface lectins, which are carbohydrate-binding attachment mediators (36). Clover roots engineered to express a pea lectin gene could be infected by the otherwise pea-specific biovar of R. Irglrrni~lns~lrnrrll ( 3 7 3 ) . Rhizobia need to reach the nodule traveling to the root cortex, and such itinerary is necessarilyan intrusion. In case of different bacteria the plant would fire off defense molecules such as phytoalexins, activate enzymes such as peroxidases, and try to isolate the unwanted microbes. In strictly hostspecific symbioses of the temperate regions the port of entry is normally a root hair. A young epidermal root hair cell experiences the accumulation of bacterial cells at its apex and is deformed by action of the chitolipooligosaccharide. The tip curls and the plant cell wall locally starts to be loosened to give place to an ingrowing channel, the infection thread, into which rhizobia proceed and multiply, well fed by plant substrates. The thread can break at any stage and prematurely reject the bacteria. as seen in many incompatible cases. Host specificity, besides relying on the submission of a specific mobile signal, is therefore exerted also a s a possibility of proceeding throughout thethread until release in the cortex.
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And a commando of invaders, be they friend or foe, need a suitable “outfit” to penetrate, a suit of polysaccharides.
IX.BACTERIALEXOPOLYSACCHARIDES, MULTIPURPOSE OUTFIT
A
Manybacteriadestinedtointeractions.eitherpathogenicorsymbiotic,with higher organisms, coat their own cellswith polysaccharides (see refs. 22 and 26). In the free-living state such a layer is helpful in holding moisture and protecting the bacteria from drying. Moreover, the presence of negative surface charges allows the adhesion, via divalent cationic bridges, to other negatively charged surfaces abundant in soil, the clay minerals. This way bacteria can anchor themselves within the micropores of soil aggregates and find shelter from predator protozoa and nematodes. Besides the role of “raincoat” and gear, when encountering the host, exopolysaccharides (EPS) can help it to identifythe microbe, playing in this case therole of a uniform, whose color isrecognized. In this respect symbiosis and pathogenesis follow different routes. A symbiont has an advantage in being recognized by the host, and adhesion is often mediated by specificity between bacterial EPS and plant lectin. A pathogen, on the contrary, will try foraslongaspossible to avoidbeingrecognized, by masking when possible, with generic EPS, the avirulence factors that would betray its identity and allow the plant to counteract it with prompt defense responses. In that sense EPS would act as a cape to cover the real identity. Alternatively lipopolysaccharides (LPS) can have a similar role in Rhi:obium otli (39). The fact that rhizobia can be aborted from infection threads shows (40) that a continuous declaration of identity is necessary throughout the travel, consisting in the exposure of specific surface EPS determinants, in the absence of which the transferis interrupted. Host specificity between rhizobium and legumes is therefore played at two main levels: a chitolipooligosaccharide password, acting at a distance, and an exopolysaccharide passportto be exhibited by the bearer in its trip to the awaiting nodule (40).
X.
HOSTSPECIFICITY AND PROMISCUITY
For a long time, host specificity, the capacity o f a particular rhizobium to nodulate defined plant species, has been regarded as a prerogativeof temperate zone environments. On the contrary, the symbioses occurring in tropical and subtropical environments used to be referred to as interactions caused by the so-called promiscuous strains, as judged by their capability to nodulate many legume species (41). This generalization results from the coincidence of some facts: there are
osis
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legume species such as cowpea (Vigtzn urzguiculata) or bambarra (Vigtzcr suhtprt - m W ) that are not very selective and can be nodulated by several different strains. In parallel there are some rhizobium strains with a broad host legume range. It is therefore statistically very likely that a strain isolated from a noduleof promiscuous V i g m is indeed one of thewidely compatible promiscuous rhizobia. In particular the Vigna isolates that were chosen in the past as reference strains of the cowpea group, when tested on a series of tropical legumes scored positive and supported the above opinion. But recent studies (42) seem to indicate that the myth of tropical legume promiscuity was due primarilyto the peculiarly wide host range of the strains initially chosen as type references. When the host specificity of 59 isolates from cowpea and other tropical legume nodules was analyzed, the average number of plant species nodulated by each strain was less than three. Indigenous tropical rhizobia appear as a mix of different symbiotic types, each one specific for itsnarrow set of hosts (42). The prompt nodulation of many tropical legumes therefore would not be due to host range width, but rather to the diffuse divcrsity of several symbiotic genotypes. The consistency of the average number of nodulating species (2.4 per tested strain) reflects a tight adaptation of the rhizobia to the local legume vegetation, supporting the hypothesis (43) that rhizobia and legumes coinhabiting a defined geographic area, be it tropical, temperate, or cold, can experience evolutionary convergenceof their respective symbiotic genotypes, and give rise to complementary gene pools whose interaction results in a functionalsymbiosis.Ontheotherhand, it can be observedhow rhizobia that are unrelated both geographically and phylogenetically may have thefortuitouscapacitytonodulatethesameplant (44). This isthe case with Prosopis chilrnsi.~,which in its native South America is nodulated by some local Sinorhi:oDiwz strains, while, once introduced in Africa, strains were recruited of a different lineage that haveAcacia s ~ r r c ~ g cas r l their home plant. This fact may suggest that the molecular combinations resulting in specificity between plant do not overlap no and bacterium are tinite in number and thatwhenhabitats mechanisms operate to avoid potential cross-specificities. Among the minority of broad-host-range rhizobia a case stands out above all others: the one of strain New Guinea Rhizobium 234 (NGR 234) isolated in P a p a in 1926. This strain nodulates more than 400 species belonging to I 10 O I Z ~ ~ ~ and Broughton, unpublegume genera and the nonlegume P N I . C I , S / J(Pueppke lished). Nearly 50% of the tested species could form nodules in its presence. NGR 234’s secret lies in the molecular signals. First, it is able to respond to several kinds of flavonoid inducers and turn on its nod genes in front of Inany different plants. The nod gene activity translates into the synthesis of at least 80 different chitolipooligosaccharide types, bearing a series of substitutions including carbamoyl, methyl-fucosyl, and acetyl groups in various combinations. Unlike narrow-host-range rhizobia, making, at the maximum, a dozen signals, this strain is apparently able to serve every host door with the right key. It is interest-
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ing that the legume species that are not nodulated by NGR 234, in addition to of nonplants from relatively ancient tribes, include some more recent species woody type, such as alfalfa, clover, peas, and sulla,used as forage or grain crops i n the temperate region. Therefore, genera such as Medicago, Trifolium, Vicia, Pisurn, and Hedysclrunl, which encompass speciesmostly living in the Mediterranean, temperate, and cold regions, constitute examplesof unsurpassed selectivity in their interaction with microbes. It is also known that the r~odgenes encoding specificity for one of those hosts cannot coexist with those specific for another in the same rhizobium background.By genetically manipulating rhizobia one can test the effect of introducing a set of genes specific for one plant nodulation into a strain already possessing those specific for another. For instance, introducing nod genes of Rlzizobiurtl “hedysari.” the symbiont of sulla (Hedyscrrum coronarium). into alfalfa‘s partner Siwor}lizobirnn mrliloti, or in chickpeas or bean rhizobia ( 4 3 , results in displays of reciprocal incompatibility and the two gene sets do not persist together for long. Typically the interaction with one of the host plants causes theloss by deletion of the group of genes specific for the other. These plants are therefore particularly exclusive. By which means and for which scopes‘? One of the peculiarities of herbaceous temperate legumes is the curled root hair mode of entry. Such an itinerary, not shared by many tropical legumes in which the entrance is by emerging lateral roots or between cracksof epidermal cells, can be seen as a strong specificity bottleneck. Which ecological reasons might havestressedseverity in partnerrecognition in herbaceouslegumesof these climates? It is common to observe in natural vegetation, such as polyphytic meadows or ruderal environments, among many nonlegumes. specimens of Medicago, Trjfolilun, and ViciLI fighting for the same habitat (15,16). I n this scenario it would be of particular importance to establish an exclusive relationship with a personal bacterial partner instead of having to compete with other plant species for a potential common resource, as a promiscuous rhizobium would be.
XI.
VENTURING INTO A LEGUME, THE PARADOX OF AN APPARENTLYDEADEND
Despite the apparently clear benefits that the symbiosis brings to both partners, combined nitrogen for the plant and a personal trophic shelter for the rhizobia, a question mark remains concerning the destiny of the latter. The fact appears as an ecological paradox. The rhizobia penetrated into their host are released from the infection thread into the nodule where they undergo an extensive morphophysiological modification, increasing their size and turning into irregularly shaped cells called bacteroids. These cease definitively to divide and devote to nitrogen fixation the energy received from the plant as carbon compounds. The point about irreversibility from the bacteroid state is controversial. Apparently
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only in particular nutrient media is it possible to restore cell division (46). In in light of the fact nature on the contrary, their recovery appears unlikely, also that they are degraded at nodule senescence. Rhizobia nodulating a plant would therefore not contribute to future generations, and the capability of establishing a nitrogen-fixing association seems to be a lethal trait for the bacterium. The mutualism would then take the tones of a slavery exerted from the plant on the microorganism. Nevertheless nature’s selective pressure has maintained the symbiotic genotype in the bacteria in spite of the apparent absence of a reproductive advantage. The problem has been considered by Jimenez and Casadesus (47). Rhizobia can experience mutations abolishing the symbiotic phenotype giving rise to mutants incapable of nodulation (nod-) or partial mutants able to form nodules but not tochangeinto fixingbacteroids(nod + fix-). It istherefore relatively frequent to find the rise of strains that are useless for the plant but capable of invading it, whichmultiply during the migration in thethreadand are released without transfornling into irreversible forms and are advantageously released in the soil. Which reason does allow nevertheless the persistence of the nod fix genotype in nature? The model considers the importance of a certain fraction of the invading rhizobia, those arriving last, the ones that at the time of senescence are still on the infection thread and would not have a chance to turn into bacteroids. These are the future survivors that, having benefited from multiplication in the thread, will then be released in soil. In this sense rhizobia having turned into bacteroids would be that massive part of a population that is clonal, having come generally from a single strain that is sacrificed to the plant for the advantage of a little rearguard destined for the future cycle.
+
+
XII. THE GENETICS OF SYMBIOSIS: A FAIR OF PLASMIDS AND A NIGHTMARE OF INSERTION ELEMENTS. WHY? The essential genes required for the symbiosis (nodulation and nitrogen fixation functions) in most rhizobia are not encoded on the chromosome but on plasmids (48,49),i.e., on autonomouslyreplicating DNA elements.whichcanbeexchanged by conjugation, also between different species, and can be eliminated as fromacell without compromising its survival. Exceptions are genera such B~cr~l~rhi~obiurn (partner of soybean. originating from the Far East) and A:orhizobilrrn (nodulating Sesbcrtzicr’s roots and stems) that carry r d and rlifgenes on their chromosome. Ribosomal RNA-based phylogeny shows that these two genera, considered more primitive, are taxonomically quite distinct from the rest of the rhizobia (SO). The majority of legume symbionts studied so far bear their characteristic symbiotic plasmids (pSym) whose dimensions vary from IS0 to over 1700 Kb. Besides the symbiotic ones, a numberof other large plasmids can
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be present. In some strains plasmid DNA can amount up to 25% of the genome. A great deal of encoding capacity of a rhizobium is therefore left toan extrachromosonlal gene pool. Another characteristic of these bacteria is the presence of repeated DNA sequences. In some strains the genes for nitrogen fixation or nodulation can be found in repeated copies ( 5 I).Hybridizations have revealed in some cases over 200 gene families per genome with an average of 3.5 elements per family. Such astrikinglevel of DNAreiterationsthatappearscommon in Rhizobiaceae is however unusual among prokaryotes, with few exceptions such as Hdobacterium and Sfreptornyc.e.7.What is the reason? One important observationis the presence of mobile genetic elements. A number of insertion sequences (IS elements) and transposons have been identified (52-54). The large (536,165 base pairs) symbiotic plasmid of strain NGR 234, the one nodulating over 400 plant species, contains clusters of IS sequences, accounting for 18% of its length (%), and appears to have worked, during its history, as a transposon trap (56). In Brcldvrl7izc)biuin jq?orlicunz several copies (upto 175) of repeated sequences (RS) have been found clustered around the nodulation and nitrogen fixation genes (57). Insertion elements, besides their capabilityof mobilizing DNA regions, are thebasis of possiblehomologousrecombinationevents that cangeneraterearrangements in sequenceorder,duplications,inversions,anddeletions.The abundance of IS elements would therefore ensure a potentially high geneticvariabilityandconfer a largeinternalplasticity to thegenome. I n addition,selfsplicing group I1 introns are being discovered in rhizobia in close proximity to IS sequences (58). Theterm“symbiosisislands,”compared topathogenicity islands, has been suggested for the nodulation loci to underline their inherent lateral genetic mobility (59). The mechanismof natural DNA amplification generated by repeated sequences has been invoked as one basis for rhizobium genome to a large DNA region plasticity, and the term amplicon has been coined, referring bordered by direct repeats (60). Mesorhizobi~rt?~ loti offers a vivid example of the potential of horizontal gene transfer. Its symbiotic genes are on the chromosome but are part of a 500-kb self-transmissible symbiosis island that integrates into a phe-tRNA gene (61). The dynamics of rhizobium genome variation can be directly observed as in the above-mentioned experiments of introduction of heterologous host specificity genes, resulting in the deletion of one set (45). It is also possible to witness the recombination between two sym plasmids ofdifferent origin that generate an hybrid replicon (62). Different rhizobia have hostspecific nodulation genes. Although their slight divergence is the key to the nodulation of different legumes, the genes nonetheless share a high homology, and that, in case of encounter of the two colinear sequences, makes recombination possible leading to a chimeric gene. The phenomenon has been observed in experiments where homologous genes encoding for interactions with clover or with pea generated hybrid combinations with new phenotypes (32).
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In light of all these observations it is possible to formulate a hypothesis to try and answer the question in this section’s title. From an evolutionary standpoint, rhizobia undergoing the speciation process that renders them unique and specific for a certain plant gain access to an exclusive niche that is the basis of their ecological proficiency. There is a problem in this quest; the specialization strategy must be always addressed toward the same resource, nodule organogenesis, but i n different plants. Since there is a limited degree of possible morphostructural variation in the way plants can make nodules, the specialization principle, from thebacterialside, cannotcount on a markedvariationlikethose observed in other ecological contexts, where one specializes for an alternative resource such as a flower with a different corollaor a larger fruit. The individuating principle for rhizobia must consist in a simple variation on the theme, sometimes in a mere nuance onit. Being the problem always triggeringthe functioning of a cortical meristem, the active principle will not have to differ greatly. For this, slight modifications on the structure of a basic molecule, the chitolipooligosaccharide, that would render it more active on a certain plant membrane will be achieved by moderate reshuffling of the proper genetic determinants, as occurs in the case of two homologous plasmids happening, upon conjugation.to temporarily share a background.In this sense carryingthe symbiotic geneson plasmids, i.e., mobile replicons, can multiply the likelihoodof encounters of corresponding gene sets and subsequent mixing. In parallel, the presenceof powerful recombinatory springs, such as the IS elements, adds an inner variation dynamics to the system. Finally, let’s not forget how that plasmid, the key element of so many molecular interactions. can also belost, and the rhizobium, dismissingits uniform and its weapon, can be drivento the state of a simplesoil saprophyte. Unfortunate replication accident? Not exactly; the amazing number of nonsymbiotic rhizobia found in soilsuggests that onceagainplasmid loss couldalsobeastrategic choice.
XIII. SILENTMAJORITIES,THENONNODULATING RHIZOBIA Nodulation-based surveys, like the m o s t ~~rohcrhle nwlher approach, havefor long time overlooked them,but soil is thehome of a large quantityof microorganisms, taxonomically indistinguishable from rhizobia, but lacking the symbiotic determinants, along with the possibility of interacting with plants. In a pea rhizosphere Rhizobitlm legur?linosarrrrrzcells lacking pSym can outnumber by sixfold their plasmid-bearing counterparts (63). These microorganisms can acquire a nodulating phenotype if they receive the plasmid by conjugation from wild-type strains. Bean roots support a nonnodulating rhizobia1 population 40-fold higher than that of regular rhizobia (64). Soils where legumes have not been sown for
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some years present twice as many sym-plasmidless rhizobia as soils where legume cultivation is continuous (65). Which possible advantages arise from living without that large extrachromosomal DNA element? For one, the saving of the energy that would have been devoted to replication, transcription, and translation of a conspicuous genome portion. The plasmidless condition is in some cases linked to increased growth rate. For recombinant plasmids loss can also be accompanied by better survivalin various environments(66). In other cases a higher resistance to bacteriophages is displayed, possibly due to the disappearance of someplasmid-encodedsurfacedeterminants usedbythe phageforspecific docking. The possibility of conjugally regaining the plasmid always exists. Therefore, a population would be ecologically advantagedif a part of it could periodically dispose of the plasmid, while another fraction would maintain it to guarantee interaction with the host. Remembering that the rhizobiasurvivingsymbiosis could be the ones that do not turn into bacteroids, one can conclude that loosing the plasmid during invasion would not be disadvantageous. It is also interesting that in Rhizobizrm leguminosarum the production of some of the quorum-sensing signals (N-acyl homoserine lactones), used by several bacteria to take a census of the population density in a concentration-dependent manner, is repressed by the presence of the sym plasmid (67). This seems to imply a mechanism aimed at monitoring the critical threshold of a specific fraction within the population in a way that is dependent on the presence of the plasmid itself. The function of the genes triggered upon reaching the quorum is not clear yet, but the plasmid involvement suggests that part of the goal is to balance the numerical ratio between plasmidless and plasmid-bearing populations. Rosemeyer et al. (68) have started to cast some light on the complex mechanisms of Rhizobium etli quorum sensing and identified bacterial genes whose expression is cell-density-dependent and leads to the restriction of the number of nodules induced on the host plant. Mutations in these genes lead to a supernodulating phenotype. On the necessityof alternating between saprophytic free life and symbiosis, it is important to add that in nature, the continuous presenceof the proper legume is surely not granted, given the legume’s attitude to colonize the environments transiently, and to exhaust rapidly its vegetative potential on a given site. As a consequence it would be convenient for the rhizobium to be predisposed for the passage to the nonsymbiotic stage of its life cycle, by promptly dismissing the heavy nitrogen fixer suit by a programmed mechanism that merely abandons a prepacked portion of the genome, which in the future, if necessary, can be acquired again. What if a rhizobium does not have symbiotic plasmids in the first place? Such is the case of Brmfyrhizohiutnjc1pponicutn.In this case the phenomenonwill not be possible but it seems that a compensative mechanism can overcome the impossibility of renouncing to the symbiotic phenotype. Tsien et al. (46) and
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Zhou et al. (69) report that B. japonicum bacteroids, unlike those ofR. legurninosclrun1, are capable of reverting to the status of dividing cells, thus surviving symbiosis. There are also cases, such as that of Sinorhizobiurn tneliloti, which has very large symbiotic plasmids, of more than 1500 kb, which are 5-10 times larger than those of most rhizobia, and whose elimination would bring about the loss of several other traits. In this respect Hartmann et al. (70) found marked genotypic differences between S. rneliloti isolated from alfalfa nodulesand from soil. Bromfeld etal. (7 1 ) were not able to find nonnodulating S. rneliloti subpopulations in soil. Moreover they also observed that the numerically dominant genotypes of soil isolates did not overlap with thoseof strains isolated from Medicago nodules. In a further work (72) the authors reported a random distribution of the pSymmegaplasmid-chromosomalgenotypecombinationsalong with a contrastingly nonrandom distribution of those with the non-Sym(pexo) second megaplasmid. In parallel Young et al. (73), searching for nonnodulating R. legurnino~clrutn, found a wide population. But as regards nodulating strains, when they compared the profiles of strains from nodules of naturally nodulated plants from soil with those from the nodules formed by axenic plants purposely inoculated with soil dilutions, the proportions appeared comparable. As a consequence it appears that S. rneliloti, not prone to the loss of its megaplasmid, would differentiate its populations between strains more adapted to saprophytic soil life (or to interactions with plants other than legumes) and strains able to efficiently nodulate their hosts. But, as indicated by the randomness of combinations, within the nodulating guild, the genetic reshufflingof the symbiotic determinants would be not as restricted as in other rhizobia. R. legurninosarurn, instead, would be able to generate an alternate population due to its plasmid instability, but within the symbiotically proficient fraction, strains would always be able to nodulate, each with respect to its own competitiveness.
XIV.
