The Rhizosphere
Agricultural Engineering Animal Science
Crops
Imgation and Hydrology Microbiology
Plants Soils
Editorial Board
Robert M. Peart, University of Florida, Gainesville Harold Hafs, Rutgers University, New Brunswick, New Jersey
Mohammad Pessarakli, University of Arizona, Tucson
Donald R. Nielsen, University of California, Davis Jan Dirk van Elsas, Research Institute for Plant Protection, Wageningen, The Netherlands L. David Kuykendall, U.S. Department of Agriculture, Beltsville, Maryland
Jean-Marc Bollag, Pennsylvania State University, University Park, Pennsylvania
Tsuyoshi Miyazaki, University of 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-Marc Bollag Soil Biochemistry, Volume 8, edited by Jean-Marc Bollag and G. Stotzky Soil Biochemistry, Volume 9, edited by G. Stotzky and Jean-Marc Bollag 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.
Humic Substances in the Environment, M. Schnitzer and S. U. Khan Microbial Life in the Soil: An Introduction, T. Hattori
Principles of Soil Chemistry, Kim H. Tan
Soil Analysis: Instrumental Techniques and Related Procedures, edited by Soil Reclamation Processes: Microbiological Analyses and Applications, Symbiotic Nitrogen fixation Technology, edited by Gerald H. Elkan
Soil-Water Interactions: Mechanisms and Applications, Shingo lwata and
A. I. Goring and J. W. Hamaker
Keith A. Smith
edited by Robert L. Tate Ill and Donald A. Klein Toshio Tabuchi with Benno P. Warkentin
Soil Analysis: Physical Methods, edited by Keith A. Smith and Chris E.
Growth and Mineral Nutrition of Field Crops, N. K. Fageria, V. C. Baligar, and Semiarid Lands and Deserts: Soil Resource and Reclamation, edited by J.
Keith A. Smith Mullins
Charles Allan Jones SkujinS
Plant Roots: The Hidden Half, edited by Yoav Waisel, Amram Eshel, and UZi Kafkafi
Plant Biochemical Regulators, edited by Harold W. Gausman Maximizing Crop Yields, N. K. Fageria
Transgenic Plants: Fundamentals and Applications, edited by Andrew Hiatt Soil Microbial Ecology: Applications in Agricultural and Environmental 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 Mohammad Pessarakli 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 Ap- Selenium in the Environment, edited by W. T. Frankenberger, Jr., and Sally Plant-Environment Interactions, edited by Robert E. Wilkinson
Handbook of Plant and Crop Physiology, edited by Mohammad Pessarakli Handbook of Phytoalexin Metabolism and Action, edited by M. Daniel and R.
P. Purkayastha
Soil-Water Interactions: Mechanisms and Applications, Second Edition, Re- vised 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 Phytohormones in Soils: Microbial Production and Function, William T.
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, Second Edition, Revised and Expanded, Photoassimilate Distribution in Plants and Crops: Sourcesink Relation- Mass Spectrometry of Soils, edited by Thomas W. Boutton and Shinichi
Management, edited by F. Blaine Metting, Jr.
proaches, edited by Amarjit S. Basra Benson
Frankenberger, Jr., and Muhammad Arshad
edited by Yoav Waisel, Amram Eshel, and Uzi Kafkafi ships, edited by Eli Zamski and Arthur A. Schaffer Yamasaki
Chemical and Isotopic Groundwater Hydrology: The Applied Approach, Fauna in Soil Ecosystems: Recycling Processes, Nutrient Fluxes, and Agri- Soil and Plant Analysis in Sustainable Agriculture and Environment, edited Seeds Handbook: Biology, Production, Processing, and Storage, B. B.
Modern Soil Microbiology, edited by J. D. van Elsas, J. T. Trevors, and E. M.