ECOLOGY AT THE SPECIES LEVEL: SURVIVAL AND COLONIZATION IN DIFFERENT RHIZOSPHERES AND SOILS
The presence of rhizobia in soil and their population density vary widely. Values ranging between 10' and 10' cells/g are typicalof sites where legumes are present or have beenin the recent past (74). Maxima of 10' cells/g of soil have occasionally been observed (75). In host rhizospheres cells can amount up to 1OX/g(76). Numbers fluctuate during the year. North American clover pastures show peaks in the period between winter quiescence and vegetative spring reprise (77). In Australia the same crop featured high numbers also at the end of the growing season after root death and nodule senescence (78), possibly due to the release of rhizobium cells that had not degenerated to bacteroids.If the temporal parame-
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ter has an influence, the spatial one has an even greater one. Rhizobia are among the microorganisms that benetit most from organic compounds released from the root. Their presence correlates with the exudate gradient, whose concentration decreases with increasing distance from the rhizoplane. In the tirst millimeters from the root surface the rhizosphere effect is exerted, and it can be quantified as the ratio between cell numbers in this range and thoseof the bulk soil. Around a legume rhizosphere the rhizobial ratio can be between I O and a million, up to the point where 10% of the total rhizosphere bacteria can be rhizobia(79). In this colonization race the prevailing nonrhizobial groupof microbes is the fluorescent pseudomonads. involved in another series of interactions with plants where protection against pathogens is the main theme. Microbial populations are also markedly affected by pedoclimatic factors. Rhizobia are negatively affected by acidic pH, due to aluminum toxicity, highly soluble at low pH (80). Salinityis a further negative agent on rhizobia (81). In this case plants are normally more sensitive than their bacterial partners, which can often survive to concentrations that are not possible for legumes (82). In the symbiosis between sulla (Hedysarwn coroI I N ~ ~ I and ~ I ~ its ) host-specific R11i:ohiurn “11ec!\arrri,” a pair found alsoin extreme environments of the saline or alkaline type. the plant shows a higher resistance to salt or alkaline stress than the respective microsymbiont.Sulla grows spontaneously in semiarid soils such as North African deserts or in alkaline environments (pH 9.6) like the pliocenic clays in Tuscany. Its rhizobia do not show a particular tolerance to those stress factors and the plant, acidifying its rhizosphere. locally moderates the severity of an otherwise extreme microenvironnlent and enables rhizobium to interact with it (82). In the presenceof another kind of stress, namely high levels of heavy metals such as chromium, it has been observed that clover can form nodules even though the concentration would be lethal for a free-living rhizobium (83). The fact demonstrates how symbiosis can offer the bacteria a permissive niche in an otherwise deadly milieu. Thewinningproperties of arhizospheredwellerare: (a) growth speed, coupled with nutritional versatility that allows a microbe to exploit a vast array of plant-released organics; (b) chemotactic responsiveness and active motility; (c) capability of adhesion to the root surface, often mediated by specific interactions between bacterialexternal polysaccharidesand plantlectins.butalready long before contact a plant can impose a selection on its rhizospheric customers via is catabothe composition of its exudates. Pea roots release homoserine, which lized by their R. Ie~~~o~lir~osarrrr?~ bv. vici(w but not by alfalfa’s specific S. irrcdiloti (84). Mimoscr and Lerrcaena exude mimosine, an antimitotic hydroxyphenyl alanine analog that inhibits the growth of several microorganisms, including rhizobia, with the exception of some, specific for those hosts, and able to degrade mimosine and use it as a carbon source (85). The concept of preferential trophic niche has been perfected by some rhizobial strains that, once inside a nodule, synthesize peculiar compounds, such as methyl scillo inosamine, that they can catabolize (86). This way, once nodules would have smesced, strains present i n
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soil that arecompetent for the use of these rhizopines will enjoy a personal advantage for survival and saprophytic growth in the absence of the host, and will be numerically favored in view of a future encounter with their plant. This rhizobia1 behavior, which implies having the host synthesize their favorite food, recalls that of the near relative Agrohactrrium tutrwfirciet1s, which transfers a fragment of its own DNA into the plants and forces them to produce opins, whose consumption becomes its privilege within the crown gall tumor. Legumes and rhizobia are intertwined for more than one function: strong evidence comesfrom studies on mycorrhizal colonizationof plants such as clover or soybean (87,88). In the latter work the authors demonstrate how rhizobia or their purified rwtl factors stimulate mycorrhizal formation. and that the fact is observed even i n nonnodulating plants. But there are not just legumes to offer selective growth chances to rhizobia. Convolvulaceae such as C d . w r e g y I s r w p i u t r r and Cornwlvolus w v e n s i s andSolanaceaesuchas Atropu l x 4 l ~ h t m ~ , unique cases among 105 species examined i n 23 families, produce calystegins, whichcan be catabolized by S. rrwliloti, thanks to specificgenes,borneonce again o n a plasmid but different from the symbiotic one(89). Therefore, rhizobia can have molecular interactions alsowith plant species other than their symbiotic partner. This could enable S. rlwliloti to cope with vegetation successions, ensuring its own survival even when the legume is not favored. I f we consider the ecological strategies of the two plants in the wild (90), for instance, Mrdictrgo l ~ ~ p u l i t ~isuan . R, S-R (between ruderal and stress tolerant-ruderal) while C. SCIPpilrru is C,C-R (between competitive and competitive-ruderal). Interacting alternatelywith one or the other plantwouldthen allow S. mdilofi t o cover two complementary ecological sccnarios.I t is likely that theseexamples of our knowledge on the interactions between bacteria and landscape, so far fragmentary, representthe tip of a complexiceberg of tieslinkingmicroto macroflora still awaiting discovery.
XV.
ECOLOGY WITHIN THE SINGLE SPECIES, SELECTION AND COMPETITION WITHIN THE SAME RHIZOSPHERE
Notwithstanding all the above differencesand preferences, a defined legume species has the biological property of being nodulated by a certain kind of rhizobia, which collectively we use to classify as well a s a distinct linnean species or a s a biovar within one species. But inside such a taxon there canabe high variability. The strains present i n a soil and able to interact with one plant can be many. When taken singularly, and inoculated as pure cultures on the plant under microbiologically controlled conditions, the individual strains may prove symbiotically sound, but in soil, in the middle of a complex microbial community, competition effects select, a s eventual nodule occupants, an enterprising minority. The per-
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centage of occupancy for each strainis therefore highly variable, and ranges from dominance to sporadic occurrence. The basis of competitiveness in nodulation has received a great deal of attention, also in light of the fact that cultivated legumes are inoculatedwith selected strains whose performance dependson their successful competition over cospecific indigenous microorganisms. An analysis of 350 clover modules from pasturesin Wisconsin (91) distinguished, by plasmid profile screening, 66 different types of R. leguminosarunz bv. trifolii, occupying their niche with widely variable percentage, the dominant case being present in 70% of the nodules. As regards B. japonicum, serological studies (92) pointed out serogroup 123as the one filling 60-100% of the soybean nodules. Nevertheless strain 123 is not numerically dominant in rhizosphere and its success does not appear due to superior colonizing properties. Which factors determine the distribution of nodules to rhizobia? From one side the competition is ruled by the soil system, by means of physiochemical and biological variables such as temperature, pH, moisture, salinity, concentration of nitrates, phosphates, and presence of other microbes or microbial products that may be advantageous or disadvantageous to the different strains in various ways. Nodulation trials in soil or in artificial media inoculated with dilutions of the same soil show that those strains that are dominant in soil-grown plants are superseded by less competitive ones when the dirt is diluted and plants are grown in agar (93). Intrinsic properties of the strain as the production of toxins active against other rhizobia have also been shown to increase competitiveness (94). On the other end, the outcome is governed by the host plant with its individual variety differences, and to one or two its preferences, that will lead to assigning a main nodule share particular strains and leaving minor shares for alternative populations whose identity may vary in time. When analyzing nodules from three Medicago safiva varieites, Paffetti et al. (95) found a significant genetic diversity among isolates nodulating the different varieties. The same authors (96) indicated that plant variety has an effect greater than soil nature in shaping the genetic structure of the Sirzorhizobiurlr populations. Handley et al. (97) examinedR. legunzirlosarum populations in relation to the host plant. Dominance by particular profiles was not the rule, and the relative supremacy of some strains was not constant throughout the years. Moreover the high diversity of rhizobia was not apparently affected by soil cultivation. Experiments on pea and vetch in Italian soils revealed the presence of a dominant strain that occupies over 50% of the nodules in both species. A genetically modified derivativeof such a strain, when massively inoculated in soil, is able to replace its parent and achieve a comparable, but not superior, nodulation score (V. Corich, personal communication). In practice, even in the presence of an excess of cells of the dominant strain, the plant would continue to assign a more or less constant nodule share to a variety of strains. The fact suggests the existence of a mechanism for partitioning a resource such as “the root” in favor of maintaining a fixed level of symbiotic biodiversity.
Rhizobium-Legume Symbiosis
XVI.
31 9
RHIZOBIUM PHYLOGENY, THE OPEN END OF A TRANSVERSEEVOLUTIONARYRACE
Significant insight into this problem is due, among that of many others, to the work of Young and Haukka (98). The comparison of 16s rRNA sequences reveals for rhizobia a polyphyletic origin. In other words, there is no branch of their evolutionary tree that bears all rhizobia and no other bacterium. Rhizobia within one branch are therefore more related to genera such as, for example, Rhodopseudornonas, Ajipia, Nitrobacter, Brucelh, and Beijerirlckia than to the is a common ancesrhizobia placed in a different branch. As a consequence there tor to rhizobia and to other bacteria. Why do these species, sharing proximity with rhizobia, have no trace of genes for interactions with legumes? A first hypothesis can be that the common ancestor was a rhizobium but subsequently the symbiotic proficiency has been lost at the differentiation of genera such as Rhocfopseuciomonu.~. This hypothesis is unlikely because, based on the molecular 16s rRNA clock, the evolutionary divergence between Rhizobium and Brudyrhizobiurn, equal to the length of the lines separating them in the dendrogram, is deeper than that between plant mitochondrial and their nearest bacterial neighbors, the rickettsiae (99). This means that higher plants, including legumes, did not yet exist at the time of the supposed common rhizobia1 ancestor. A second hypothesis is that the genes for symbiosis evolved independently several times in some of the dendrogram lines. It is not very likely how sequences such as the nocl genes, which share outstanding homology throughout different rhizobium species, could have possibly arisen in an independent manner. A parallel, unrelated origin could instead by invoked in the case of the actynorhizal symbioses of Frankirc with nonlegumes, in which the search for nod gene homologs has so fargivenscantyevidence.The third hypothesisproposes thatthe symbiotic genes originated after separation from the common ancestor during plant age, in one of the lines, and successively have been transmitted by lateral gene transfer to some of the other, generating the present lineages. The plasmid location and the abundance of insertion elements, creating solid grounds for a horizontal evolutionary short cut, make this hypothesis the most likely to explain rhizobium origin.
XVII.
CONCLUSION
Modernnitrogenfixationistheresult of coevolution of atmosphere andlife. Dicots’ morphological attitude made many of them prone to elect symbioses as a stable part of their physiology by establishing a mutual carbon-versus-nitrogen compromise. Nonsymbiotic plant families can take advantageof ecosystem comor succeeding them in time. plexity when sharing space with symbiotic ones,
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Nevertheless, cereal plants can interact with endosymbionts, capable of nitrogen fixation in other species, and be stimulatedin their productivity. The odds of soil life are balanced for some bacteria by their interactivity at rhizosphere level, and a realm of exchanged signals dictates entry into hormonally reprogrammed root sites. Specificity for partner plant species is part of a fine speciation process that actively involves the bacterial nodulation genes, and continuesto drive their variation dynamics. The evolutionary history of symbiotic nitrogen fixers is therefore a tale of coevolution, which occurred in the shadow of their hosts, chasing their growing roots, and striving for adaptation. It is an example of how bacterial genetics has managed to keep pace with the creative power of eukaryotic sexual recombination. Mobile replicons, insertion elements, and symbiotic islands prone to move have helped rhizobia to succeed in their pursuit. The race, naturally, is not over and, looking at it from a distance, what we have seen, comparedto what we have yet to see, is probably just a cloud of dust.
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D. Tepfer, A. Goldlnann. N. Pamboukdjan. M. Maille. A. Lepingle, D. Chevalier. J. Denari6. and C. Rosenberg. A plasmid of Rhizohitrrrl rmliloti 4 I encodes catabolism of two compounds from root exudate of C d y ~ e ~ q i serepiwtz. cr J . Rttctc,rio/. 170: 1153-1 161 (1988).
90. J. P. Grime, J. G. Hodgson.R.Hunt,
Tlre Ahritl,ycd Cf)/?l/?c/rt/filJC Plttrrt Ecology,
Chapman and H;dl London, 1990. 91. Y. Moenne-Loccoz, D. Sen. E. S. Krause, and R. W. Weaver, Plasmid profiles of
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92.
93. 94.
95.
96.
97. 98. 99.
Squartini
rhkobia used in inoculants and isolated from clover fields. Agron. J. K6: 117-121 ( 1994). H. A. Moawad, W. R. Ellis,and E. L.Schmidt,Rhizosphereresponse a s a factor i n competition among three serogroups of indigenous R h i x ) / h mjaponicurn for nodulation of field-grown soybean. Appl. Blvirorl. Micrnbiol. 47: 607-61 2 (1984). D. H. Demezas and P. J. Bottomley, Influence of soil and nonsoil environments on nodulation by Rltizobiunt trifolii. Appl. Enviror~.Microbiol. 53: 596-597 (1987). E. A. Robleto, A. J. Scupham, and E. W. Triplett, Trifolitoxin production in Rllixlhiurn et/i strain CE3, increases competitiveness for rhizosphere colonization and root nodulation of phaseolus vulgaris in soil. Mol. Plont Microbe httertrt. 10: 228-233 ( 1997). D. Paffetti, C. Scotti, S. Gnocchi, S. Fancelli, and M. Bazzicalupo, Genetic diversity of an italian Rhizobium rneliloti population from differentMedicago sativtr varieties. A/>/>/.Ernjirort. Microbiol. 62: 2279-2285 (1996). D. Paffetti, F. Dauguin, S. Fancelli, S. Gnocchi, F. Lippi, C. Scotti, and M. Bazzicalupo, Influence of plant genotyped on the selection of nodulating Sir~orhizobiurn rtteliloti strains by Metiicccgo sativu. Antorlie Vtul Leedwc)rlhoek 73: 3-8 (1998). B.Handley, A. J. Hedges,and J. E. Beringer,Importanceofthehostplants for detecting the population diversity ofRhi:obirtrn legrtrlliflostrncrrl biovar viciae in soil. Soil Rio/. Biochern. 3 0 241 -249 (1988). J. P. W. Young and K. E. Haukka, Diversity and phylogeny ofrhizobia. NOWPh)’fo/. 133: 87-94 (1996). B.L.Maidak, N. Larsen,M. J. McCaughey,R.Overbeek, G. J. Olsen, K . Fogel. J. Blandy, C. R. Woese, The Ribosomal Database Project. N d c i c Acids RCS. 22: 3485-3487 (1994).
11 Modeling the Rhizosphere Peter R. Darrah and Tiina Roose University of Oxford, Oxford, England
1.
INTRODUCTION
The rhizosphere is a highly complex environment where almost every process involves multiple interactions between the soil-root-microbe triumvirate, as other chapters in this volume document. Understanding rhizospheric processes is therefore extremely challenging. especially as many of the questions to be studied require quantitative answers. For example, the rate of nitrogen influx into the root system is an important determinant of plant productivity, and yet explaining why this flux differs between different experimental treatments requires a very detailed knowledge of plant physiology, soil physics, chemistry, and biology as it relates to the N cycle (1). The value of mathematical models is that they do generate quantitative answers and allow the integration of many individual plant and soil processes to predict a single outcome. However, such models require parameters to run the simulations and the experiments to measure them may be extremely difficult to do. of the rhizosphere are gradiThe most striking, indeed the defining, feature ents of solute concentration extending from the root surface intothe surrounding soil. The zone encompassed by this gradient is, in modeling terms, the rhizosphere, and it defines the volume of soil that is influenced by the root. This is in stark contrast to the practical definition of the rhizosphere used elsewhere in thevolume-i.e.,thesoil adhering to gentlyshakenroots. To talk about the rhizosphere is rather meaningless, as each solute under consideration will have a different gradient encompassing a different soil volume, and strictly the rhizosphere is the totality of all the solute gradients surrounding a root. Thus there will be gradients of ammonium, nitrate, phosphorous, and each of the other 13 327
Darrah and Roose
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essential mineral nutrients (MN) required by higher plants, and most of these gradients will be depletion profiles (i.e., the solute concentrations will be lowest at the root surface). There will also be gradients of the 200 or so soluble organic solutes that roots release into the soil (2); these will typically be accumulation gradients (i.e., concentrationsof each solute are highest at the root surface). There may also be gradientsin volatile compoundsin the gaseous phase (e.g.,O? depletionprofiles and CO? accumulation profiles due to root respiration as well as gradients of compounds such as ethylene and other volatile organic compounds). The spatial scale over which these gradients are formed varies tremendously, from a few micrometers to perhaps meters, but the typical scale for most soluble inorganic and organic compounds would be the millimeter scale. A typical idealized root rhizosphere is shown in Fig. I , which illustrates how much the environment of a rhizosphere bacterium dependson its spatial locationin the rhizosphere. The existence of cylindrical multiple gradients in very close proximity to root surfaces in part explains the extraordinary difficulty of studying the rhizosphere.Ratherfewexperimentaltechniquesareappropriate for sampling soil with sufficient precision to explore these gradients, and many sampling strategies simply average the gradients and compare these averaged concentrations to those o f bulk soil (see Chaps. 6 and 12). Yet in doing so, most of the detail that drives
Glucosc
Amino acid Citric acid
0
SO
IO0
1'
Figure 1 Diagrammatic representation of thc nondimensional profilcs of mineral nutrients and organic compounds in thc soil surrounding a root. Note that thc width of the rhizosphere (arbitrary units) differs for different solutes.
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rhizospheric processes is inevitably lost. Where appropriate techniques are used, gradients around roots have been demonstrated (Fig. 2). The rhizosphere defines the soil volume from which plants extract their mineral nutrient requirements. It also defines the zone where plant-microbial interactions occur. Eachunit length of root has its own unique, dynamic rhizosphere gradients, its own unique flux of nutrients into and out of the root and its own unique microbial interactions. The whole plant integrates all these unique fluxes into a global nutrient acquisition rate and adjusts its growth rate, physiology, and allocation patterns accordingly (3). Adjustments may involve major changes such as a new root architectural form or increased root turnover or may be subtler, involving an increase in uptake capacity at the local rhizosphere scale (see below). However such adjustments are likely to have implications at the rhizosphere
tU
12
l.l~lO-~
Herrenhausen u S
L
1.5~10-~
Briinddn 0
1
0.l
0.2 0.3 0.4 Distance from root, cm
I
0.5
Figure 2 Gradients in Rb around a maize root as visualized by autoradiography. The depletion of Rb depends on the diffusion coefficient (D,) which in turn depends on the soiltexture. (From Ref. 12.)
and
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level as whole plant adjustments feedback to the activities of individual roots. Anymathematicalconsideration of the rhizospheremustthereforerecognize these two spatial scales: the root scale and the plant scale; and consider how they are interconnected. Ultimately, mathematical models of the rhizosphere will have to consider even largerscales-e.g., field, community, landscape scales; thefinal section of this contribution deals with these aspects.
II.
MODELING SHORT-RANGE TRANSPORT AND UPTAKE IN SOILS
What processes drive the formation of solute gradients around roots? Why does the rhizosphere exist? It exists for two main reasons: ( I ) Soil is a heterogeneous medium consisting of a solid phase interspersed with a pore space that is filled withbothair and water. Convective mixing of boththeairand water phases hardly occurs and both these phases are effectively stationary in an undisturbed soil. Any concentration imbalances in the mobile phases are therefore dependent largely on diffusion for their resolution. (2) Roots are active biological organs and remove some solutes and deposit others frotdinto the soil at their surfaces. Such activities generate concentration imbalances. Rhizosphere modeling therefore simply consists of identifying and mathematically describing two typesof processes: those responsible for perturbing soil solution equilibria, predominantly the biological activitiesof plant roots and soil microorganisms; and processes responsible for restoring soil solution equilibria, predominantly physical and chemical processes involving transport and chemical phase reactions. Mathematical modeling of these two processcs led to the development of the tnodel (4) that forms the basis of most mechanistic models of nutrient uptake from soil.
A.
Diffusion of Solutes
The developtnent of the theory of solute diffusion i n soils was largely due to the work of Nye and his coworkers in the late sixties and early seventies, culnlinating in their essential reference work( 5 ) .They adapted the Fickian diffusion equations to describe diffusion i n a heterogeneous porous medium. Fick’s law describes the relationship between the flux of a solute (mass per unit surface area per Unit time, and the concentration gradient driving the flux. I n vector terms, .lI,)
J,,
-D,VC,
(1)
where the proportionality constant, DL, is the diffusion coefficient in pure water at the ambient temperature and pressure and C , is the concentration of the SolUte
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in solution. For a soluble compound diffusing in soil, three additional factors must be taken into account (S): first that the cross-sectional area available for diffusionisreducedbecausethesolutecan only move in thesolutionphase. Second that the distance that the compound must travel in moving from a point .xI to x ? is not simply the difference between xI and x?. In an unsaturated soil, an individual solute molecule must follow a tortuous path through the moisturefilms around the soil particles, which increases the effective path length between x l and x ? . This introduces a tortuosity or impedance factor. And third, that diffusive movement is only significant in the solution phase and only concentration gradients in the soil solution drive diffusive movement. To account for these factors, all of which act to reduce the rate of diffusion in soil compared to water, the solution diffusion coefficientmust be modified (5) to represent the effective diffusion coefficient in soil:
J
=
-DLBfVC/.
(2)
where 8 is the volumetric moisture content and ,f defines an empirically determined impedance o r tortuosityfactorwhichaccountsfortheincreased pathlength. Cl,defines the soil solution concentrationof the solute: some solutes occur entirely or predominantly in the soil solution. This would include most anions in most soils and uncharged organic molecules such as glucose. However, many solutes display positive charge and will interact electrostatically with the negatively charged surfaces found in most soils. Others, such as phosphate. may interact with surfaces via ligand exchange mechanisms. Such inner and outer sphere surface complexes (6), jointly termed eschrrngectblr solutes, do not contribute directly to diffusional flux and are not considered to be part of soil solution. In the simplest case, a linear relationship may exist between solutesin soil solution and those on the solid phase, such that C, = hC,,, where C, is the solute concentration on the solid phase and b defines the buffer power. In perhaps more typicalsituations.therelationshipmightbedefinedbyamorecomplicated, concentration-dependent relationship such as a Freundlich or Langmuir expression: here the instantaneous buffer power may be defined as the tangent to the exchange isotherm i.e., h = rlC,/dCL.Some care is needed when defining this buffer power (7). By convention. m w i l c h l e solutes are defined as those that are in, or can equilibrate with, soil solution on a timc scale defined in minutes; thisis essentially an arbitrary definition but i t is intended to encompass solution and exchangeable ions and to exclude every other solute form. Thetotal concentration, C, of available ions in a given soil volume is then
c = c, + W,= (e + W,,
(3)
Fick’s law, as modified to describe the diffusion of available (diffusible) solutes in soil and written in Cartesian coordinates is therefore
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The effective diffusion coefficient of a solute in soil is thus
and it should be noted that this parameter may be concentration dependent over the range of concentrations found in the rhizosphere via the buffer power term,h. To describe the diffusionof solutes in the rhizosphere, where concentration gradients change with time, t , aswellas space, mass conservation is invoked with the spatial geometry appropriate for the cylindrical root (8):
Here the spatial coordinate, r , defines the radial position in the cylinder of soil surrounding the root: the root surface is at r,,. This parabolic, second orderpartial differential equation describes how the concentration of available solute at an arbitrary point in the rhizosphere changes with time in response to the concentrationsoneitherside of it. Note thatthis equation is stilleffectivelyonedimensional because concentration gradients along the root lengthcan be neglected on the basis that roots are long and thin and concentrations around the root are assumed to be radially symmetrical. If concentration gradients along the root are thought to be important, then two-dimensional equations and solutions are required (9).
B. Convective Transport As water moves through the soil pores in response to water potential gradients, it moves with it the solutes dissolved in soil solution. In a rhizosphere context, water moves radially toward the root to replace water taken up by the roots for transpiration. The flux of solute dueto water movement ( J , v )is simply the product of the rate of water flow at that point and the concentration in soil solution:
In a cylindrical system, because equal amounts of water are being transported across successive concentric radial increments, the rate of flow of water must vary across the rhizosphere, with the fastest flows at the root surface. It is
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conventional to use a single parameter for rhizosphere water flow, v , l , which is defined as the water flux (volume per unit area per unit time) at the root surface, I’~~.
C.
DiffusionandConvection
If convection is included as a transport process comes
in the rhizosphere, Eq. (6) be-
D h/Pe or h < Pe. Inspection of the tableof standard parameter values (Table 2) showsthat this isnever the case for realistic soil and root conditions. Inspection of Table 2 also reveals that the term relating to nutrient efflux, E, can also be ignored because E < Pe > I , C, >> 1 , etc.) and found a solution that was valid for all values of h and C, and at all times larger than the natural diffusive timescale which typically ranges from a few seconds to a few hours (Table 2). Subsequently, a more direct solution for the nutrient flux at the root surface was derived (39), again valid for all values of h and C,, which is given below:
This equation gives the instantaneousflux where y is Euler's constant (~0.5772). into unit surface area of plant root at any time point in the simulation period.
and
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Table 2 Standard Parameter Values for the Uptake of Mineral Nutrients by Maize'
P Mg
5.0 0.046 0. I 2.9 X 1 0 1 . 0 x IO
cil
0.8 x I O
NO,-N K S
I
39
'
2 239 1.2 I S6
I .o x 10 3.0 x 1 0 "
3.0 x 10-7 3.26 X
IO-"
4.0 x 1 0 1.0
x
'I
10-6
0.02s 1.4 x 1 0 1.0 x 1 0 S.8 X I O
s7x 17.467
0.04 14
24
0.15
1,020 106,355 666
4.0
69.466
21
2
'
0.06
88
"The soil i s representative of a silt Ioanl. Par;umeters arc delined In the text. V;~lucsconmon to all MN arc 8 = 0 . 3 . f = 0.3. D , = 1 . 0 x
The cumulative uptake can easily be calculated by numerical integration of Eq. (29). Comparison of the numerical and analytical solutions for a range of nutrient parameters showed very close agreement, indicatingthat the asymptotic approximations were valid. An important advantage of the analytical approach is that is allows more sophisticated sensitivity analyses to be carried out. A traditional sensitivity analysis (e.g., Ref. 22) shows how step changes in a single model parameter changes the cumulative uptake into the plant after a certain time. A typical plot is shown for P in Fig. S and reveals that uptake is most sensitive to changes in root radius with changes in E and v ( , having a negligible effect (as predicted above). However, these typesof analyses can only present a single snapshotof the importance of the parameters at a particular time. They may also be slightly misleading in that they imply that the response is linear over the interval where the parameter values are halved or doubled: there is no reason to believe that the responses are in fact linear. Figure 6 shows sensitivity analyses of flux ( F ) of P into the root over time for similar parameter values asFig. S but calculated for infinitely small parameter changes (e.g., dF/dcr, where (I is any given parameter). If the value at a particular time is negative, then this indicates that the flux decreases as the parameter value increases while conversely a positive value indicates that the flux increases as the parameter increases. The figure illustrates how dynamically root different parameters affect uptake during the uptakeperiod.Initially,the radius has a very strong influence on uptake, but this effect declines as the s o h tion concentration at the root surface falls close to zero. Conversely, factors affecting the diffusion coefficient (€If) become more important over time as uptake becomes increasingly controlled by transport processes. Km is seen to have a
345
Modeling the Rhizosphere
k
Raub soil
V'
0.5
1'
1.o
1.6
2.0
CHANGE RATIO
Figure 5 A sensitivity analysis for the Barber-Cushman model for the uptake of P by maize in Raub soil. The sensitivity was analysed by halving and doubling each parameter value in turn while keeping all other parameters at their standard values. (From Ref. 10.3.)
transitory effect on flux during the period that soil solution concentrations are relatively high at the root surface.
of P
3. Other AnalyticalApproximations One of the earliest approximations studied is to assume that the solute concentration is small or rapidly becomes small in comparison to the affinity constant for uptake. This then allows the nonlinear Michaelis-Menten equation to be approximated by:
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CO
~ _ _ _
K
Theta,f a b
Fm I
10
Km
Figure 6 Sensitivity analysisof maize seedlings to some model parameter values during the first I O days of uptake. The curves show how phosphorus flux (F) into the roots responds to differential perturbation to the parameters, a (i.e., 6F/6a). (Model parameters are given in Table 1.)
which is linear in the concentration term and can be solved analytically. The ratio, I,,,,,,lK,,, was termed the root absorbing power ( 5 ) , who also discussed the implications of this simplification for the accuracy of prediction. Another commonly used approximation for which there is an analytical solution is to assume that the root acts as a zero sink for uptake. Here the solute concentration at the root surface is taken to be zero and uptake is therefore completely controlled by the diffusive flux to the root (21,40,41). The implicit assumption is that root uptake is very rapid in comparison to resupply by transport and hence the root very rapidly depletes the solute concentration at the root surface to zero and maintains it there. The validity of this assumption depends on the value of h and it is inapplicable unless h is greater than or about I O (38). For such large h, there is a nondimensional critical time (t,) after which it is reasonable to assume a zero sink (38,42). Approximate values of t , are
or
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Using the standard parameter values for maize presented in Table 2, the dimensional critical times after which a zero-sink approximation is valid are 180 S for P and 89 S for K. The zero sink assumption is not valid at all for NO3- N ( h = 0.04). Such an analysis indicates that the zero-sink assumption must be used with extreme caution if accurate flux calculations are required at the local root level. Potassium, for example, is close to the limiting value of h for the zero sink assumption to be fulfilled, and simulationswith larger roots or larger buffer powers could well lead to inaccurate simulation results. Any zero-sink model involving nitrate should be treated with some suspicion. The zero-sink assumption is also widely used in root architecture models (see later).
G. Validation of the Local-Scale Model of Nutrient Uptake The measurement of spatial gradients around roots at a resolution sufficient to provide an acceptable test of model predictions is very challenging and few studies have attempted it. Figure 7 shows the measured and predicted depletion of K for 4-day-old rape seedlings grown as a planar mat in contact with a column
0
0
0.4 0.6 0.8 Distance from roots, cm 0.2
1 1.0
Figure 7 Comparison of experimental and modeled potassium depletion in the soil close to a planar mat of rape roots for three soil Kt levels. The modeled lines were calculated using the Barber-Cushman model. (From Ref. 104.)
Darrah and Roose
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of soil. This method, although not representative ofrhizosphere geometry, at least allows adequate spatial resolution to be obtained by sectioning the soil column. An alternative method for nutrients with radioisotopes is to scan the film density of autoradiographs. Figure 2 shows the results obtained from a cross section of a maize root growing in a sandy soil with "Rb. These and other published studies (reviewed in Refs. 5 and 12) generally show good agreement between observed and predicted gradients; however, only rarely have such approaches been used to validate local scale and plant scale models in the same experiment.
111.
MODELING THE DYNAMICS OF MICROBIAL POPULATIONS AROUND THE ROOT
The rhizosphere was first defined (43) as a zone of increased proliferation of nitrogen-fixing bacteria associated with the roots of legumes. This definition was later widened to encompass increased proliferation of any micro-organisms in the soil around any living plant root (44). Many scientists still reserve the term rhizosphere for this microbiological phenomenon. Carbon from root exudates, sloughed root cells, and other root debris is thought to fuel this enhanced microbial growth, although it is possible that some species may respond to the altered mineral nutrient, 0 2or , COz concentrations or to other perturbations in the physicochemical environment surrounding the root. So far, modelers have concentrated on describing the microbial response in terms of a microbial biomass response rather than a species-specific response: this mostly reflects the uncertainty about the niche requirements and the identities and competitive abilities of the vast majority of microbes that may inhabit the rhizosphere. The exception to this rule is where the population dynamics of root pathogens have been modeled (45,46). Newman and Watson (47)first applied mathematical modeling to thep o p lation dynamics of rhizosphere microorganisms growing on root exudates. Later their equations were modified (9,48) to account for microbial death, C recycling, and the deposition of insoluble C. The basis of these models is the description of microbial growth using classic bacterial growth equations coupled with transport equations describing how microbial growth substrates are transported from the root surface through the soil. Microbial growth is generally described in terms of biomass carbon, and it is assumed that a single specific growth rate can describe the response of all the microbial species in the rhizosphere (49). The specific growth rate of total soil biomass, p (h") is given by (48):
p =P
lllilI
~
C
+ K, -
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349
where C is the concentration of carbon growth substrate in soil, M(, and M are the initial and current microbial biomass, U,,:,, is the maximum specific growth rate and K , is the Monod affinity constant. Some carbon substrate is required for maintenance of the existing biomass, where Z is the maintenance requirement and Y is the growth yield. Any biomass dying because of insufficient substrate (i.e., p < 0) is assumed to enter a necromass pool, which can be decomposed to recycle carbon for further growth. The growth of microbial biomass is then described by -
pM
"
at
(33)
Therefore if the carbon substrateis present at sufficiently high concentration anyp = p,,,,), the microbial biomass will increase where in the rhizosphere (i.e., exponentially. Most models have considered the microbes to be immobile and so Eq. (33) can be solved independently for each position intherhizosphere provided the substrate concentration is known. This,in turn, is simulated by treating substrate-carbon as the diffusing solute in Eq. (32). The substrate consumption by microorganisms is considered as a sink term in the diffusion equation, Eq. (8). The outer boundary condition is that given in Eq. 1 I . The inner boundary condition must describe the flux of carbon from the root into soil as a function of time. In the earlier models, this flux was either treated as time invariant or assumed to be maximal at the root tip and decline linearly over time corresponding to a reduction incarbon efflux with root age (47,48). Solutionof these models revealed that the population dynamics of microbial biomass around roots was surprisingly complex (Fig. 8). In the early stages of rhizosphere formation, the influence of the root extended several millimeters from the surface. As the root matured, the extent of the rhizosphere influence diminished as the population closer to the root consumed all the root exudation, hence depriving outer shells of a source of substrate. However, the intensity of the rhizosphere effect close to the root increased enormously, with the ratio of M:M,, reaching a peak of 14 (47). The model revealed that the time course of root exudation had profound consequences for rhizosphere population dynamics. In Fig. 8A, the root was asIO-day period while in Figure 8B, sumed to exude C at a constant rate over a the same amount of C was exuded but all within a l-day period. In turn, these different patterns of biomass proliferation could have important impacts on root functioning: for example(3), roots in Fe-deficient soils release phytosiderophores from near the root tip to chelate and transport Fe. These organic compounds can also serve as carbon substrates for rhizosphere microorganisms: the impact of microbial consumption on Fe nutrition will depend on the size and speed of response of the microbial community around the root tip ( S O ) . These simulations
350
Darrah and Roose
53
54
a C
J Figure 8 The simulated distribution of (a) soluble carbon and (b) microbial biomass in the rhizosphere of maizeover a period of 10 days. The X axis represents distance (cm) from the root surface and the Y axis represents time (h). A uniform exudation rate (S3) is compared to the situation where the same amount of exudate is released in the first 24 h (S4). Note that the Z axes have different scales although both represent pg C cm-3. (From Ref. 48.)
suggest that the spatial and temporal patterns of exudation could have a major effect on root acquisition efficiency. More recently, it was realized that rhizosphere carbon dynamicsare more complex than these early models envisaged. Jones and Darrah (51-53) showed that roots actively scavenge their root exudates and that the re-uptake of exudates was selective. In most situations, sugars and amino acids were scavenged by roots, while organic acid exudates were not (54). The authors also found that exudation losses were largely passive. This active involvement of the root essen-
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351
tiallyinvolvessubstrate-specificcompetitionbetweentherootandtherhizosphere biomass. This more complex situation was modeled using the root boundary condition ( S S ) :
(34) where C,,, and Cl. are the exudate-carbon concentrations in the root cytoplasm P describes the permeability of theroot to and soil solution respectively and exudates. The second term describes the reuptake of C in terms of MichaelisMenten kinetics. It was found that, in sterile soil, exudation losses declined over time and eventually reached a pseudo-steady state where the reuptake of carbon matched the efflux. When microorganisms were included in the simulation model, total carbon losses increased due to microbial consumption, but roots were nevertheless surprisingly good competitors for their exuded carbon, being capable of reacquiring about 50% of the their exudation losses. A sensitivity analysis of the model (Fig. 9) revealed that the exudate loss was most dependent on the factors controlling passive loss and active reuptake-i.e., C,,, and V,,,,,, respectively. Exudation was surprisingly insensitive to the growth rate of microorganisms, implying that the rate of microbial accumulation had little effect on efflux. These and similar findings have revealed that roots are capableof exerting subtle effects on the carbon spectrum that are made available in the soil. This may then mean that the microbial composition of the rhizosphere is to some extent controlled by the root. The importance of including soil-based parameters in rhizosphere simulations has been emphasized (56). Scott et al. used a time-dependent exudation boundary condition and a layer model to predict how introduced bacteria would colonize the root environment from a seed-based inoculum. They explicitly included pore size distribution and matric potential as determinants of microbial growth rate and diffusion potential. Their simulations showedthat the total number of bacteria in the rhizosphere and their vertical colonization were sensitive to the matric potential of the soil. Soil structure and pore size distribution was also predicted tobe a key determinant of the competitive successof a genetically modified microorganismintroducedintosoil (57). The Scott (56) model also demonstrated thatthediffusive movement of root exudates was an important factor in determining microbial abundance. Results from models that ignore the spatial nature of the rhizosphere and treat exudate concentration as a spatially averaged parameter (14) should therefore be treated with some caution. The recent report of wave-like patternsof bacteria and water soluble carbon associated with wheat roots (58) which were not strongly correlated with each other orwith lateral root formation pose a challenge for models. So do the reports of highly dynamic and erratic population fluctuations of individual pseudomonad clones on sugar beet roots (59).
352
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Q
l
2
Change mu0
Figure 9 A sensitivity analysis for exudation losses by maize roots. The sensitivity was analyzed by halving and doubling each parameter value in turn while keeping all other paranleters at their standard values. Parameters shown on the graph are defined in the text. (From Ref. 55.)
IV. MODELING UPTAKE AT THE PLANT SCALE The objective of the local-scale simulation of uptake into roots is normally to allow prediction of nutrient acquisition by the whole plant. This is true even where the objective is to validate the local-scale model because of the difficulties of measuring experimentally the cylindrical gradients around plant roots to the necessary precision and resolution to validate the local-scale model adequately. Typically the models are applied to growing plants with continually expanding root systems. Nutrient acquisitionat the plant scale is then achieved by integrating root uptake at the local scale and root growth. Barber ( 2 2 ) reviewed the general approach. He made the following assumptions about the growth of maize root systems:
I.
Rootsgrewexponentiallyovertheexperimentalperiod.Thegrowth rate of the roots (g) was calculated as In(L, - L l , ) / ( t ,- tl,), where L,
Modeling
2.
353
denotes the total root length at the end of the experiment at t , and Lo the initial root length at the start of the experiment at fo. In a typical experiment, to would occur 6 days after germination and t , some 2-3 weeks later. A single average root radius could approximate the radius of the roots for the entire experimental period. T o calculate this radius, the total root length and the total root weight at harvest were measured. The latter was converted into a root volume, V,, and the equation below applied:
The cumulative uptakeinto whole plants ( N , ) could then be calculated as a function of time using the equation:
which integrates the flux into the root, J L (expressed on a unit length basis), by anexponentiallygrowingrootsystemovertime, T. The main feature ofthis model is the exponential growth rate of the root system with a constant coefficient, g, and it is therefore hereafter referred to as the uptake with convection and diffusive model with constant growth (UDC-GC) model. This equation has beenused,mainly by the original authors to compare predicted and observed uptake by a range of plant species. A typical outcome for maize seedlings up to 22 days old is shown in Fig. IOA. Generally, these models have shown good correlation between observed and predicted uptakes in both pot and field experiments. It is true that the imposition of a constant growth rate forces the model to accumulate nutrient and therefore some degree of correlation between observed and predicted uptakemust be expected if intermediate harvest data is used. Moreover, examination of the regression coefficients often indicates that there is substantial under- or overprediction of plant uptake indicating a systematic error in the formulation of the models. Without an independent test of the local-scale model, it is impossible to be sure whether this systematic bias derives from the local-scale or from the plant-scale as both scales make assumptions that may not be appropriate.
A.
The Assumption of Constant Root Radius
One obvious deficiency of the treatment of root growth in the UDC-GC model is the assumptionof a constant root radius. Root systems consist of several orders
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Darrah and Roose
Observed P uptake @mol)
Figure 10 Relationship between observed and predicted P uptake by two tomato cultivars grown in soil of differing P supply. Uptake was predicted by the original BarhcrCushman model (a) or by the UDC-GV model (h). From Darrah’s (Backhuys Publishers).
of root, with each order branchingoff the parent root and having a smaller diameter. Therefore onewould predict that the average root radius would decrease over time as the root system consisted of proportionately more higher-order roots. Becauselocal-scaleuptake of nutrientsishighlysensitive to rootradius(see Fig. S ) , the average root radius simplification might be expected to be rather inaccurate.
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Recent developments have led to the formulation of mathematical models capable of describing the three-dimensional architecture of plant roots growing i n soil (60-64). Most of the models operate by applying setsof rules-e.g., which define the distance between an existing lateral branch and a new branch or the branching angle and orientation for each root order-or by imposing the rules of fractal geometry. Most are also capable of visually displaying the spatial distribution of the evolving root system (see Fig. I I ) . The application of different branching parameters leadsto the development of different root architectures that are particularly efficient for the exploitation of large soil volumes (herringbone architecture) or for the intensive exploitation of small soil volumes (dichotomous architecture). Model simulations (65) suggest that the herringbone topology is efficient for acquiring rather mobile nutrients such as nitrate, while the much more immobile nutrients like P are most efficiently exploited by a dichotomous form. Here efficiency is measured by comparing the carbon cost of construction to the amount of nutrient acquired. Virtual root systems can now be inspected on line (65) and the general field of morphological modeling of plants was recently reviewed (66). Architectural models explicitly specify the distribution of roots in space. An alternative approach. which is also useful for rhizosphere studies, is the continuum approach where only the amountof roots per unit soil volumeis specified. Rules are defined that specify how roots propagate in the vertical and horizontal dimensions, and root propagation is usually viewed as a diffusive phenomenon (i.e., root proliferation favors unexploited soil). This defines the exploitation intensity per unit volume of soil and, under the assumption of even distribution, providesthenecessaryinformationfor theintegrationstepabove.Acockand Pachepsky (68) provide an excellent review of the different assumptions made i n thevariouscontinuummodelsformulatedandshowhowsuchmodelscan explain root distribution data relating to chrysanthemum. In one of the more recent continuum models (69). an approach first suggested by Leonard0 da Vinci in the fifteenth century, from his observations that the cross-sectional area (CSA) of the main stem of a tree equaled the total crosssectional area o f all the tree branches, was used. The more generalized rule was considered:
d” =
a((/:+ c/$)
(37)
where d , and (l2represent diameter values before and after branching respectively, A is the diameter exponent and a is a proportionality factor, which allows for unequal branch diameters to be formed. By assuming that each segment of root had to attain a minimum length before branching could occur and assuming that therewasaminimumrootdiameterbelowwhichnofurtherbranchingcould occur. a computer program was developedthat could produce very realistic computer representations of real roots (69).
Roose 356
a
and
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b
B
Figure 11 Root systems as simulated by the computer program Simroot. The systems display herringbone architecture(a); dichotomous architecture (b); and bean root architecture (c). The bean root system (c) is shown in (d) with simulated phosphorus depletion zones around the root system. (From Ref. 105.)
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Maize roots typically have three to four orders, with zero-order roots having the largest diameters and being formed from the seed and shoot (61). Each order can be divided into three regions: a basal nonbranching region ( l b ) , a branched zone ( I ) , and an apical nonbranching zone ( l ( , ) . New branches can form on this root if l > l,, l , and these branches arise at regular intervals defined by the internodaldistance, l,,. Hence a root of length l hasamaximumnumber of branches given by [(l - l,, - /,,)/l,,].The lengths l,,, l/,, and l,, depend primarily onthe branch order but could also depend on age, nutritional status or other physiological or environmental factors. Roose et al. (38) extended an existing theory of variable root elongation (61) and constructed a model describing the length and time development of the populations of roots of different orders in the form of a hyperbolic partial differential equation. Each root order was also considered to have a maximum attainable length and the elongation ratesof roots were assumed to decline as this length was approached. The investigators could then calculate the average root radius as a function of time during the development of a maize seedling and compare these valueswith the fixed, average value used in the classical approach above. The results are shown in Fig. 12, which
+
0.05 -
0.04 ~-
0.03.-
a
0.02
-I
l
Figure 12 The predicted changes in average root radius of maizc root systems during 20 days of growth. The subscripts on the curves denote differing ways of calculating thc average (by volume. by surface area or by root number). (From Ref. 38.)
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demonstrates the highly dynamic nature of root radius during the early development of seedlings, where the volume-averaged root radius halves in magnitude during the first few weeks of growth. The changes suggest that the approach of estimating root radiusas a single fixed point calculated from theroot size distribution at the end of the experiment is likely to yield inaccurate cumulative flux estimates. Once the root size distribution had been successfully modeled, the total uptake by the plant could then be predicted (38) by integrating the flux equations for each root order separately and then summing the contributions of each root order. Figure 13 shows the relative contributionsof each root order to total uptake over a 20-day period. Initially all uptake is by growing zero-order roots, which do not reach their maximum lengths until about 50 days. From around day 6 , first-order roots start to be produced, and these are responsible for the increase in uptake observed at this time. The simulations shown are for phosphate uptake, where it is possible to assume that interroot competition between individual roots in the time scale is unimportant. Second-order roots are relatively unimportant simulated but would start to have a major impacton uptake at a later stage. These simulations can also be compared withthe “classic” UDC-GC approach, and these simulations are shown in Fig. 14: it is obvious that the Barber-Cushman method of radius averaging significantly underestimates the uptake.
,Total
Order 1 Order 0
Figure 13 The predicted contribution of roots of different order to the uptake of phosphorus by maize over a 20 day period. Parameter values are given in Table 2.
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50’
40
lot
/
Figure 14 The predicteduptake of phosphorus by maizeovera 22 dayperiod. The Barber-Cushmanmodel (Fh),usesanaverage root radiuscalculated at the end o f the simulation period. while the F,,, model uses a population of roots of differentradius. Identical parameter values were used byboth simulation models with the exception of root radius.
B. The Assumption of Exponential Root Growth with a Constant Coefficient The local-scalenlodelpredictsnutrientuptake by plantsasafunction of the nutrient concentration in the rhizosphere: it should be equally accurate for both high and low nutrient concentrations provided that the buffer capacity term has been described accurately. It has indeed been used to validate the uptake model for a range of soil types with wide ranges of nutrient availability (22). However, this introduces a complication at the plant scale.If the rates of uptake at the plant scale are low, it might be reasonable to expect that the rate of plant growth might also decline as a consequence of nutrientdeficiency. In fact, in anysituation where the yield potential of the plant is constrained by a limiting nutrient which is itself the nutrient being simulated, the assumption of constant exponential root growth seems a priori to be unacceptable. In these nutrient-limited plants grown from seed, one might instead predict a gradual decline in plant (and root) growth with time as the seed reserves become diluted by growth and the uptake mechanisms cannot keep pace with the MN demands of growth. The lack of feedback
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mechanismsbetweencumulativenutrientacquisitionandsubsequentgrowth seems to be one of the main deficiencies of the classic approach. Inclusion of feedbackbetweenuptakeandgrowthobviouslyrequires knowledge of the relationship between MN content and growth, and such relationships form the basis of many whole-plant growth models (70). Whole-plant growth models typically relate above-ground parameters-such a photosynthetically active radiation, humidity, leaf area, and net assimilation rate-to predict rates of carbon fixation. Their below-ground submodels are usually empirically based and “predict” water and MN uptake. When combined with developmentally controlled descriptions of resource allocation and ontogeny, these models aim to predict biomass or grain yield. Such models are typically management tools, used for guidance on when and where to apply fertilizer or irrigation, and most are “models without roots.’’ Such empirically based models are of limited use in the currentcontext. Other models (7 I ) specify the nutrientflux as a parameter and then model the allocation of this resource and photosynthate within the plant: there is obviously considerable scope for amalgamating this approach with the mechanistic modeling of nutrient uptake. One approach that has been used (28) is totry to assess the potential importance of feedback while keeping the description of the whole plant model as simple as possible. The relative addition rate (RAR) technique is an experimental procedure(72) thatallowsplantstobemaintainedataconstantexponential growth rate in nutrient-limited conditions. Thisis achieved by supplying nutrients at an exponentially increasing rate such that the supply of nutrients is always just sufficient to maintain a constant tissue concentration of this growth-limiting nutrient. If the tissue concentration departs from this constant value, then the plant growth rate changes. In a series of papers (see Ref. 73 for review) Ingestad and ¥ derived a linear relationship between internal nutrient concentration and relative growth rate, or RAR, extending from zero growth at some threshold internal concentration to the maximum specific growth rate at a concentration termed optimal. Above this optimal internal concentration, plants grew atthe maximum specific growth rate. This then gives the simplest possible relationship between nutrient uptake and subsequent plant growth (Fig.15). The only modification required to solve the equations is to allow the growth rate, g, to depend on internal nutrient concentration in Eq. (35). This approach was used to reanalyze some data (74) on the growth of young tomatoes in soils of differing P status. These data had previously been analyzed using the UDC-GC approach, with a fixed growth rate calculated from theroot system at the end of the experimental period. Figure 16 shows the local-scale predicted P uptake for the two tomato cultivars in the P-sufficient and P-deficient soils and shows that the deficient soil is capable of delivering P to the plant at a much lower rate than the sufficient soil. Therefore, unless the plants are indulging in “luxury” uptake in both cases, one would expect the growth rates of the plants in the different condi-
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I RGRrnax .""""""
Cnmin Cnopt Internal nutrient concentration (Cn)
Figure 15 Therelationshipbetweenrelativegrowthrate (RGR)andinternalnutrient conccntration (Cn). Growth ceases at non-zero Cn (at Cn,,,,,,)andthe ~ n a x i ~ n urate n ~of growth is reached at Cn,)n,.(From Ref. 73.)
tions to be different: this was indeed found, and the UDC-GC model was validated (74) with post-experimentally determined average growth rates of 0.0096 and 0.0085 h " for the sufficient and deficient soils respectively. Note that these are the constants in an equation describing exponential growth, so that the differences revealsubstantiallydifferentgrowthpotentials.However,theseedlings were pre-germinated and maintained under identical conditions prior to starting the experiments.Therefore it isreasonable to assumethat,immediatelyafter transplanting. all the seedlings were growing at the same rate irrespective of soil treatment. In the new analysis (38), a constant growth rate was used for all seedlings initially and these growth rates were allowed to vary as a consequence of their P uptake history. Figure 17 shows the predicted growth rates for both cultivars in bothsoiltreatments overtimeandreveals an approximatehalving in growth rate over the experimental period for the deficient soil. However, when the predicted uptake was compared with the experimentally observed uptake (Fig. IOB), the correlation coefficients were identical for the UDC-GC and UDC-CV (growth variable) models, andthe accuracy of the predictions were actually better for the UDC-CV model. WhattheUDC-CV modelhas shown isthatplants grown in nutrientlimited conditions could downregulate their growth rate in response to nutrient deficiency and still produce predictions that validate the model at least as well a s the original analysis. I t is extremely unlikely that the very simple and direct
Darrah and Roose
l
0
l
I
I
I
1 0 0 # 1 0 3 0 0 4 0 0 5 0 0 8 0 0
(h)
Figure 16 Cumulativephosphorusuptakecurves (per unit root length) predicted for two tomato cultivars (‘Knox’ and ‘C37’) grown in a soil fertilised to two P-levels. (From Ref. 28.)
feedback between growth and uptake used in the UDC-CV model is correct. For example,there islikelytobesometimedelay in response to deficiency and probably some internal reallocation of P to enable growth to be maintained at a of whether agreement between higher rate for longer. This then raises the question observed and predicted values at the plant scale can be considered to provide adequate validation at the root scale. If two models with very different root dynamics produce similar results, how does one judge which is correct? The most complete mechanistic model to include both local and plant-scale processes is ecosys (75,76). This includes a Barber-Cushman mineral nutrient submodel, a mineralization by microbial biomass submodel, and explicitly models mycorrhizae and root growth. It also includes a whole-plant growth model based on the functional equilibrium concept (77). The model is driven by around 50 parameters and requires supercomputing facilities to execute. Validation is primarily at the plant scale. The model has been used to investigate P uptake i n barley in two soils over the whole growing season (78). Reasonable agreement was found between observed and predicted variables such as water-soluble P in the soil, root length density with depth, shoot P, and shoot dry matter. The
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0.015
0.014 0.013
i
I
i
0-01*
i 0
O.Ol1
K 0.010
0.009
Figure 17 Predicted changes i n the relative growth rates of two tomato cultivars grown under 'sufficient' and 'insufficient' levels of phosphorus. The inset shows the changes in root mass: plant mass ratio of the cultivars in thc low-P soil. (From Ref. 28.)
correspondence was much better for plant variables than soil variables. Ecosys represents the first serious attempt to employ fully mechanistic principles to construct an agronomic management model.
V.
PLANTUPTAKEINHETEROGENEOUS ENVIRONMENTS
Soils are spatially variable at allscalesfromthemicroscaletothelandscape. Variability on the field scale has led to the development of precision agriculture (79,80), where fertilizers or other agrochemicals are applied at different rates at different locations to compensate for local patchiness. Within the rooting zone of an individual plant or n small group of plants, spatial heterogeneity is also present (81,82). The most obvious is a general decline of nutrient content with depth, which is often matched by a similar decline in rooting density. Drew ( 8 3 ) conducted a much cited experiment where the roots of barley were exposed to an artificially provided patch of nitrogen. The plants responded by proliferating
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extensively in the patch and reducing root investment in the unamended soil. A second response to nutrient patches is a stimulation of uptake per unit of root (increased The proliferation response seems to be largely unspecific, whereas the I,,,:,, response seems to be much more nutrient specific and the magnitude of the response seems related to the concentration of the nutrient (84). Recently a list of the responses of 27 uncultivated forbs and grasses differing in inherent maximum growth rates to localized nutrient patches has been compiled ( 8 5 ) . It was found that fast-growing species displayed more plasticity within root systems and responded more quickly to patches than slow-growing species. However, they pointed out that root proliferation within a patch cannot always be expected to result in greater nutrient capture. In fact, for a mobile ion like nitrate, such proliferation may have little effect on nitrate inflows, and with isolated plants, there is only a weak link between proliferation and nitrate capture. Recently it hasbeen shown (86) thatproliferationconfers an advantage only when roots are competing for the N resource in N-limited plant communities. It is also suggested that attempting to increase the proliferation response in crops grown in well-fertilized monocultures would have little benefit (86). The genetic basis of at least some aspects of the proliferation response has Arnhidopsis mutantshasshown thata recentlybeen uncovered.Workwith nitrate-inducible gene (ANRl) encodes a member of the MADS box family of transcription factors (87). Repression of this gene resultedin plants that no longer responded to nitrate-rich patches. Jackson and Caldwell (88) usedthe Barber-Cushman modelto examine thesignificance of localizednutrientpatchesandrootproliferationforwhole plant uptake by Agro/>yrondesertorurn. They divided the simulated soil volume into 25 soil cells either all at the same nutrient concentrations (homogeneous) or with one cellwithanelevatednutrientconcentration(heterogeneous). In both cases, the total amount of nutrient in the soil volume was the same. Roots were either assumed to exploit the soil volume with evenly spaced roots (r?orlplmtic.) or were allowed to proliferate preferentially i n the enriched cell (plcrstic). Their simulations showed that plastic roots in the heterogeneous environment acquired more N and P than i n the homogeneous state while nonplastic roots acquired less in the heterogeneous state compared to the homogeneous. They also simulated (N03-N) variations observed experithe response to the 3-fold (P) and 12-fold mentally in a 0.25-m’ area in the field. Preferential proliferation in nutrient-rich patches was estimated to increase acquisition by 28% for P and 61% for N where roots were allowed to colonize unoccupied soil (Fig. 18). They attributed these gains to increased root surface area in the patches together with higher patch concentrations, allowing faster uptake per unit surface area of root. However, when roots were allowed to proliferate in patches already containing some roots, the gains for nitrate largely disappeared as the new roots essentially competed for N with the existing roots.
I
l!
t
’*om
**O 1.5
je
0.5
0.0
conhot
+plesticity
Figure 18 Simulated uptake of nitrate from a “patchy” soil. The relative nitrate distribution from field data is shown in D. In A, the plant roots were allowed to respond in a “plastic” fashion to local resource availability whileB shows the “nonplastic” response: both plots show the relative nitrate uptake over the 0.5mZarea C. The total nitrate acquired in each plot. Plastic roots acquired 61% more nitrate during the 2-day simulation. (From Ref. 88.)
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In a further paper (89), the effect of a highly heterogeneous environment on nutrient uptake was simulated with Agropyron desertoruml. Nutrients patches were allocated using Monte Carlo techniques and the volume was divided into 10 or IO00 such patches. Root density per cell was constant and adaptive root proliferation was therefore not examined.The results showedthat root acquisition of P and N 0 3 - N was always lower when the nutrients were unevenly distributed. However, the reduction in uptake could be largely offset by allowing the uptake capacity per unit surface area of root (I,,,,) to increase with increased nutrient concentration (i.e., within nutrient-rich patches). Such modification of uptake capacity has been shown to occur for many plant species (90) as well as for Agropyron desertorurn in direct response to nutrient concentration (88). Localization of P in nutrient patches increased the predicted uptake by maize even without invoking root proliferation or altered uptake kinetics(91). In this case, thebenefit was due to a highly nonlinear buffer power, which increased the soil solution concentration of P, allowing a much more efficient uptake of P per unit root surface area within patches. In the Jackson and Caldwell (88) study, the buffer term was essentially linear, which minimized the uptake efficiency gains. Such studies illustrate the power of mathematical models to resolve apparently contradictory results. Gleeson and Fry (92) developed a model that simulated optimal foraging strategies for roots with respect to root proliferation. Uptake was not simulated mechanistically. They assumed that the root system would operate by investing roots in patches such that the marginal return (differential uptake rate divided by differential root allocation rate) would be equal across all patches and that marginal return was correlated with patch quality. Container-grownSorghurti vulgare fine root biomass showed behavior consistent with the foraging model; however, overall much of the root behavior remained unexplained by the model.
Vi.
FUTURE PROSPECTS
Most mineral nutrient uptake models have considered resource capture by isolated plants or by monocultures. Elsewhere in the ecological literature are models dealing with interspecies interactions and particularly competition. For example, a model (93) has been developed based on the area of influence (AOI) principle, detined as the circular area around a plant where it is effectively able to acquire resources. Overlapping AOIs indicate that competition is occurring. This model was used to show that plants that grow their root systems asymmetrically to avoid the roots of neighboring plants (compensatory behavior) outcompete noncompensatory plants and have a greater biomass. They had no mechanistic description of resource acquisition and each soil volume (pixel space) was assumed to provide an equal amount of resource. The marriageof such models with mechanistic models of the rhizosphere could provide powerful tools to aid in the understand-
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ing of below-ground competition in natural communities. This is important because below-ground competition is predicted to be much more symmetrical than above-ground competition for light and may therefore to actpreserve biodiversity in natural communities (94). Many studies have shownthat the rhizosphere microbial community is different from the bulk soil community and differs between plant species (see also Chaps. 4 and 12). For example, FAME analysis hasbeen used to show that gramnegative species, particularly pseudomonads, were favored in the rhizosphere of soybean, while numbers of Bacillus spp. were lower than in bulk soil (95). Both plant genotype and micronutrient status havebeen shown to affect root colonization by soil bacteria (96). Individual members of the rhizosphere community have been shown to promoteplant growth (plant growth-promoting rhizobacteria (97) (PGPR); or to have deleterious effects without being pathogenic (98) strictly speaking. Microbial rhizosphere models have thus far largely dealt with the community as a single entity using biomass as the state variable. Future models will have to attempt to understand the rhizosphere at the species level. This will involve understanding why particular species are selected as rhizosphere colonists, how microbial species interact between themselves, their population dynamics, and how their activities influence and are influenced by the root. Advances i n techniques (99,100)are making such questions more experimentally tractable and rhizosphere modelers may soon be able to address these important issues. Global climate change is predicted to have major consequences for plant growth and this will provide a fresh impetus for the mathematical modeling of the rhizosphere (101).
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(1991). 5 1. D. L. Jones and P. R. Darrah, Resorption of organic-components by roots of Zecr rrltrys and its consequences in the rhizosphere: I . Resorption of “C labeled glucose, mannose and citric-acid. Plnrlr Soil 143:259 (1992). 52. D. L. Jones and P. R. Darrah. Re-sorption of organic-compounds by roots of Zecr rnngs L. and its consequences in the rhizosphere: 2. Experimental and model evidence for simultaneous exudation and re-sorption of soluble C compounds. P l m t Soil /53:47 (1993). 53. D. L. Jones and P. R. Darrah, Influx and efllux of amino-acids from Z w rrluys L. roots and their implications for N-nutrition and the rhizosphere. P l m r Soil 156:87 (1993). 54. D. L. Jones and P. R. Darrah, Influxandefflux of organic-acidsacrossthe soilroot interface of Zecr r m v s L. and its implications in rhizosphere C flow. P l e ~ r Soil ~t 173:103 ( 1995). 55. P. R. Darrah, Rhizodeposition under ambient and elevated COz levels. Plnrlt Soil 187265 (1996). 56. E. M. Scott, E. A. S. Rattray, J. 1. Prosser. K. Killham, L. A. Glover, J. M. Lynch, of bacterial inoculants colonizand M.J. Bazin, A mathematical model for dispersal ing the wheat rhizosphere. Soil. Biol. Biocher?~.271307 (1995). 57. N. van der Hoeven, J. D. van Elsasand C. E.Heijnen, Amodelbasedon soil structural aspects describing the fate of genetically modified bacteria in soil. Ecol. Modellirlg 8Y: 161 (1996). 58. A. M.Semenov,A.H. C. vanBruggen,and V.V. Zelenev.Movingwavesof bacterial populations and total organic carbon along rootsof wheat. Microb. Ecol. 371 l6(1999). 59. V. Goddard. Population dynamics of fluorescent pseudomonads in the rhizosphere, D. Phil. thesis, Oxford University, 1999. 60. A. J. Diggle, ROOTMAP-a model in three-dimensional coordinates of the growth and structure o f fibrous root systems. Plant Soil 105:169 (1988). 61. L. Pagks.M.D.Jourdan.and D. Picard, A simulation model of the three-dimensional architecture of the maize root system. Plnrlt Soil 119147 (1989). 62. A. H. Fitter, T. R. Stickland, M. L. Harvey. and G . W. Wilson. Architectural analysis of plant root systems: 1. Architectural correlatesof exploitation efficiency.Nc>n. Ph.vtol. 119375 (1991). 63. K. L.Nielsenand J. Lynch,Fractalanalysisofbeanrootsystems. Agron. Abstr. 150: 1 ( 1994). 64. G. M. Berntson, Topological scaling and plant rootsystemarchitecture:developmental and functional hierarchies, Neb\, Phytol. 135:621 (1997). 65. A. H.Fitter,Anarchitecturalapproach to the comparative ecology of plantroot systems. New Pkyrol. 106:61 (1987). 66. P. M. Room, J. S. Hanan, and P. Prusinkiewicz, Virtual plants: new perspectives for ecologists, pathologists and agricultural scientists. Trerlris Plunf Sci. 1:33 (1996). 67. M. T. Michalewicz, Plur~tst o Ecosystems: A c l ~ w ~ c eirls Corrlputoriorlal Life Scier~ces,Vol. I . CSIRO Publishing,Melbourne,1997.
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R. F. Grant, Simulation model of soil compaction and root growth: 1. Model development. Plnrtf Soil 150:IS (1993). 77. J. H. M. Thornley,A balanced quantitative model for root: shoot ratios in vegetative plants. Ann. Bot. 36:43 I (1972). 78. R. F. Grant and J. A. Robertson, Phosphorus uptake by root systems: mathematical modelling in ecosys. Plant Soil 3883279 (1997). 79. S. E. Cook and R. G. V. Bramley, Precision agriculture-opportunities, benefits and pitfalls of site-specific crop management in Australia. Altst. J. Exp. Agric. 38:
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ammonium and potassium on the growth of the seminal root system, and the shoot, in barley. New Phytol. 75:479 (1975). in root systems in relation 84. D. Robinson, Variation, co-ordination and compensation to soil variability. Plant Soil 18757 (1996). to nutrient patches 8 5 . D. Robinson andM. M.I. Van Vuuren, Responses of wild plants in relation to growth rate, I t t h m v t t Vm-iotionin Plrott Grotvtlz: Phyriologicrd M ~ I arlisrrls ar~dEcological Cor~.sequer~cc~.s (H. Lambers, H.Poorter and M. M. 1. van Vuuren, eds.), Backhuys Publishers, Leiden, 1998, p. 237. 86. D. Robinson, A. Hodge, B. S. Griftiths. and A. H. Fitter, Plant root proliferation
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in nitrogen-rich patches confers competitive advantage. Proc. R. S o c ~ . H. 266:43 I ( 1999). 87. H. Zhang and B. G. Forde, An Arcrbitlo/~si.sMADS box genc that controls nutrientinduced changes in root architecture. Scieucc. 279:407 (1998). 88. R.B.JacksonandM.M.Caldwell,Integratingresourceheterogeneityandplant plasticity: modelling nitrate and phosphate uptake i n a patchy soil environment. J. E d . 84:891 (1996). 89. R. J. Rye1 and M. M. Caldwell, Nutrient acquisition from soils with patchy nutrient distributions as assessed with simulation models. Ecology 792735 ( 1998). 90. D. T.Clarkson.Factorsaffectingmineralnutrientuptake by plants. A m f r . Kc w. P l m t Ph~~.siol. 36:77 (1984). 91. J. L. Kovar and S. A. Barber, Reasons for differences among soils in placement of phosphorus for maximising predicted uptake.Soil. Sci. Soc. Am. J. 5 3 : 1733 ( 1989). 92. S. K. GleesonandJ. E. Fry,Rootproliferation and marginalpatchvalue.Oikos
79387 (1997). 93. J. Brisson and J. F. Reynolds, Effects of compensatory growth on population processes: a simulation study. Ecology 78:2378 (1997). 94. B. B. Casper and R. B. Jackson, Plant competition underground. Artrlu. Rev. E d . Systetttcrt. 2570 ( 1997).
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Methodological Approaches to the Study of Rhizosphere Carbon Flow and Microbial Population Dynamics J. Alun W. Morgan and John M. Whipps Horticulture Research International, Wellesbourne, Wanvick, England
1.
INTRODUCTION
Loss of carbon compounds from roots, o r rhizodeposition, is the driving force forthe development of enhancedmicrobialpopulations in therhizosphere in comparison with the bulk soil.Although rhizodeposition is a general phenomenon of plant roots, the compounds lost from different species or even cultivars can vary markedly in quality and quantity over time and space. of compounds lost from the root are Details of the types and quantities discussed elsewhere (Chap. 2), but briefly, rhizodeposition is measured as root loss and root respiration together with the accompanying microbial biomass utilizing the root-derived material, the COz generatedby these microorganisms and the portion entering the soil organic matterpool. It takes the formof water soluble exudates;polymericcarbohydratesandenzymes;lysatesincluding cellwalls, whole cells and, with time, whole roots; and gases such as ethylene and respiratory COz ( l ) . Rhizodeposition commences during seed imbibition.often as watersoluble exudates and gases, and continues as the plant grows. Cells arelost from the root cap continuously as the root grows through soil, accompanied by losses of exudates and polysaccharide gel. The zone just behindthe root tip appears to be the site of maximum exudation with both the zone of elongation and the cell junctions also indicated as localized regionsof greater exudation in some species (2-4). However, eventually whole roots or parts of root systems die, and thisroot
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loss with COz derived fromroot and the accompanying microbiota respiration are the major sources of rhizodeposition. Such differences in the amount and type of rhizodeposition that occur on the root with time result in concomitant variations in microbial populations in the rhizosphere, both within the root (endorhizosphere), on the surface of the root (rhizoplane), and in the soil adjacent to the root (ectorhizosphere). The general microbial population changes and specific interaction of individual compounds from specificplants or groups of plants with individual microbial species are covered in detail elsewhere (Chap. 4). Consequently. this chapter is restricted to consideration of methodologies used to study carbon flow and microbial population dynamics in the rhizosphere, drawing on specific plant-microbe examples only when required. The methodologies for studying rhizodeposition, with one or two exceptions highlighted later, have changed relatively little over the last few years, and detailed reviews of most of the approaches, their advantages and disadvantages, have been published (1,5-9). Consequently, the concepts and procedures are described but references to the topics will be limited. In contrast, huge advances in the methods for studying microbial ecology and population dynamics have been made as molecular techniques became available; therefore greater emphasis is placed on these evolving technologies.
II. METHODS FOR THE STUDY OF RHIZOSPHERE CARBON FLOW Experiments to examine rhizodeposition can vary markedly in scale and complexity depending on the information required, the equipment available, and the plants concerned. In general, experiments to study exudates and other material lost from young roots are the simplest and are carried out in the laboratory under controlled conditions. Plants are grown in nutrient solution culture, sometimes with sand or other solid support systems, and compounds released into the culture solution are collected and analyzed chemically. The experiments are mainly short-term to and the roots can be kept sterile if required. Techniques are also available label plants growing in these systems with IJC and to monitor the presence of the isotopes in the rhizodeposits. Plants grown for longer periods in solid supports such as sandor soil represent the next level of complexity and, although other techniques are available, carbon flow is most frequently estimated using ‘‘C labeling experiments. In the laboratory, ‘ T O 2can be suppliedto shoots either as a short pulse or continuously, and the carbon flow can be monitored. In the field, due to technical limitations, only IJC02 pulse labeling procedures are possible.A final approach, termed c r v studies, involves the measurement of components of crop growth from which
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estimates of rhizodeposition can be made. This remains the only technique available for some ecosystems. Recent examples of all these procedures are given below.
A.
Measurement of Rhizodeposition in Solution Culture
At its simplest, rhizodeposition can be measured chemically by growing plants in solution culture, although the presence of a soil microbiota or soil particles can influencerhizodeposition. In theshortterm,rhizodepositioncomprises largely water soluble exudates, but, gradually, higher-molecular-weight materials including sloughed cells may accumulate, and all of these have been measured routinely. Gases are occasionally quantified but, because of the relatively short periods of nutrient solution experiments, dead roots are seldom considered. Seeds can be placed in water, and as imbibition occurs, the materials lost from seeds can be collected and analyzed qualitatively and quantitatively (IO). Once germinated, seeds are commonly placed on some form of grid or support above the nutrient solution and, as the roots grow in the nutrient solution, it can be collected at intervals and the rhizodeposits analyzed. Seeds are often surfaceor alteration of the materials derived from the sterilized to prevent utilization seed by contaminating microorganisms (e.g., Ref. 11). The type of sampling employed reflects the target of the experiment and can range from detailed analyses of a single class of chemical to much more general observations. For example, in sterile solution culture, dicarboxylic acids were quantified in one study of exudates of wheat and flax (12), and isoflavanoids in exudates of white lupin were measured in another (13). In contrast, changes in gross classes of compounds such as low- and high-molecular-weight materials and particulate matter have been examinedin exudates derived from maize grown axenically in solution culture (14). The ability to change and control the composition of the nutrient solution and the relatively small size of the microcosms used enables manipulation of environmental variables and time-course studies of rhizodeposition to be made relatively easily. The influence of nutrient availability, mechanical impedance, pH, water availability, temperature, anoxia, light intensity, CO, concentration, and microorganisms have all been examined within a range of plant species (9). A few examples to illustrate the continued interest in examining the effect of such variables on rhizodeposition in nutrient culture are given in Table 1. Besides enabling the identificationof the effects of a range of variables on rhizodeposition, plant culture solution studies have recently helped to highlight the the reabsorption of organic compounds by roots, causing a reappraisal of quantification of carbon loss (19-23). Using maize, reabsorption was demonstrated by a combinationof techniques. These included monitoring uptake of‘“Clabeled glucose, mannose, and citric acid from the nutrient solution; quantifying
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Table 1 Examples of TreatmentsInfluencingRhizodepositionExamined Culture Solutions
i n Nutrient
Treatment ~~
Iron deficiency Mechanical impcdence Phosphate level Plant mutants and bacteria Root density Sterility
Chickpea (Cicer lrrictinuul) Maize (Zrcr t t ~ c r y s ) Rape (Brcr.s.sicu rlupts) esc~rlerlt~rrlr) Tomato (L~copersic~ot~ Maize (Zrcc I I I ~ ~ S ) rape Forage (Brnssiccc r l c r p s )
IS 16 17 18 14 II
differences in absolute levels of soluble carbon compounds in the nutrient solution when left unchanged or changed regularly; manipulation of the nutrient levels i n the nutrient solution; use of sterile and nonsterile systems; and the application of metabolicinhibitors.Mathematicalmodelswerealsoemployedto interpret and predict release and uptake of carbon compounds i n the rhizosphere. Rather than measuring rhizodeposition in nutrient solutions chemically, another approach has been to expose shoots to “CO2 for a short period of time and to followthespread of the through theplant,intotheroots,andtheninto the nutrient solution. Kinetics of carbon flow and quantification of rhizodeposition can then be obtained (e.g., Ref. 24). Advantages and limitations of this approach are discussed more fully in Sec. 1I.C.
B. Measurement of Rhizodeposition in Solid Supports Rhizodeposition in nutrientsolutionculture isrelatively easy to measureand readily altered. It may not reflect what happens in vivo due to the absence of a solid substratum for root growth. Simply including glass beads in nutrient solution can significantly increase rhizodeposition i n maize (16). and such an effect must be considered when estimating rhizodeposition i n relation to the natural environment. Consequently. a range of systems using various solid supports have been developed. Rhizodeposition in soil is considered in Sect. I1.C. The simplest system devised comprises a microcosm where seedlings with roots sandwiched between Millipore menlbranes are in contact with agar containing plant nutrients (25). The shoots can be exposed to ‘‘CO?, and the loss of “C into the agar via the roots is then monitored. The design enables the effect of microorganisms on rhizodeposition to be examined easily (26) but really lacks the complexity of substratum to allow data obtained to be related to rhizodeposition i n soil.
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The next level of sophistication is to use microcosms containing sand with nutrient solution to provide the physical conditions of soil but without its inherent complexity. Such systems have beenused to measure rhizodeposition ranging root from exudates to total carbon budgets. For example, soluble fractions of exudates from 5- and 7-week-old mycorrhizal and nonmycorrhizal maize seedlingswerecollectedbypercolatingthesandsubstratuminatubemicrocosm with distilled water (27). Similarly, forage rape (Brassica n r p u s ) was grown in a syringe-based microcosm containing sand and nutrient solution and, via a series of root-zone Bushing systems, amino acids released over specified time intervals were collected and quantified chemically with plants upto 60 days old (28). Such microcosms were adapted to enable IJCO7 pulse-chase experimentsto be carried out with forage rape and full-carbon budgets were obtained (29). Unusually for such studies, through the repeated sampling system employed, an estimate of the proportion of T O 2 respired by the root and the proportion respired by the accompanying microorganisms was made. Other sand-based systems using 'JC07pulse-chase procedures have been used to produce carbon budgets forFestuclt ovirla and Plantagolmcrolata seedlings (30) and white lupin ( Orpi nus a1b~r.s)(3 l ) . Significantly, ''CO2 pulse labeling of proteoid roots of white lupin under phosphate-deficient conditions showed that high levels of dark fixation of I' CO2 by the roots took place and that 66% of this root-fixed carbon was exuded from the roots (31). Clearly, dark fixation of CO7 by roots and subsequent rhizodeposition is an area that deserves further study i n the future. Sand-based systems can gradually be made to mimic soils by addition of other solid materials or nutrient sources used in agriculture. For example. rock phosphate and mica were added to sand microcosms containing mycorrhizal or nonmycorrhizal pine ( P i n u s sylvrstris) and beech (Fcqps sy1varicu.s) to investigate the effect of added microorganisms on rhizodeposition (32). The effluents from the tube microcosms were collected continuously, and after I and 2 years the tubes were harvested. Total organic carbon in the sand-that is, the soluble fraction from the sand and in the rhizosphere sand adhering to the roots-was measured. Estimates of sugars, amino acids, organic acids. and phenolics were also madc. This work clearly demonstrates that, depending on the plant species involved, sand-based microcosms can support plant growth suitable for rhizodeposition studies for long periods of time if required.
C.
Measurement of Rhizodeposition in Soil
Despite the advantages that sand-based systems provide for estimating rhizodeposition,studies in soil are stillrequired.Thesearefarmoretechnicallydemanding and are heavily biased toward the use of radioactive tracer methods,
particularly the use of I4CO2,to produce estimates of rhizodeposition and carbon budgets. Examples of a range of these techniques are given below.
L o n g - t e ~measurement of the release of exudates is impossible in soil; two techniques have been devised to address this problem. In the first, the plants are removed from soil and carbon loss is monitored from roots placed in solution for brief periods of time on the assumption that exudation remains the same as that occurring in soil. In the second, exudates are physically captured from roots, which are exposed in purpose-built microcosms using agar or filter-paper strips. 0th types of procedure risk creating artifacts in both quality and quantity of exudates measured because of the disturbance involved, but at least they provide an approximation of potential exudation if appropriate controls are included. For example, using the first technique, 18 species of plants were grown in either acid or alkaline soils for 8-12 weeks, freed from the soil by washing, and placed in aerated water (33). After 24 h, the water was replaced and the exudates collected over the next 24 h. Alternatively, using the second technique, sheets of agar- or quartz-filter paper strips were placed on roots of mycorrhizal plants grown for v ~ y i n glengths of time in microcosms to collect e~udatesdirectly. ~ o r with k agar sheets involved chemical analysis of the exudates collected from mycorrhizal maize grown in special microcosms with nylon mesh separating roots and ~buscularmycorrhizal fungi (34). In contrast, work with quartz filter paper strips involved monitoring 14Clevels in exudates at a range of sites on roots of mycorrhiu ~ a 14C02p~lse-labelingexperiment (35). Interestingly, zal C a ~ s i c au ~ n u after in the latter study, a bioluminescent fluorescent pseudomonad ( P ~ e u ~ o ~ o ~ ~ u o r e s c e 2-79 ~ s RL) was used as a general indicator of the amount of nutrients u could u ~ be useful for this purpose available in the rhizosphere of C. a ~ ~ and in other plant-soil systems. ~icroautoradiographyhas also been used to monitor distribution of I4C in roots of ponderosa pine (Pinus ~ o ~ ~ e ~following o s a ) a 5day pulse with “C02 (36). A technique utilizing genetically modified bacteria to ‘‘report’’ the presence of particular compounds in soil has also been developed (3’7). These bacteria respond to the presence of specific compounds in their environ~entby producing ic~-nucleationproteins that enter into cell membranes, enabling cells to be detected by means of a droplet freezing assay. The presence of trytophan in 1-10 p molar concentrations has been detected using reporter bacteria in a study examt a soil (38). ining loss of amino acids from roots of Avena ~ a r ~ ainto
L a b e ~ i nTechniques ~ for CarbonTwo different approaches to the use of I4CO2labeling have been applied to measure carbon flow within plant-soil systems-pulse-chase and continuous labeling.
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Each has advantages and disadvantages, and these have been reviewed (7). Essentially, pulse labeling results in the labeling of nonstructural labile carbon pools, whereas continuous labeling homogeneously labels the whole plant. Pulse labeling is relatively quick and easy to carry out and allows plant responses to rapid changes in environmental conditions to be determined, which is impossible with continuous labeling. Pulse labeling also allows determination of carbon flux to the root, whereas continuous labeling gives only cumulative data regarding carbon transport. Nevertheless, continuous labeling is the only way to determine total flux of carbon through the root-soil system. Consequently, different qualities of information are provided by both labeling procedures. As yet, pulse-chase and continuous labeling experiments have not been carried out simultaneously, but they should prove feasible in the future. The requirement for dedicated and sophisticated equipment for continuous labeling will probably mean that this procedure will remain laboratory-based, whereas pulse-chase technique can be used in the field as well. Some examples of the applications of the various ‘jC02 labeling systems used in the laboratory and field are given in Table 2. The “CO2 labeling apparatus can range from a polyethelene bag (48) to more complex Plexiglas canopies (45,49) to highly sophisticated dedicated growth cabinet facilities such as the Experimental Soil Plant Atmosphere System in The Netherlands (57,58). Nevertheless,thekeyfeature in allthesefacilitiesistheseparation of theshoot compartment,wherethe1JC02 isreleased,fromtherootcompartment,thus to be allowing the spread of the “C through the plant and into the rhizosphere monitored. One of the major advances made in recent years in these studies has been the developmentof techniques to separate root respiration from that of the associated soil microbiota utilizing the carbon compounds coming from the root. Earlier studieshadattemptedtocomparesterilewithnonsterilesystems,butthese workedfor only shortperiodsbeforesterilitywaslost(64). In otherstudies, microbial inhibitors had been added to nutrient solutions, but side effects on the plant could not be ruled out and the lack of soil as substratum compounded the problem (65). Thus, three systems to address this problem in soil have been devised. The first, by Johannsson (60), calculates gross rhizodeposition in continuously 14C-labeled plants from the stable V-residue remaining in the soil after of a long-term incubation. The former is calculated from the latter through use stabilizationfactor,which is estimated by comparison withhumificationof known organic materials (glucose, grass shoots, and roots). Thus, C from microbial respiration is the difference between the calculated gross rhizodeposition and that measured in soil immediately after plant growth. The second, by Cheng et al. (66,67), involves saturatingthe soil with “C-glucose immediately beforeI4CO2 labeling to eliminate “C microbial respiration. This isotopic Trapping system requires that the measurement period be short (4 h), which means that 14C root
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Carbon Flow Microbial Population and Dynamics
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respiration is underestimated. The third, by Swinnen (68) involves the injection of model 'JC-labeled rhizodeposits (glucose, root extract, or root cell-wall material) into the rooted soilof an unlabeled plant simultaneously with the ',CO2 pulse labeling of a similar but separate plant. Hence, the assumption is made that the model rhizodeposits create a model rhizosphere and that microbial transformation of the introduced compounds is representative of the transforniation of "C-labeled rhizodeposits in the "'COz pulse-labeled system. One practical aspect of the procedure for monitoring carbonflow following ',C labeling is the need to separate roots from the soil for analysis. Incomplete removal of roots can lead to an overestimation ofrhizodeposition, but overzealous washing of soil may lead to leaching of ',C or loss of fine roots. This problem has been examined in detail for wheat and barley, and procedures to correct for these errors have been developed (69). Another relatively recent innovation has been to include the measurement of '"C present in themicrobialbiomassfollowingIJCO2labeling of plants to allow differentiation of this carbon pool from the remainder of the ',C labeled material present in the soil. Generally a chloroform fumigation is carried out and the microbial content is measured using direct extraction, incubation, or centrifugationmethods(36,40,41,50,58,59).Thevaluesobtained still depend on the method of calculation and the Kc factor (the fraction of biomass C extracted by K2S0.,converted to CO2 after fumigation according to the fumigation-extraction, or fumigation-incubation method, respectively) chosen,if used (59). Other methods have been utilized. For example, hyphal biomass of the mycorrhizal fungus HoDdornn c.r.u.stiliil$iw~w has been estimated using light microscopy with fluorescein diacetate as an activity stain (70)and bacterial biomass by [?H] thymidine incorporation procedures (7 l ) . Currently, there is some discussion about the most appropriate technique to use (69,72), and further work is needed t o resolve this problem.
3.
OtherIsotopicTechniques
I3C is gradually being introduced into carbon-flow studies in plants. It has the advantage that it is naturally occurring, stable, and nonradioactive, and it is less discriminated against than ''CO2 during photosynthesis. The natural abundance of "C is higher in C, compared with C3 species (73), and by growing C, plants on soils previously exposed only to C >plants or vice versa, this difference may be utilized to study carbon partitioning i n plants and the rhizosphere (74-77). Recently, pulse-labeling has been carried out with "CO2or '3C-depleted COz to monitor carbon allocation both within and between mycorrhizal plants (78,79) and with I3CO2to estimate carbon flux to roots of wheat and into the soil (80). "C-depletedCO?levels in field experiments have also beenusedto examine carbon turnover in cotton and wheat (81). Nevertheless, the use of '?C isotopic
Table 3 Examples of Experiments Employing Crop Study Techniques to Examine the Effect of Treatments Influencing Carbon Flow to Roots or Soil System
Treatment examined
Artificial Controlled-environment laboratory
Elevated C02: plant density
Glasshouse Glasshouse
Elevated C02; mycorrhizal status Elevated CO,; N level
Polyethylene house Rhizolab facility Solardome facility Field
Elevated COz Elevated COI Elevated CO, Fertiliser; irrigation Fertiliser; imgation Management practice N level P level Tillage
Plants Perennial ryegrass (Loliurn perenne),white clover (Trifoliurn repens) Loblolly pine (Pinus taeda) Loblolly pine. Ponderosa pine (Pinus ponderosa) 15 tropical species Perennial ryegrass JuncudNardus sward; Fesruca turf Barley (Hordeum distichurn) Wheat (Triricuwi aesfivum) Sugar beet (Beta vulgaris). wheat Barley, lucerne (Medicago sariva), meadow fescue (Fesruca prarensis) Snowgum (Eucalyptus pauci’ora) Sorghum (Sorghum bicolor), soybean (Glycine ma). wheat Barley, wheat
References 84 85 86
87 88 89 90 91 92 93 94
95
1
4 tn
P)
3
51
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labelingtechniquedoeshavedrawbacksincomparisonwith'"C-basedtechniques. For example,a mass spectrometer is needed for sample analysis involving large capital costs anda need for skilled backup. Generationof "C for experimental use is also relatively complex and time-consuming. Overall, '"-based techniques are less user-friendly, more time-consuming and more expensive than "Cbased techniques. A final, highly specialized procedure for monitoring carbon flow in plants involves the use of "CO1. The use of this positron gamma-emitting isotope of C, with a half-life of 20.3 min, allowed several physiological parameters of mycorrhizal and non-mycorrhizal plants of Panicum colorarum to be measured simultaneously i n real time (82). However, the technical problems associated with using "CO2, particularly the ability to produce isotopes of carbon, and with handling this short half-life are likely to limit this approach to specialised facilities.
4. Crop Studies Crop studies involvethe measurement of components of crop growth, fromwhich carbon budgets, including rhizodeposition, can be estimated either continually or over a whole growing season. These can include carbon gained by photosynthesis and carbon lost by respiration from roots, shoots, and soil microbiota as well as that lost by harvesting or tissue death. In perennial systems, yearly accumulation in the standing vegetation is also considered. Sequential harvesting and monitoring of all these components are required to obtain the data needed, and such procedures are technically demanding and difficultto use to achieve realistic estimates of rhizodeposition. Thus, in forest ecosystems, it is impossible to obtain respiration data from roots of a single tree, and soil-coring procedures are likely to miss fine roots and associated mycorrhizae, leading to an underestimation of rhizodeposits (83). Nevertheless.it is theonly procedure available for many forest ecosystems. Recently, such crop study approaches have been applied to plants grown under controlled laboratory, glasshouse. or rhizolab conditions to assess more easily the effects of potential climate change. Some examples of experiments employing crop study techniques are given in Table 3.
111.
SIGNIFICANCE OF CARBON FLOW IN MICROBIAL POPULATION DYNAMICS
The loss of carbon compounds from roots can influence microbial populations in various ways. Sincethe presence of readily available carbon sources is thought in soil (96), rhizodeposition to be the most limiting factor to microbial growth acts at a gross level to stimulate microbial populations. This generatesthe "rhizosphere effect" (97), where the number of microorganisms in the rhizosphere (R)
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is increased relative to the number in the bulk soil (S). The R:S ratio measured by isolation and culture for unicellular bacteria is generally between I O and 20, but the figure can be greater than 100. For other groups of organisms the R : S ratio decreases i n the following order: unicellular bacteria > actinomycetes and fungi > microfauna (98). Increasednumbers of microorganisms intherhizosphere have been observed microscopically (99) and microbial cells nearerto the root have been shown to be more active metabolically than those in the bulk soil.Forexample, O? consumptionmeasured usinga micro-oxygenelectrode decreased with distances from the barley root surface (loo), and specific activity changes in microbial biomass nearer the root were greater than those in the biomass in the bulk soil following “CO2 pulse chase experiments with ponderosa pine (36). Significantly, recent studies (101) have suggested that microbial respiration is not limited by available carbon in the rhizosphere, indicating that other factors rather than simply organic carbon may be important i n regulating microbial growth in the rhizosphere. Besides a general stimulation of microbial populations due to rhizodeposition,theactualqualityandquantityoforganicniaterialcontainedwithinthe rhizodeposition may also be important in controlling the behavior and development of specificmicroorganisms. For example, root exudatesorsinglecompounds within the root exudates can stimulate germination of fungal propagules and subsequent directed growth of the mycelium of a range of fungal pathogens (102). In addition, therole of individual chemicals in signaling between plant and microbial biotrophic symbionts, particularly in legumes, is well established. These aspects are considered in detail in Chap. 7.
W.
METHODS FOR THE STUDY OF MICROBIAL POPULATION DYNAMICS
In soil. ectorhizosphere and the rhizoplane microbial populations develop that can vary in size, complexity, and activity. The interactions between the microbial populations in these environments and between thesc populations and plants can have important consequences for plant and soil health. An understanding of the microbial population is therefore central for soil management. The main obstacles to increasing our knowledge in this field are methodological (103). Microbial cell enumeration techniques and identification procedures are often difficult or tedious, and the collection of relevant samples or the simulation of natural conditions in the laboratory can be problematical. However, the development of molecular approaches for the study of microbial populations can contribute to solving these problems. Considering the vast array of techniques used and presented in the literature, onlya selection of these methods is discussed in this review.
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Cell Culture
Traditional methods for counting microbial colonies on laboratory media all require the growth of cells. The number of colonies counted is used to indicate the size of the total population. Selective media can also be used to build up profiles of microbial populations or to monitor groups of microorganisms within a population, thus providing greater detail on the types of microorganisms present than total counts alone (Table 4). Once a colony has been subcultured on laboratory media, it is a relatively simple task to obtain further information on its identity and metabolic capacity (106-109). The main limitation to this type of approach is the number of colonies that can be analyzed. Normally less than 200 colonies are included in any study, and this represents less than 0.001% of typical soil and rhizosphere populations. Generalizations about the total microbial population based on theresultsfromsuchasmallsamplemaynotbepossible. A novel approach to obtain further information on the types of colonies isolated during a study on the behavior of Pseuclomoncrs species has been developed by DeLeij et d . ( I IO). In this method, by measuring the rate of colony development on the culture media the population is split into I’ and K strategists (fast-growing opportunists and slow-growing specialists, respectively). This simple alterationin the dilution plate count can provide useful additional information on a microbial community without becoming a labor-intensive exercise. To overcome the need for isolating individual colonies and then profiling their metabolic capacity, Garland and Mills ( 1 1 1) developed a method to profile the whole population. Cell suspensions taken from samples are placed directly on media with single carbon sources and a metabolic indicator (tetrazolium). If any members of the population hadtheabilitytoutilizethe carbon source, a positive reaction was recorded. I n this way the carbon-source utilization profile
Table 4 Media for the Enumeration and Isolation Total bacteria Total fungi and yeasts P.selt~lorr~or2rt.s species Enterobacteriaccac Spore formers Streptomyces species Chitin or cellulose utilization Denitrifying or nitrate-reducing
of Soil Microorganisms”
Nutrient agar, R2 agar, Tryptone soya agar Potato dextrose agar PI, P2 etc., P.seltriornor~o.7isolation agar MacConkey agar, XLD agar, brilliant green agar Heat treatment of samples, 75°C for 15 min. e.g., for the detection of Bacillus species RASS agar Clearing zones on agar plates Most probable number methods
bacteria .‘The composition of the media can he found Rcfs. 104 and 1 0 . 5 .
In
many microbiologymanuals and paperssuch as
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of the total population would be determined. Initially a simple bacterial identification system (Biolog) was used to profile samples ( 1 11); but since then, the types of carbon sources used has been broadened to include those likely to be found in the environment under scrutiny ( 1 12). However, this method still requires the growth of microorganisms; consequently, nonculturable strains will not be included. Conditions within the test strip will inevitably favor certain types of bacteria, such as pseudomonads, andso bias the results. To overcome the need for cellculture in thistype of study, Morgan and Pickup (1 13) developeda method for profiling total populations on the basis of their enzyme activity. This method was used to investigate water samples and would require the development of a suitable cell extraction method before soil samples could be studied. In addition, during the required incubation period, cell growth or the induction of enzymes is likely to have taken place that could bias the profiles of enzyme activity.
B. Techniques for Direct Microbial Detection All cell culture-based methodologies are essentially limitedwhen studying whole microbial populations, since the dominant proportion of microbial biomass of soil, rhizosphere and rhizoplane, and other environments cannot be cultured on standard laboratory media ( 1 14). T o obtain information on the composition and activity of the nonculturable fraction and to aid the study of the culturable fraction, direct detection methods are needed. 1. Microscopy A direct microscopic analysis of microbial cells can provide information on cell size, numbers,and biomass (1 15). Modifications of this basic method can be used to obtain additional information on cell viability and activity or to identify types of microorganisms present. Table5 outlines a range of these methods. For example, the identificationof individual cells may be possible using one o f a number of immunologically based methods. Fluorescently labeled antibodies are commonly used in conjunction with microscopy to allow the study of cells in soil. In most cases the results are affected by background fluorescence levels and the nonspecific binding of all antibodies to nontarget cells or particles. Specialized confocal microscopes that use a laser light source can reduce these problems and may also penetrate a small distance into the sample to view cells below the surface (126). This is a particularly useful feature for observing microbial films, including those that develop around roots and soil particles. The use of another laser-based system, the flow cytometer, may also overcome some of the methodological limitations that prevent the analysisof a large numberof samples by direct microscopy. The flow cytometer can analyze samples atarate of over 2000 cells S" and
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Table 5 Examples of Microscope-Based Methods for the Detection Environmental Samples
of Cells in
Target DNA RNAIDNA
Ethidium bromide, DAPI Acridine orange
RNA
Fluorescent oligonucleotides
Proteins Membrane potential Outer cell-wall structures Others
FITC Rhodamine123 Fluorescent antibodies
Viable cells
Direct viable count
Esterases Respiration systems
Fluorescein diacetate INT. CTC (tetrazolium compounds) Calcafluor white M23
Polysaccharide
Molecular probesstain all, vital stain
Stains all cells
116
May be affected by membrane changes 16s rRNA probes, specific for a species, genera or kingdom General strain Vital stain Specific antibodies must be available Vital stains particularly useful for total viable cell counts Detection of viable cells by elongation Vital stain Vital stains
1 l7
Direct staining
I18
1 l9
I20 1 1 s . 121
122
123 124 116, 125
I l7
measure the physical and chemical characteristics of individual cells as they move past optical and electronic sensors. This method has been applied to the detection of cells in water and sewage samples where i t can also be used to separate cells from samples ( 1 27) and may also be applied to soil samples. Once isolated, cells can be analyzed using techniques designed for monocultures. It is expected that furtherdevelopments in lasermicroscopyand flow cytometry willbe needed before their routine use i n rhizosphere studies is seen.
2. Biomarkers A range of biomarkers (biological markers) have been developed for the detection of microorganisms using both their genetic (DNA and RNA) and biochemical components. Most methods have originated from studies on pureisolatesand have been adapted to identify and quantify either the total o r a subset of the microbial biomass in a sample. In these methods, specific taxonomic or pheno-
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typic features are used to provide information on the presence of specific tnicroorganisms or the composition of the total population ( 128- 130).Detection of these compounds and translation of the results into theories on the microbial structure of soil can be difficult due to the presence of soil and other particles and highly complex microbial populations. A group of chemicals especially useful as biomarkers are fatty acids. These have been of great value in determining bacterial phylogeny and also provide a useful set of features for characterizing strains ( 1 3 I ) . Specific fatty acids, especially phospholipids, which are the major constituents of the membranes of all living cells (with the exceptionof archaebacteria), have theuseful properties that they are degraded rapidly following cell death, are not found in storage lipids or in anthropogenic contaminants, andusually have a comparatively rapid turnover ( 132). Bacteria also contain phospholipids as a relatively constant proportion of their biomass. This makes the analysis of the phospholipid fraction of mixed communities a useful measure of the viable cellular biomassand complementary to other traditional methods such as enzyme profile, muramic acid levels, and total adenosinetriphosphate-ATP(133,134). Lipid markers can berecovered from isolates and environmental samples by a single-phase chloroform/methanol extraction, fractionation of the lipids on columns containing silicic acid, and derivatization prior to analysis by capillary gas-liquid chrotnatography/mass spectroscopy (GC-MS). Quantitative estimates of microbial and community structure by means of on sediments, water analysis of the phospholipid fraction have been performed ( I35), and dust ( 1 36) as well as soil (137-141). The method is applicable to the study of mixed populations of varying degrees of complexity and is relatively straightforward to perform. A selection of studies involving the analysis of fatty acid profiles of environmental samples are outlined in Table 6. Changes in the environment that result in an alteration in the physiology of a microorganism can sometimes be detected in differences in the lipid profile. This factor is often mentioned when using fatty acid profiles for identification purposes where growth conditions must be kept constant. However, these changes may be exploited to determine the metabolic status of an organism and the environmental conditions it encounters (149). The presence of trrrns fatty acids has been associated with the physiological statusof the microorganism ( 1 13,150)and the ratio of trurzs fatty acids to cis isomers of monounsaturated fatty acids has been used as a measure of physiological stress (150). Changes in fatty acid composition of phospholipids have been observed as bacteria enter a nonculturable but viable condition ( 1 13), where cells are unable to grow on laboratory media but are able to take up vital stains. These changes in the fatty acid composition may be linked with a survival strategy where alterations are occurring to a cell to ensure the efficient uptake of nutrients or for strengthening the membrane.
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Table 6 Examples of Some of the Fatty Acids Used for the Detection Microorganisms in Environnlental Samples acid
Fatty
(f.a.)
of
References detected Component
biomass Bacterial Palmitic acid (16:O) 143 142, 141, Actinomycetes Is0 and anteiso f.a. Microeukaryotes Polyunsaturated f.a. Aerobic prokaryotes Monounsaturated f.a. Gram-positives and anaerobes Saturated & branched f.a. (C14C16) Sulfate-reducing bacteria Saturated & branched, f.a. ( C 1 6 C19) Desulfoirbrio 10 Me 16:O Gram-positive and gram-negative 134 Branched and monounsaturated bacteria f.a. Gram-negative bacteria 18: lw7 cy17:O bacteria i 15:O. I O Me 16:O and1X:lwY:137 Gram-positive sediments 145. Bacteria in146 Cyclopropyl f.a. Cl7 and C l 9 Terrestrial organic 147 matter 134, Long chain f.a. (e.g.. 24:O) 148 “Chemotypes” Straight chain , Straight chain cis rnonoenoic Branched chain saturated Straight chain t m . v monoenoic Cyclopropyl Branched-chain monoenoic
131
I44
144
Changes in the fatty acid profiles of mixed microbial communities may be used in a similar way to detect stress responses or periods of activity. Ergosterol is a predominant sterol limited to the true fungi (IS l ) , and it is possible to use measurements of ergosterol content as an indicatorof fungal biomass (152). The content of ergosterol in fungal membranes is species-dependent and can also vary with physiological state. Factors such as age, developmental stage, and general growth conditions can all cause variation in ergosterol levels. Suberkropp et al. (153) compared adenosine triphosphate (ATP) levels with ergosterol content as indicators of fungal biomass associated with decomposing leaves (Liriodemhn ttrlipfeln L.). They concluded that ergosterol concentrations provided a more accurate measureof fungal biomass when other organisms were present and likely to contribute to the ATP pool. Stahl andParkin ( 1 54) proposed a method to estimate living fungal biomass from soil ergosterol content by compensating for variabilityin ergosterol concentration and accounting for nonliving hyphal tissue. At best, ergosterol detection is limited to coarse determinations of
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fungal biomass and cannot be used to distinguish fungal species or define differences in fungal communities. Quinones, lipid-soluble substances involved in electron transport, can also be used as biomarkers. Lipski et al. (155) used quinone analyses, physiological tests, and fatty acidprofiles to differentiate Gram-negative non-fermentative bacteria isolated from biofilters. Quinone type was found to be an efficient method to group isolates prior to the analysis of results from the physiological tests. The detection of quinones appears to be restricted to the discrimination of isolated colonies and has limited potential to the analysis of mixed populations. Lipopolysaccharides (LPS) are major constituents of the outer layer of the cell walls of Gram-negative bacteria. LPS consistof an outer 0 antigen, a middle core of L,D-heptose and occasionally D,D-heptose, and an inner lipid A region, which consists of a phosphorylated glucosamine disaccharide with covalently linkedfattyacids.Both L p h e p t o s e and 3-hydroxyacid levels associated with LPS are potential biomarkers. Indeed Fox et al. (1 56,157) used GC-MS analysis of dust to study bacterial levels by targeting the hydroxy fatty acids (3-OH 12: 0 and 3-OH 14:0), L-glycero-D-mannoheptose andmuramicacid(achemical markerforpeptidoglycan). It maybethatvariability in LPS,lipoproteins, or outer membrane proteins-which has been used to distinguish between bacterial isolates (158)"will be exploited to study natural populations in the future. A number of biochemical markers not associated with the cell envelope allow the specific detection of individual microorganisms in environmental samples. These include secondary alcohols.For example, Mycobacterium xenopi can be detected through the hydrolysis of wax ester mycolates, which liberates 2docosanol, a characteristic and dominant secondary alcohol, which can be detected at low levels by GC-MS. This biomarker was found to be very useful for the rapid detection of M. xenopi in drinking water (159,160). Results from the GC-MS detection of 2-docosanol were obtained within 2 days compared to the 12 weeks required for culturable detection of M. xenopi. The detection limit for this type of approach was foundto be IO3 colony-forming units (CFU) m " drinking water. Volatile fungal metabolites can also be used as indicators of fungal growth in samples such as stored cereals and wheat. Metabolites 3-octanone, I-octen-301, and 3-methyl- 1 -butanol, 3-methylfuran, or total concentrations of a group of compounds such as carbonyl compounds havebeen used for this purpose ( 16 I 164). In general, the production of a volatile metabolite that is to be used as a biomarker must not change with substrate type or level, an essential feature that needs to be addressed in such studies. The metabolite l-octen-3-01 is produced during the breakdown of lipids; its quantity can vary and is dependent on the lipid content of the substrate (164).This limits its use as a biomarker of biomass. However,thistype of method will provide usefulinformation on thetype of substrate being attacked in undefined substrates. Since the production of volatile
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metabolites can be influenced by the duration of fungal growth (161), it could be essential to consider the stage of fungal growth in any study when these products are used as biomarkers. Borjesson et al. (161) indicated that 3-methylfuran could be used as a good indicator of fungal growth with little variation related to fungal species or growth substrate. This study also illustrated that terpenes (molecules composed of C, isoprene units) were the most suitable compounds for differentiating between fungal species and could be used to differentiate between fungi growing on different substrates. The value of these approaches is questionable, however, since studies on mixed bacterial and fungal populations were not performed and the soils need to be characterized for the presence of adsorbing matrices. Pyrolysis mass spectroscopy has been used for the rapid classification of microorganisms (165). The complex molecules present i n a microorganism are degraded through cleavage at their weakest point to produce smaller volatile fragments such as methane, ammonia, water methanol and H$. These are separated in a mass spectrometer to produce a pyrolysis mass spectrum, which can be used as a chemicalprofile or fingerprintof the complex material analyzed. This method has been used in microbial identification (165). Further advances in the application of this method to the analysis of complex microbial mixtures present in soil could provide methods that may make a significant contribution to the study of natural communities in the future.
3. D N A and R N A Studies The previous biomarkers relate to phenotypic assessmentsof microbial diversity and most will probably measure a restrictedpart of the total microbial pool, since not all markers will be expressed uniformly by every cell. In contrast, methods involving the detection of nucleic acids may be directly applicableto all microorganisms provided that the complete extraction of DNA (lysis of cells) or permeabilization of cells can be achieved. The diversityof single-stranded DNA in a sample can be assessed by studying its reassociation kinetics, and this approach has been used to study the diversity of microbial populations. The method involves the extraction of DNA from samples followed by heat treatment to make it single-stranded. As the sample is cooled, its optical density is monitored and, as the DNA reassociates, a change i n optical density occurs. The rate of change is used to provide a Cot 1/2 value (50% reassociation). DNA with reduced complexity reassociates quickly, while more complex DNA takes longer to reassociate. Torsvick et al. (166) found that in soil the major part of DNA isolated from the bacterial fraction was very heterogenous, with a Cot 112 of 4600, the equivalent of 4000 completely different genomes. The method also indicated that there may be populations of plasmids and/or bacteriophages that show very rapid reassociation (5% of the total DNA).
and
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The results also suggested that most of the diversity was located in the part of the population that could not be isolated by standard techniques. Recently this technique has been used in a range of environments (167-170). Holben et al. (171) used a method for obtaining a profile of a bacterial community by studying the % G C content of DNA. A sample of DNA from a bacterial community is mixed with the dye bisbenzamide, which binds preferentially to A T base pairsand alters the buoyant density. After equilibrium density centrifugation the DNA profile of the gradient is recorded, which reflects the relative proportions of microorganisms with differing % G C content in the sample. The gradient can also be extracted and each fraction analyzed furtherby polymerase chain reaction (PCR) or reassociation studies. Ultimately a detailed profile of the community based on the % G C gradient can be obtained. Cot and % G + C methods can provide informationon the whole microbial population but not onthetypes or identity of cells present in the population. This can be overcome using methods involving the analysis of genes encoding ribosomal RNA (rRNA). Sequence divergence among rRNA molecules hasprovided a framework for a natural classification of microorganisms and has served todefinetheir primary lines of evolutionary descent. The largest data sets of complete sequences have beenconstructedforthe 5s and 16s rRNAs.Total rRNA can be isolated directly from the environment and the various types of 5s rRNAseparated by high-resolutiongelelectrophoresis.ThentherRNAtypes are extracted from the gel, and sequenced and the phylogenetic affinities of the organisms in the community can be determined from sequence comparisonswith databases. These studies have been restricted to bacteria in environments of limited species diversity such as hot springs ( 1 72), marine invertebrates in a hydrothermal vent (173), bacterial populations in a copper recovery pond (174), and a bacteroplankton population (175). The larger 16s rRNA gene (- 1600 nucleotides) allows a broader phylogenetic analysis and a range of methods using 16s rRNA are available for the study of diversity within natural microbial populations. Four of these are addressed below.
+
+
+
+
(I. rRNA Seylrencr Atzuly.si.s. TotalDNAisextractedfromthesample, the 16s rRNA genes are amplified by PCR and the products are cloned into a suitable plasmid vector. One colony developing on a selective plate containing a single 16s rRNA gene insert (clone) therefore represents an individual rRNA gene present within the total population. T o distinguish clones from each other in a rapid fashion, theclones are restriction-mapped; for more detailed studies, the clones can be sequenced. In this way comparisons of DNA profiles or sequence information are used to determine either the diversity or the identity of clones (176). Each individual clone is acting as a biomarker for an individual cell. Organisms that are dominant in the total population will have a better chance of
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being represented among the clones, and a picture of the population is achieved through the analysis of numerous clones (> 100). One complication associated with this method is the generation of 16s rRNA molecules that did not exist in the original sample but were created by the PCR process (177). These products can be created by an error in the copying process so that the incorrect nucleotide is inserted into the sequence. This occursat a predictable frequency dependingon the type of enzyme used and the number of cycles. In general, this is considered a minor problem in the context of the scale of these studies. However, during PCR amplification, two different 16s rRNA molecules can combine to produce a hybrid (chimeric) moleculewith areas representing partof each of the original molecule. The frequency of production of chimeric molecules within a standard reaction is approximately 10% for templates that are 82% similar but rises to 30% of the final product with coamplificationof two nearly identical sequences (177). I n DNA samples isolated from mixed populations, a diverse sample of genomes is present, and the formation of chimeric molecules leading to the description of nonexistent species is a problem that still needs to be addressed.
b. RFLP Atlnlysis of 16s rRNA Cetze Pool. PCRamplications of 16s rRNA genes leads to a mixed PCR product containing genes of a similar size. The product is digested with restriction enzymes that cleave DNA at specific sequences and the banding pattern of the DNA analyzed is analyzed by agarose gel electrophoresis, which separates the DNA fragments on the basis of their size. The complexity of the banding pattern is used to determine the complexity of the original population (177). In this simple approach, many more samples can be processed, since only limited gel analysis is required for each population. However, sequence information to aid identification or assess species similarities will not be obtained, and the resolution of this method has not been fully determined. c. It1 situ DNA Hybridization of 16s rRNA. In situ hybridization is often used to confirm the presence of microorganisms in samples where 16s rRNA sequence analysis has indicated their existence. This methodis especially important when sequences have been obtainedthat do not resemble any microorganism that has been cultured. The method is also used to design fluorescently labeled synthetic olignucleotide probes ( 1 5 to 22 DNA bases long), which are hybridized to the rRNA present within fixed cells. Those cells containing homologous 16s rRNA regions to the labeled probe retain the fluorescent tag and are detected by microscopy or flow cytometry. The probes can be designed at different levels of identity: kingdom, genus, species ( 1 78- 180). Embley and Findlay ( I 8 1) used such an approach to analyze the relationship between methanogenic archael endosymbionts withintheir anaerobic ciliate hosts. Such applications illustrate the exciting potential for this technique, which can provide information on both the number and activity of cells. Confocal laser microscopyis a new version of fluo-
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rescence microscopy that uses a series of lasers to provide a light source at a specific wavelength and focal distance. This method allows thequantification of the fluorescence of individual cells, three-dimensional imaging, and reduced background noise. With these advantages, this method could bean important development to the use of fluorescence tags i n microbial studies.
cl. Derzaturir~gGrctdierzt Gel E1ectrr)l~hore.si.s. Muyzer et ctl. ( 1 82) have of PCR products obtained developed an interesting alternative to the analysis from samples by using denaturing gradient gel electrophoresis (DGGE). In this method, PCR-amplified fragments are separated from one another on the basis of their DNA composition. As the PCR product migrates through gelacontaining a chemical gradient of formamidehrea, the two DNA strands begin to melt at the denaturant concentration equal to its melting temperature. PCR products with slightly different base compositions melt at different locations within the gel. The position and number of each band on a gel reflects the sequence diversity within the PCR product. Initially this method was developed to identify mutations in specific genes. Muyzer et al.(1 82) used DGGE to differentiate amplified products from 16s rRNA genes where the products from the strains where identical in length but different in sequence composition. This method can also provide information on the sequence diversity of a wide variety of other genes that produce PCR productsof a similar size. Therefore,this method provides an interesting alternative to use of restriction-enzyme site analysis or DNA sequencing, which are commonly used to differentiate PCR products and screen 16s rRNA clonebanks. The method has also been applied to the direct analysis of soil microbial communities in situ, where the initial mixed PCR product was analyzed directly (1 82- 185). Using DGGE in this way ( 182), i t was possible to identify members representing only 1% of the total population. Although there are many methods that use the 16s rRNA gene as a target for such studies, this approach is limited by the nature of this gene. Often there is little 16s rRNA gene divergence between closely related species, while other areas of the microbial chromosome show greater variation. For use i n PCR-based detection systems, the only prerequisites are ( 1 ) that target genes should contain conserved regions from which oligonucleotide primers canbe designed; (2) variable regions are present that give a measure of diversity; and (3) that the target gene is universally present in all individuals of a species or strain. The sequence between the 16s RNA and 23s rRNA gene has been used as a suitable variable target. Conserved areas within the rRNA genes are used for primer binding and allow amplification of the variable spacer region between the genes. This method gives better resolution between strains than the 16s RNA gene alone( 1 86). Alternatively, variable coding sequences have been used as targets for PCR. Numerous examplesincludegenesencodinginterestingmetabolicactivitiesorvirulence
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factors-e.g., polychlorinated biphenyl degradation genes in Et-rvinia carntovora (187), the hypersensitivity-pathogenicity genes in phytopathogenic Xanfhonwnas ( 1 88), the invasive associated protein (iap) gene encoding an extracellular protein (p60) in Listeria species (189,190), and a virulence gene in Aerornorzas salnlonicida (191). PCR target genes can also involve other variable genes such as the flagellin gene, which encodes the structural protein present in bacterial flagella (192). Diversity can be assessed by variations in product size, restriction sites and DNA sequences. These methods can beused for cultured isolatesor directly, using DNA obtained from the environment, the advantage being that only DNA from the target population is amplified.
4.
Release of GeneticallyMarkedStrains
Methods of DNA manipulation now make it possible to insert DNA into prokaryotic, eukaryotic, or viral hosts, creating versatile marker systems that allow assessment of the survival and spreadof strains, studieson gene transfer, and determinations of cell activity. A potential marker gene must be absent from the strain used in the study, and either absent orin sufficiently low abundancein the microbial population under study to allow detection of marked cells at an appropriate level. Potential markersincludeantibioticresistancegeneswhich, in specific combinations, canbe used to overcome ahigh background of naturally occurring antibiotic-resistant bacteria. However, selection of a strain carrying antibioticresistance markers on highly selective media can reduce plating efficiency, and the introduction of antibiotic resistance into natural systems may not be considered desirable.For these reasons, it has been necessaryto develop marker systems that do not rely upon antibiotic resistance. Such marker systems can vary considerably. For example, the .rylE gene from Pseudotnotms puticllt ( 193) encodes the enzyme catechol 2, 3 dioxygenase, expression of which can be detected by a simple colorimetric test. Bacterial colonies marked with this gene can be identified after application of catechol, whichis colorless. The appearance of a yellowcolored product (2-hydroxymuconic semialdehyde) indicates expression of.yylE. In a similar way the lacZY genes from Escherichia coli can be inserted in other species unable to metabolise lactose and used as a colorimetric marker (colorless to blue) for their detection (e.g., P . s c ~ u d ~ ) t t ~ ospp.) t ~ ~ ~(194). .s More recently the pglucuronidase gene (gusA) from E. coli has become a very useful marker (195). This is in part due t o the range of fluorescent substrates available for the enzyme p-glucuronidase and its rare occurrence in environmental samples. This has enabled the detection of single cells in situ by fluorescence microscopy ( 196). The prsA genehasalso been introduced intofungalstrainssuchas Arstrri~rmO,Vysporunl ( 197). The luciferase-encoding genes (~LLYAB) isolated from a species
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of Vibrio (198), when expressed under the correct conditions andi n the presence of an aldehyde, allow bacteria to produce light. This marker has proved to be very useful for in situ detection when combined with luminometry or specialized light sensitive cameras fitted to a microscope. The luxAB marker is widely used, since it is uncommon to find large numbers of indigenous microorganisms that emit light. Reporter genes can be manipulated to give an indication of the activity of a desirable phenotype. King et al. (199) constructed a bioluminescent reporter bacterium for the monitoring of naphthalene and salicylate bioavailability and catabolism. The system involvedthe use of a reporter plasmid encoding naphthalenedegradationand the bioluminescencereporterfunction. A transcriptional fusion between theluxCDABE gene cassette of Vibrio,fisckeri and the tlclhC gene from the naphthalene degradation lower operon ensured that, in the presence of salicylate or naphthalene, the lower operon is induced, leading to bioluminescence. On-line monitoringof catabolic activity in waste streams can be achieved by using an optical whole-cell biosensor to monitor light output continually (200). Such methods offer the potential to monitor a variety of environmental samples for a rangeof chemicals for whichan understanding of gene regulation for associated proteins is available. Nevertheless, there are limitations to the use of reporter genes. I n the case of the lacZY marker genes, culturesof the microorganism are essential for detection, while the use of the lux marker system may be restricted by the low metabolic activity of cells in situ. In some environments, the presence of q1E-conof the catabolism of aromaticcompounds tainingpseudomonadscapable precludes the use of any PCR-based method for the detection of released strains marked with this gene. However, the inherent limitationof all introduced genetic markers is that they are limited to studies on culturable microorganisms that are amenable to genetic manipulation.
V. SUMMARY AND FUTUREDEVELOPMENTS It has taken time for methods to be developed that meet the desire of microbial ecologists to learn more about carbon flow and microbial communities in natural environments. In recent years techniques have developed at considerable speed that offer the potential to answer some of the commonly asked questions. Use of "C techniques for measuring carbon flow is one example. While questions still remain about the interpretation and relevance of some modern methods regarding the real makeup and interactions in mixed populations, there is no doubt that the use of biochemical or molecular signatures providesthe best opportunity to advance our knowledge in this area.
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ACKNOWLEDGMENTS This work was supportedby the Biotechnology and BiologicalSciences Research Council (BBSRC), UK.
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Index
Actrcia serwgal, 309
Acetosyringone, 106 Acetyl-coA, 59 Acidification (see ulso Rhizosphere acidification) root induced, 149, 334, 335 pH, 334 nitrogen source, 334 Acinetohacter, 247 Aconitase, 58, 64 Actinomycetes, I 14, 115, 232, 298, 300, 384, 389 Adhesion, 267, 275 Aerornontrs cuviur, 1 IO, 395 Agrobacterium, 6, 7, 214, 236 Agrohacteriuntrudiobacter, 109 Agrohocteriurn tumefuciens, 1 1 , 106,
109, 317 Agrocin, 84, 109 A/cnligenes, 6, 236 Alkaloids, 197 Allelochemicals, 20, 23, 27, 30 isothiocyanates, 175 nitriles,175 Allelopathy, 20, 30, 33 Allium cepu, I 74 Aluminium, 26-28, 3 I , 32, 34, 284 complexation, 7 I , 72 phosphates, 53, 54, 57, 281 toxicity, 7 1-74
Amino acids. 42, 96, 98, 99, 106, I 12, 117,120,121,279,280 adsorption in soil, 50 as microbial substrate, 185 exudation, 44, 51, 52, 59, 70, 73-75 microbial degradation, 47 nonprotein amino acids, 197, 2 14 retrieval, 50, 51 transporters, 5 1 Amids as microbial substrate, 185 Ammonia, 105, 108,176 volatilization, 182 Ammonification, 278 Ammonium, 277-280 assimilation, 61 Anion channels, 52, 59, 64, 65, 67, 68. 72, 73 Anthoxanthurn odorututn, I65 Antibiotics, 2, 7, 50, 108, 109, 247-249 Antimutagenic effects, 207 Aphtrno~nyce.~, I 12 Apoplasm (see ulso Root apoplast), 52, 64, 72 Appressorium, 268 Arabidopsis, 13, 150, 364 Aromatics, 106 Arthrobacter, 7, 103, 236 Arthrohactrr~a~~e.sce?~s, 235 Ascomycotina, 264 ATP, 12, 59, 388, 389 41 1
Index
412
Cadmium, 68, 69, 7 l , 73. 284 toxicity, 73. 74, 284 Caffeic acid, 20, 30, 33, 42. 62, 203, 206.231 CeIJ'crr~rrs ceIJ'c111,60
B ~ l ~ i l l / /I .15, ~ . 214, 236, 245-247. 367
Bacteria-like organisms, 285 Bacteroid(s), 105, 310, 311, 314, 315 Barley, 6, 7, 13, 26, 32, 66-68. 74. 98, 100, 103,114,117, 118, 178. 182, 225, 232, 234, 235, 237, 240, 242-244 Basdmnycotina. .' 264 Benzoate,106 Bicarbonate, 23. 32. 70 B ~ ~ I ~ ) h n r t c , r i r244 ~rr~, Biflavonoids, 203 Biocontrol,104. 108-1 IO, 283 of plant disease, 224, 246-249, 253 Biofertilizers, 286 Bio-indicators (SCY, Biomarkers) Biolog, 6, 184, 214. 386 Biomarkers DNA/RNA, 184, 39 1-394 ergostcrol, 389 fatty acids, 184, 388, 389 lipopolysaccharides, 390 phospholipids,184 quinones, 390 Biotin (vitamin H).216 Black locust, 303 Black root rot. 109. 1 1 0 B o t r y i s cirlc~rcYl.2 I6 Brcrcl~r/~i-obircrr~, 8. 104- 106, 208, 250, 306, 31 I , 319 Brctc/~r/~i:oOi~o,l japonicrrrr~,198, 200, 203, 209. 246, 3 12, 3 14. 3 15 '
Calcareous soils, 31. 32, 53. 65, 66, 69, 70, 239 Calcicolc plant species, 54. 223 Calcium, 343 phosphates, 53, 54. 57. 107, 281. 333 role i n membrane stabilization, 44 role in exocytosis, 53 C ~ r l ~ ~ s t c ~. sg ~i c~l/ ~ i l l 3l l lI7 , Canavanine, 215 Carbohydrates, 55. 97, 100- 102. I I8 catabolism, 55, 57-59 shoot-to-root allocation, 55 Carbon ( s c v crlso Rhizosphere) assimilation, 18I available. I60, 176.177.297 belowground,166 distribution. I 65 decomposability effect of allocation. 171 deposition, 97, 160 discrimination.178 lixation, l66 fluxes,165,166.300 effect of CO!, 164 pulse-labeling. I65 immobilization, 1 59 inputs, 165 isotopes (SW d s o Isotopes). 165, 167 doublelabeling. I86 microbial,168 mineralization,159 partitioning effect of COz. 166 respiration. 165. I66
413
Index
[Carbon root-d-rived, I77 seque!.tration. 17 1 storag: i n soil, 166 translocation,179 Carbon dioxide (COz), 12 I , 160 elevatd concentrations, 7.5 non-pl~otosynthetic fixation,55. 57 Carbon metabolism, 280 Carbon/Nitrogen (CIN) ratto, 175, 181. 297, 300, 301 Carbopenem. I O Carboxyiates, 45. 54-62, 282 adsotytion i n soil, 50 exudalion, 50-52, 60. 63. 64, 69-71 metal complexation. 45. SO, 54. 57. 63-65, 69-71 microbial degradation, 47, 48 retriecal, S 1 root-tc,-shoot allocation, 59 shoot-.()-root allocation, SS, 61 Cnrriers.148, 152 Caseinas: (see Proteases) Ctr.s.sitr torcl. 72 Casuarinsceae, 45 Cation/a81ion uptake ratio. 58 Cell wall. 271-275 Cellulosc~I I I , 171,271. 300 Cereal cyst nematodes, 125 Chalconc S. 203 Chelate.146.147, 152, 224, 226. 227, 2.!9,230,238,240.246,248.252
Chemoattractant, 62, 119, 306 Chctnoattraction. 267 Chclllodif~erentiation.268 Chemotaxis,7, 105, 200 Chenopodiacene, 55 Chickpea. 54-56, 64, 310 Chitinast, I I O . 271 Chitolipc.oligosaccharide. 307-309, 3 13 Citrate. :4, 31, 44, 47. 54, S S , 57-59, 6 i-65, 69-74, 23 I , 241. 246. 249-25 I , 335 lyase, S9 synthax, 57 OverexprcSsIoIl, 73
Cluster roots, 45, 46, 54-59. 69, 377 Cobalt, 68 Coll~~ototric.hltr,l orl?icrdtrrc3 1 I 1
Compost, 172, 225, 247 Corlvo1wlrr.s t r n w s i s . 3 I7 Copper, 68. 69. 232 toxicity, 74 Covy1rt.s c1\~ell~111n. 275 Coryneform, I 15 Cotton, 44. 109, 247, 381 Coumestans, 203 Coumestrol, 199, 268 Crop rotation 108, 122, I23 Croppingsystem122, 123 Crown gall, 109 Cucumber, 33, 74, 147. 231. 235. 239, 247 Cytokinins, 24, 107, 1 1 I Cytoplasmic acidosis. 59
Dadzein, 199, 268 Damping-off, I09 I~fllltllorlitrr i ~ ~ ~ l ~ l r ~ lI85 sl~llii, Dealninases (see Histidinase)
Denaturing gradient gel electrophoresis (DGGE) (see DNA) Denitritication, 22, 176- 178 2,4-Diacetylphlorogluci1~ol, 109, 2 12 Dicarboxylic acids ( s w trlso Carboxylates), 375 Diffusates. 3, 41. 51, 52 Disease-suppressive bacteria, 247249 bacterial wilt and, 249 siderophores and. 247-249 Disease-suppressivesoils, 108 DNA DGGE. 241-243. 394 % G+C composition, 392 hybridization, 393 manipulation. 395 rcassociation, 391 D ~ L I ~fir, Iw 267 DRB (sec, Rhyzobacteria deleterious) Drought stress, 74 Durum wheat, 71, 102
414
Ectoenzymes (see Enzymes) Ectorhizosphere, 4, 96, 115, 374, 384 EDDHA, 234 Electrochemical transmembrane potential gradient, 51, 52, 62, 63, 149 Electron microscopy, 5 , 29, 99 Endoplasmic reticulum (ER), 53, 67 Endorhizosphere, 4, 96, 113-1 15, 304, 374 Enterohacter, 245, 247 Enterohacter agglomerans, I06 Enzyme(s), 20, 23, 30, 97, 110, 160, 175 ectoenzymes, 5, 27, 28, 34, 53 effect of organic amendments, 172 effect of plant age, 174 immobilized, 171 persistence, 30 potential vs. actual, 171 rhizosphere effect, 174 rhizosphere vs. bulk soil, 172 sources, I7 I state in soil, 17 1 Envinia, 2 14 Erwinia carotovorcc, 10, 1 I , 395 Escherichicc coli, 217, 235, 245, 395 Ethanol, 23, 75 Ethylene, 23, 63, 269, 328, 373 E ~ c ~ l y p t268-270 ~.~, Exocytosis, 53, 65, 67 Exopolysaccharides (EPS), 308 Exudates (see also Root exudates), 9599,101-104,106, I IO, 112114,116,122,124,125 Farmyardmanures,125 Ferrihydrite, 50 Ferrioxamine, 232 Festucarubra, 165 Festucn ovinu, 328 Fibronectins, 275 Fimbriae, 106 Flavanones, 42, 198, 267 Flavins, 63 Fluvobacteriurn, 6, 236 Flavones, 42, 198, 267
Index
Flavonoid(s),8, 50, 105,106,197,198, 267, 306, 307, 309 as allelopathic compounds, 62 as signaling compounds, 60, 62 exudation, 60, 62 Flavonoid-NodD complexes, 8 Flax, 69 Flooding, 75 Flow cytometry, 386 Forest ecosystems, 277-280 Formononetin, 268 Free radicals, 71 Freezing injury, 118 Fulvic acid(s) (see Humic substances) Fungal spore germination, 267 Fuscoiunl, 247-249 Fusarium o.ry.sporu~n,109, 1 IO, 395 Fusarium solmi pllccseoli, I 12 Fusariumwilt,125,247-249 Gneurllorl,lomyce.sgran~inis,247 Gc~eurnarlnonlyce.sgran~inisvar.
tritici, 109,214 Genetic diversity, 264 Genetically modified microorganisms released into the environment, 187 Genistein,199 Gibberellins, 107, I 1 I , 150 Gigasporn rnurgcrrita, 285 Glomus, I72 Glomus vers~jbrnle,270, 273, 282 p-1.3 Glucanase, 1 IO, 1 1 1 Glucose, 20, 42, 59, 331, 379, 381 respiration,168 utilization,163 Glucosinolate,175 Glutamate dehydrogenase, 280 Glutamate synthase, 280 Glutamine synthetase, 280 Glyceollin, 200, 268 Golgi apparatus, 53 Greenmanures,122,125 Growthexperiments,177,178 Growth yield, 100 Gutierrezicc srcrothrcle, I2 1 Gymnoascaceae, 28 1
415
Index
H'-ATPase (see also Plasma membrane),149,171,172,282 H +-ATPasegene, 12, 13 Hokaa undulrta, 60, 69 Hartig net, 271 Heavy metals, 68-7 I , 284, 316, 381 Hehelorno erlrsrrrlirlifonlle, 28 1 Hehelornn c:\lirldro.si~nrrrrn,267-269 Hexose, 280 Histidinase,174 Holclts Irrrmtus, I65 Hordelyrn[t.s europaem. 69 Hormones (see c r l s o Phytohormones), 97, 150-152, 303 Host specificity, 210, 306-309, 3 12 Humic acid(s) (see Humic substances) Humic molecules (see Humic substances) Humicsubstances,2,141,142, 146 effect on nutrient accumulation, 147 effect on plant growth, IS0 fulvic acid(s), 142, 225, 231 humic acid(s), 53, 70, 142,144.149, 225. 231 hurnins, 27 in soil solution.l43 metal complexes, 145- 147 molecularstructure,142,143 molecularweight,143,144,147, 149 nutrientsin,143 solubility,142,143,145-147 Hyacintlnrs, 267 Hydraulic lift, 75 Hydrocarbons,125 Hydrogencyanide (HCN), 108, 109 Hydrolases, 17 1 extracellular. 5 Hydrolytic enzymes, 281 Hydrophobins. 275 Hydroponics, 46, 239 2-Hydroxyphenazine, 2 12 2-Hydroxyphenazine- 1 -carboxylate, 212 Hydroxyproline-rich glycoproteins, 1 I I Hymer~oscy~lu~s ericrre, 28 I Hypaphorine. 270 H~~)hor~licrohiurl1, 244
Hyphosphere, 264, 280 Hypoxia, 75 IAA (see Auxins) Ice-nucleation reporter, 240, 241 Ice-nucleation proteins, 47, 378 Indolacetic acid (see Auxins) Indole-3-acetic acid (see Auxins) Induced systemic resistance. 1 I I , 247249 Inositol hexaphosphatcs, 281 Insertion elements, 3 12, 3 19, 320 Integrin, 275 Intergenic spacer, 266 Internal transcribed spacer, 266 Ion channels, 52, 53 Iron, 26, 31, 32, 34 amorphous, 50 chlorosis,147,234 deficiency, 45, 62-69 humatecomplexes,147 mobilization in soils. 45, 63-66, 146 oxidedhydroxides, 50, 66 phosphates. 53, 54, 57. 60 plant-microbial competition, 233, 234 reduction, 63, 64, 147 root-to-shoot translocation, 64, h7 stress response, 63, 146, 224, 229, 239-241, 249, 251 uptake mechanisms, 230, 233 Isoflavones, 203, 267 Isoflavonoid(s), 61, 198, 375 estrogeniceffect,198 Isoliquiritigenin,199 Isotopes (see ulso Carbon isotopes) "C, 178 "N,178 dilutiontechnique,178, 180, I83 labeling "C, 381 "C, 178, 376-381 "CO2, 97, 99, 376-381. 384 P-Isoxylinonyl-Alanine (P-IA), 215
416
Index
krcclrritr hicolor, 267
Lactate. 59, 75 Leaching,177 Lectins.106,307, 316 Legumes. 8, 98. 105, 120,123.184, 197, 306, 3 10 Lefcctrerlcf.8 Ligand exchange, 50, 54, 65 Light intensity, 3, 74, 121 Lignin. I 1 I Lignolysis. 28 I Lipo-chitin-oligosaccharides(LCOs), 8 Lipopolisaccharides, 308 Liquiritigenin, 9, I99 Litter, 278, 279 L o l i f r r r r , I64 L o / ~ I / I/> C J ~ CS. S~CCU /O~ , III I~ I ~ I U( s c , ~ t
t l . To~ ~
mato).117,121,248 Lysate(s). 3. 4. 6. 41, 97, 98, 225, 252, 373 Maize
22-24, 3 I , 32.66-68.74,143,149, 150, 177, 216, 234, 235, 245, 357, 375-378 Malate, 24, 47, 54-58, 69, 71-73, 147 Manganese, 21, 25, 26, 30, 32-34 oxidation. 69, 70 oxidedhydroxides, 21, 26, 30, 32. 33. 50. 69 reduction, 69, 70 toxicity. 69 Mechanical impedance, 3, 43, 54 ( s e c c/lso Zm rtltrys),
Medicugo / / I / M I ~ I I3~17 Medictcgo strtivtr. 8 M ~ d i c t / g otrur~ccrtula.282
Membrane(s) permeabilty, 31. 32. 44, 52. 59, 149, 172 stability. 44. 70, 71 Mesor/li~ohi/rr?r, 208 Mesorlri~obiurrlloti, 3 12 Metal chelate transport, 230 dissolution. 229 Methyl scillo inosamine. 3 16 Microarthropods, I23 Microautoradiography. 378 Microbial communities, 224. 239. 24 1-243 ecology, 224, 230, 251 iron nutrition. 224. 229 substrate assimilation direct route. 183 rDNA 16s. 236, 241 -244 rhizosphere competence. 223. 224. 244-246. 253 Microbial activity. 47 statefunction,176 Microbialbiomass. 159, 349 C/N ratio.175,181 culturablemicrobes.186 dormant vs. active.184 estimation,180 nutrient content, 176 specific growth rate. 100 Microbial colonization, 45, 47 Microbial diversity, 4 ecosystemfunction, I84 effect of plant residue, 184 molecular techniques. 186 ribotypes TGGE, I85 Microbial growth, 350 0 Microbial population (see ~ 1 1 . ~Rhizosphere) detection cells, 386 CFU.385 DNA, 39 1-395
41 7
Index
[Microbial population] fatty acids, 388 marked strains. 395 dynamics, 97 Microfauna, 101. 123,263,384 Micronutrients.68-71.145,146 Microorganisms, 45 Microscopy, 283. 285, 381. 386 Microsuction cups, 46 Mimosine, 8, 316 Mineralfertilizers.122 Mineralization, 98, 99. 101, 104,121, 123- 125, 278 Model(s), 226, 227, 229 chemical speciation, 226 kinetic processes iron, 226 Model assumptions constant root radius, 353 exponential root growth, 359 Model compounds I ,S-diphosphate carboxylase. I83 Model solutions analytical solution, 341 by analytical approximation. 345 by numerical approximation, 339 validation, 347 Modeling Barber-Cushman model, 357, 362 boundary conditions, 336 convection, 332 diffusion, 330 finite-difference, 340 finite-element.341 microbial population dynamics. 348 PCclet number, 343 sensitivity analysis, 345 solubilization of P, 335 short-range transport and uptake, 330
uptake in heterogeneous environments, 363 uptake at the plant scale, 352 Molecular cross-talk, 3 Molecular signals ( w e crlso Signaling molecules), I , 2, 307, 309 Mucigel, 96
Mucilage, 23-25, 27-29, 45, 60. 7274, 96, 97. 115, 118, 160 hydraulic conductivity, 29 permeation of soil, 28. 29 secretion, 29, 3 0 Mugincic acid(s) ( S W n l s o Phytosiderophores), 225 Mutualistic plant-fungus association. 263 Mycorrhiza(e), 3, 24.104,107.108, 235, 239, 264, 362 arbuscular, 60, 265-270 arbutoid, 265 ectomycorrhiza(e), 60, 265 ectomycorrhizal fungi, 268-27 I , 279-282 endomycorrhizae. 265-270 endomycorrhizal fungi, 282 ericoid, 265, 266, 270 ericoid fungi, 270, 27 I , 279-28 I monotropoid, 265 orchid mycorrhizae, 265, 270 symbiotic bacteria, 270 vesicular-arbuscular (VAM), 24 Mycorrhizal fungi (SLJC Mycorrhizae) Mycorrhizal helper bacteria, 283 Mycorrhizal symbiosis, 263 Mycorrhizoplane, 263, 277 Mycorrhizosphere. 4. 263 Myrosinase (see P-Thioglucoside glucohydrolase) Myxotrichaceae, 28 I N-(3-oxo)-dodecanoyl-L-honloserinelactone(ODHL), I I N-(3-oxo)-hexanoyI-L-homoserinc lactone(OHHL), 10, 1 1 N-(3-oxo)-octanoyl-L-homoserine lactone(O0HL). 1 1 N-acyl-L-homoserine lactone nucleotides, 1 1 N-acyl-L-homoserine lactones (AHLs). 10
NAD, 57, 58 NADH. 57. 58, 64, 66 NADPH, 64. 66
418
Naringenin, 9, 199 Nematodes. 123-1 25 bacterial-feeding population dynamics,177 C/N ratio,177 rhabditid, I77 Nickel. 68 Nicorianamine, 67 Nitrate, 150, 279,280 assimilation, 58, 60, 61 transporter, 1 SO transporter gene (ArNTRI), 150 uptake, 58, 149 Nitratereductase,149, 150 Nitrification, 176, 278 inhibitors,178 Nitrifers, 244 Nitrogen,60,98, 1 0 1 . 104, 105. 107, 108, l IO, 119-124 acquisition, 277 assimilation. 280 ammonium vs. nitrate, I79 direct rou~c.181 plant vs. microbes, 181 plant-microbialcompetition,179 preferentialforms,179 deposition, 98 different pools. 178 immobilization,22. 159, 175,178 in humicsubstances,144 metabolism. 278 mineralization, 159 enzyme activities, 175 protozoangrazing.175 root-induced,175 minel.alization-inllnobilization turnover. 181 netmineralization,I75 residue C/N ratio, 182 residue chemical composition. 182 rhizosphere effect, 175 organic. 278, 279 plantavailable,I75 root-derived,175 supply,160 transformation processes, 178
Index
[Nitrogen] transforrnationmodels,175 uptake microbial vs. plant, 178 Nitrogenfixation. 100, 105, 107,108, 235, 249 Nitrogen fixing bacteria, 250 Nitrogenase, 249, 297-299 Nitro.sococw.r,6, 244 r ~ o c lexpression, 8 Nod factors, 208 r~odgene-inducer, 8, 62 r ~ o genes d (see Nodulation genes) r~odDgene, 8 NodD proteins, 8 Nodulation,8,62. 105, 108, 199 gene products, 21 I factors, 209, 2 I O Nodulationgenes. 105. 106 Non-cultivable bacteria, 386, 388 Nuclear magnetic resonance, 282 Nucleic acids, 97 Nucleotides,20,106 Nutrient solution (see solution culture) Nutrient(s), 119 accumulation.147 acquisition, 21, 23, 28. 30, 150 availability,22.141 content, 141 gradients, 328 immobilization, 22 160 uptake.142.149,152, Oat, 66, 239 Oidiorierldrotl, 28 1 Oil-seed rape, 54. 55. 65 Opins. 3 17 Opportunistic microorganistn. 5 Organic acids, 20, 23, 27, 30-32, 96, 101, 114.120,143,146,147 a s microbialsubstrates.185 oxidation in soils. SO persistence. 30 Oxalate, 44, 54, 55, 60, 7 I , 72 Oxygen deficit, I19
Papaya, 73 Parasitism, 1 1 0
index Pos/~lllurllr l o r c l t t c r : l , I00
Pathogenesis-related proteins, 1 I I , 27 1 Pathogens,104,108-1 13, I 19,125 PCR-DGGE (see also Denaturing gradient gel electrophoresis), 6 Pea (see LIISO Pisltrrr), 28, 215, 313, 316 Pectins, 27 I PEP-Carboxylase. 55, 57, 58, 64, 73 Peptides as microbial substrates, I83 Peroxidase. 20, 42, 53, 1 I I , 307 Pesticides,122,125 Petunidin, 9 PGPR ( s c v Plant growth-promoting rhizobacteria) pH107, 117,119,120,122,334 PH-stat, 58, 61. 62 Phctseollts twigoris, 9, 58, 59. 199.307 Phenazine- 1 -carboxylate, 109, 212 biosynthetic operon (ph;ABCDEFG), 213 Phenolics, 30, 33, 42, 106, 120, 377 adsorption in soils, 50 antibiotic properties, 60, 62 exudation, 50-53. 59. 60, 62-64, 69, 70 oxidation in soils, 50, 60, 62 root products. 27 secretion. 34 Phenylpropanoids, 267 Phorna Detu, IO9 Phosphatase, S . 30. 144. 172, 174 acid, 42. 53. 57. 60, 61, 75, 283 Phosphate transporter, 282 fungal transporter gene (GvPT). 13 high-affinity Pi transporter gene (LePTI). 13 Phosphodi-esterases, 281 Phosphohydrolases role in P mobilization. 60 root secretion, 60. 75 Phosphornono-esterases. 28 1 Phosphorus, 104.107, 281. 327 deficiency. 24. 30. 32, 45. 53-60. 69 in humic substances. 144 mobilization in soils. 50. 53-55. 57, 60 metabolic bypass reactions, 55. 60 organic. 30. 60
419 Photohacrrriwrl fischeri, 10, 1 1 Photorl~izobi~trrl thortlp.sor~icrrlrr:t~, 299 Phyllohac.teriurrl. I I 5 Phytase, 42, 53, 60, 174 Phytates, 60 Phytoalexin(s), 5 , 27, 28, 30, 200, 307
resistance, 202 Phytoactive compounds persistence, 2 I Phytohormones (.SL,P a l s o Hormones), 5 , 24, 27, 42, 63, 104,107, 112, 152, 269 Phyrophorfl, l 12 Phytophorcl cir~ar:~or~i, I 12 Phytosiderophores (see d s o Mugineic acid), 6, 23. 24. 26, 28, 45, 49, 50, 65-69, 120,146, 223, 225227, 230-235, 240, 24 I , 25 1253, 349 biosynthesis, 66, 67 diurnal production, 232, 233 exudation. 52. 53, 65-69, 73 metal complexation, 65, 66. 68. 69 uptake, 51, 66 Pigeon pea (sec Cajurlrrs c u j m ) Phytotoxins, 2, 20, 27. 112 Pigments, 97 Pili, 106 Piscidic acid, 60 Pisolitlnrs tirlcrorius. 268. 270, 275, 279 Pi.sttr:r (.see nlso Pea), 307, 3 10 Pisfrrll .scrtiv1rrn, 70, 1 I9 Plantage,102, 103 Plant growth-promoting bacteria. 245 rhizobacteria (PGPR), 104. 1 I O , 250. 263, 284 Plant growth regulators, 2 Plant redisue C3 1's. C4plants.167 Piar~tflgo ktr~c~cwlutu. 75, 328 P~asmalemma(see Plasma membranc) Plasma membrane. 52, 23 I HI-ATPase, 52. 53. 59, 63. 64, 150152, 171 reductase, 63. 64, 15 I Plasmid(s), 249. 3 I 1-3 15, 3 17-3 I 9, 392 Polyamines. 15 1 Polygalacturonase. 20, 28 I
Index
420
Polypeptides, 32, 73, 97 Polyphenolics. 281 Polyphosphates, 282 Potassium, 51, 52, 60, 62, 63, 65, 72, 108, 333. 347 mobilization in soils, 63 Potato.72.184.185 Predation. 116, 122,123 Prosopis chilerrsis, 309 Proteaceae. 45, 54. 69 Proteases, 172.174,18 I , 279 Proteindecomposition,183 Proteoid roots (sec Cluster roots) Protons. 23, 45 Protozoa.122-124, 305, 308 population dynamics, I76 P n r d l u , 267 Pseudobactin, 234, 235. 245, 246, 248, 249 Pseudomonads, 108, 1 15, 185, 212, 233, 236-241, 245-247. 249-253, 284, 367, 386, 396 P.sc~l~~lorlrorlcl.s, 6, I IO, I I I , 1 15, 233, 236-240, 242. 245-249 Pserrdorrlorm aereqfircietls Q2-87, I09 P.seur1omorltr.sc r c w g i r r o s o , 7, 1 I , 73, 103.106 Pscwdornotms jluorescens. 101 , 106, 112, 1 15, 116, 213, 285 P.seurlorrlotlr/.sj1uore.scerI.s 2-79, 109, 378 Pseudorrlonus jluore.scevl.s CHAO, 108.109 Pse/rtlonlorrcrs jhorescerls F1 13, I09 Pselrdorrlorlcrs fluoreseem M. 3.1., 7 Pseuciortwrlus ,fluorescctIs Pf-5, 109 PseLtdonrorus JIuorescms SBW25, I l6 Psertdorrrorlcls kdIrwrrurls, 106 Pseudornorm putickr, 106. 395 Pseudotnorlcrs svrirlguc), I 10
Pterocarpans, 203 Pulse-labeling,170 Pyocyanine, 2 I2 Pyoluteorin, 109, 2 I2
Pyoluteorinl, 109 Pyrrolnitrin,109, 212 Pythiurn. 1 10, I 12, 247 Pvfhirorr Icltitnurn, 109, 1 12, 216
Quercetin, 268 Quercus suber, 276 Quorum sensing, 1 0 , 314
r and k strategy, I 16, 306. 385
Red clover, 54 Reducing agents persistence, 33 Reductase (see r r l s o Plasma membrane), 230-233 Relative addition rate. 360 Reporter bactcria, 47. 378, 396 Reporter gene, 240 Respiration. S8 cyanide-resistant. 58, S9 cytochrome pathway, 58 Rhizobacteria, 3, 7. 98, 99, 104, 108, 110, 112,114, I I S , 285 Rhizobacteria deleterious (DRB), 104, 112.113.24.5 Rhizobactin. 249, 250 Rhizobia, 3, 21, 28, 60, 62, 105. 107, 108, 197, 284. 297, 299, 300. 304, 306-3 16, 3 18-320 Rhi:obirrrtl, 7, 8, 104- 106. 2 14, 250, 251, 297, 309, 313 Rhizohirrrrl efli, 9. 308, 3 14 Rhi:ohiton kcdysnri, 3 10, 3 16 Rhizobium I~.Xrtrrlitlo.strnrrrl, 9, 285, 304, 307, 3 14, 3 15, 3 I8 ~ h i ~ O ~ J i l lnC'[;/Ofi. Utl 106
Rhizobiunr tropic;, 2 I O Rhizobium-legume symbiosis, 62, 105,
197. 250. 297, 298. 300, 301, 31 1 Rhizo-box, 43, 46 Khtocrorlicl solorri, 109, I I O . 2 I6 Rhizodeposition, 5. 7, 22, 24, 25, 9599, 121,123 composition of, 4 I definition, 373 factors affecting release, 41, 75
421
Index
[Rhizodeposition] mathematical model, 376 measurement, 375. 377, 379, 383 in soil, 377 in solid supports, 376 in solution culture. 375 process. 373 Rhizodeposits. 22 decomposability,169 effect of plant species. 165 exudationrate.163 Rhizodermis, 63, 64 Rhizoferrin. 66, 23 1-233, 256 Rhizoplane, 4, 64, 95. 263, 306, 3 16, 336. 374, 384, 386 Rhizopus, 235
[ Rhizosphere(s)]
R1Iiwpu.s stolon~feru. 109 Rhizosphere(s). I , 19, 21 acidification (see crlso Acidification), 57, 58. 62-64. 69 biodiversity, 159. I83
Right set of circumstances, 2 I , 30-34 Kobiuin l~.sc~~ctioc~coeirr, 303 Root age,162 clumping, 22 competition between. 338 decomposition, 166 elongation/differentiation zone, 160 function, 2 1 lateral, 23 mass, 160 meristematiczone. 160 morphology. I 50, 160 order, 353, 357 plasticity, 364 radius, 337, 354 tip (see Root apex) Root apex, 45, 54. 160 Root apoplast (see d s o Apoplasm), 337 Root cap, 96 cells, 23, 24 Root exudates (see NISOExudates and Root products), 224. 225. 239. 252, 253, 335 collection in trap solutions, 42-45 collection media, 44 collection techniques, 42-5 I , composition, 20, 42 concentration techniques, 44, 45 dialysis, 45
biologicalcontrol, I76 carbon budgets (see also Carbon). 377 carbon flow ( s c ~ 1 1 . ~Carbon), 0 374 colonization, 22, 25 definition, 19, 327 dimension. 160, 162. 175 effect. 159, 160, 383 exchange of genetic material, I87 feedback, 329 gradients longitudinal, 22 radial, 22 metabolic diversity effect of CO2concentration.185 microbial community structure, I85 microbialcomposition, I84 microbial population (see c11.so Microbial population), 162, 383 microcosm, 375-377, 382 mineral weathering, 161 nitrogentransformations.178 nutrient availability. 22 nutrient cycling, 176 nutrientdistribution, 160
nutrient flow, 159 overlapping, 22 pH,107.120, I22 products, 46 respiration, 24 soil, 46 extraction, 46 separation, 46 solution, 46, 54 volume, 162- 164, I86 water potential, 160 Rhodotorulic acid, 23 1 Riboflavin. 20, 23, 216, 231 Rice, 66, 70, 144, 235. 240. 304, 335 Kicir2us cortzmurlis. 59
422
[Root exudates] lyophilization. 44 precipitation, 44, 45 solid-phase extraction, 44 ultrafiltration, 45 vacuum evaporation. 44 diffusion, 5 l, 52 localized collection in soil culture, 43, 46, 47 solution culture, 43, 46 localized release, in cluster roots, 45, 54, 56, 57 in (sub)apical root zones, 45, 54, 66, 7 1-73 release mechanisms, 5 1-53, 64, 65, 72. 73 microbial degradation, 45, 47-50, 54 recovery, 47-5 1 retrieval mechanisms, S O , 5 1 re-uptake, 350 temporal changesin release, 46.66.74 Root growth, 2 I , 22, 27, 45, 54, 161, 162 Root hairs, 160. 336 Root hair branching, 208 Root injury, 5 1 Root products (see ulso Root exudates). 21, 23-28, 34 clnssitication, 23, 27 debris, 23-25 exudate(s). 20. 23, 26 diffusate(s), 23. 25 excretion(s), 23, 24 secretion(s), 23-28, 31. 34. 41 vesicles, 53, 67 Rootsensing.12 Root windows, 46 Roses.109 Rotenoids. 203 S-adenosyl-methionine (SAM). 67 Salicylic acid (SA), 248 ScIwotirli(1. 247 scl~~roiill~ ro/jiii. ?l I 10 Secretions (.sec d s o Root products), 4 I persistence. 27, 34
Index
Serine, 106, 121 Scw-atin mursesc'ens, 1 10
Siderophores (see d s o Ferrioxamine, Pseudobactin, Rhizoferrin, Rhodotorulic acid), 2, 3. 6, 7, 65, 66, 108, 146, 223, 230, 235, 244, 246, 249 bioassays, 235 catecholate type, 235, 250 concentrations in rhizosphere. 230, 235, 237, 252 degradation, 234 extraction from soil, 229, 230, 235 hydroxamate type, 235, 250 production by bacteria, 237 schizokinen, 235 utilization by plants, 233 Signaling molecules (see a l s o Molecular signals), 267 Sirlorhi,obiut,l mdiloii, 9, 2 17, 3 I 5 , 3 17 Sitzorhtobi~trrlsaheli. 2 IO Sirrorhizobium rercrngcc, 2 1 0 Sloughed cells, 3 Soil buffer power, 33 1, 346 bulk,46.160 culture. 43-47 extraction, 46. 47 planted vs. unplanted, 163 respiration. l67 Soil organic matter decompositionrate,I78 microbialturnover,175 refractory. 160, 169 Soil organisms mutualistic, I86 saprophytic, I O 1. 186 Soil stabilizers, 2 Soil structure, 27, 28 heterogeneity, 22 poor, 22 Soil-plantmicrocosms, I80 Soil-plantsystems. 161, 178 Soil-plant-microbesrelationships, 159, 176, 187 Soil-root interface, 160. 277
Index
Solutes available, 331 exchangeable, 33 1 unavailable. 33 1 Solution culture, 43-46 features, 2 I failings, 22, 33, 34 reabsorption in, 26 relevance, 23 S o r g h m , 33, 66, 285 sorghloll \ d g a r e , 366 S/~h~tl~~orlrolln.s pnlicirllohilis, I I 5 S p i ~ ~ r t wolerrrcetr, r 54, 69 Spinach (see Spirlncrcr olercrcen)
Sporopollenin, 270 Stonefruit. 109 Strategy I plants, 63-65. 146, 148. 231 Strategy I1 plants, 65-69. 146, 148, 233 Strawberry, 74 Stress factor, 3 Stress response, 25, 26, 31, 32 Substrate(s) distributioninsoil,160 high molecular weight cellulose, I7 I chitin, 17 1 humic molecules, 17 1 lignin, I7 1 pectin. 17 1 litter, 161 movement,163 particulate.162 interaction with extracellular enzymes, 17 1 physical behavior soluble v s . particulate.169 residencetime,160 soluble, 162 utilizationprofiles, 185 transformation,160 Sugarbeet, 109, 1 12 Sugars, 42, 96, 99, 106, 112, 114, I19 adsorption i n soil. 50 exudation, 44. SI, 52, 59, 70, 74, 75 microbial degradation, 47 retrieval, S0
423
Sulphur, 3 in humic substances, 145 Superoxide dismutase, 7 1 Sustainable agriculture. 286 Symbiosis-regulated acidic polypeptides, 275 ss.sitllbrilrlll OfJicirlclla, 55, S6 Take-all, 109, 125 Tartaric acid, 20. 42, 71 Temperature, 74, I 15, 1 17, 1 18 Thiamine (vitamin B,), 20, 216 P-Thioglucosideglucohydrolase, I75 Tllirlviopsis hnsicoltr, 109, I I 2 Tobacco. 73, 109, 1 10, 214 Tomato(es), 13. 33, SS, 56, 60, 74. 179, 216. 248, 360 Transfer cells, 63 Transgenic plants, 73, 187, 270 Transporter genes, I2 Tricarboxylic acid (TCA) cycle, 57-59. 64 Trijolim tr/e.uc~nrlrit~~tnl, 174, 304 T r ~ ) l i u n reperls l 9, I 17, I2 I Trifoliwtl,subte,.rntlc~lr~ll,I98 Triticrtrrr w s t i v u r n , 174 Trrhar I I I U ~ ~ ~ I ~ L 275 ~ I I I ,
Urea, 250 direct assimilation, 182 high-aftinity uptake system. I8 1 turnover rate. I8 1 Vigtlcr slthterrcrrlen, 309 Vigr1cr rrtlglticlrlelrtr, 309
Virulencegenes, 106 Vitamins.20, 97, 124, 197. 216, 245. 284 Vitronectins, 275 Water availability, I 18 Weathering, 279 Wheat, 55, 56, 66, 72, 73, 98. 101-103, 109, 114-117,120,122,123, 232, 249
424
White lupin (see nlso Lupine and bpifiI4.s cllblrs), 24, 30, 31, 45, 74, 375, 377
Yang cycle, 67 y s - l maize mutant, 69
,vs-3 maize mutant, 67
index Zerr m r y s . I 17, 1 19, I2 I , I67
Zinc, 232 deficiency, 52. 68-7 I. membrane stabilization, 7 I mobilization, 68-71, 74. 75 toxicity. 284 uptake, 69 Zygomycetes, 270 Zygomycotina, 264