Growth and Mineral Nutrition of Field Crops: Second Edition, N. K. Fageria, Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Plant Pathogen Detection and Disease Diagnosis, P. Narayanasamy Agricultural Systems Modeling and Simulation, edited by Robert M. Peart 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 Arthur Wallace 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 Mycotoxins in Agriculture and Food Safety, edited by Kaushal K. Sinha and Plant Amino Acids: Biochemistry and Biotechnology, edited by Bijay K. Singh Handbook of Functional Plant Ecology, edited by Francisco I. Pugnaire and Handbook of Plant and Crop Stress: Second Edition, Revised and Ex- Plant Responses to Environmental Stresses: From Phytohormones to Ge- Handbook of Pest Management, edited by John R. Ruberson
Environmental Soil Science: Second Edition, Revised and Expanded, Kim H.
Microbial Endophytes, edited by Charles W. Bacon and James F. White, Jr.
Plant-Environment Interactions: Second Edition, edited by Robert E. Wil- Microbial Pest Control, Sushi1 K. Khetan
Soil and Environmental Analysis: Physical Methods, Second Edition, Re- Second Edition, Revised and Expanded, Emanuel Mazor
cultural Production, edited by Gero Benckiser by Teresa Hood and J. Benton Jones, Jr.
Desai, P. M. Kotecha, and D. K. Salunkhe H. Wellington
V. C. Baligar, and Charles Allan Jones Defense Mechanisms, P. Vidhyasekaran
and R. Bruce Curry
and Radhey La1 Kushwaha Deepak Bhatnagar
Fernando Valladares
panded, edited by Mohammad Pessarakli nome Reorganization, edited by H. R. Lerner
Tan
kinson
vised and Expanded, edited by Keith A. Smith and Chris E. Mullins
Interface, Roberto Pinton, Zen0 Varanini, and Paolo Nannipieri
Additional Volumes in Preparation
Woody Plants and Woody Plant Management: Ecology, Safety, and Envi- Handbook of Postharvest Technology, A. Chakraverty, Arun S. Mujumdar, Metals in the Environment, M.
N.
V. Prasadronmental Impact, Rodney W. Bovey and G. S. V. Raghavan
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
M A R C E L
MARCEL DEKKER, INC.
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The research on plant-soil interaction is focused on the processes that take place in the rhizosphere, the soil environment surrounding the root. Many of these processes can control plant growth. microbial infections, and nutrient uptake.
The rhizosphere is dominated by organic compounds released by plant roots and microorganisms. Furthermore, stable components of soil organic matter. namely, humic and fulvic substances, can influence both plant and microorganism metabo- lism. A variety of compounds are present in the rhizosphere, and they range from low-molecular-weight root exudates to high-molecular-weight humic substances.
The chemistry and biochemistry of these substances are becoming more and more 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 and updated 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.
the book presents a good opportunity to summarize information concerning agronomy, soil science, plant nutrition, plant physiology. microbiology, and bio- chemistry. The book is therefore intended for advanced students, and researchers in agricultural, biological, and environmental sciences interested 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 rhizo- sphere, 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 plant roots to modify the rhizosphere in order to cope with unfavorable stress-reducing conditions. These aspects are dis-
iii
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 mycor- rhizae 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 iron at the rhizosphere; a wealth of literature is already available on this subject and many studies on the production of siderophores by microbes are being carried out. Their potential use by plants and their relationship to other plant- borne iron-chelating substances are still a matter of debate (Chapter 8). The ful- fillment of the nutritional requirement of plants and microorganisms also depends on the processes leading to mineralization and humification of organic residues (Chapter 6). The presence of humic and fulvic substances can have a considerable effect on root habitability, plant growth, and mineral nutrition (Chapter S).
Knowledge of these aspects needs to be reconsidered at the rhizosphere (Chapters S and 6). The development of specific models can shed light on the events taking place at the rhizosphere (Chapter 1 l ) . Validation of the models and a better under- standing 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 contributions will stimulate further interest in and work on this fascinat- ing topic.
Roberto Pitltotl Zerm V ~ m r l i r l i P m l o N m n i p i e r i
111 ...
v r r
I .
2 .
3.
4.
S .
6 .
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 Types, Amounts, and Possible Functions of Compounds Released into the Rhizosphere by Soil-Grown Plants Nicholas C. Urerz
The Release of Root Exudates as Affected by the Plant’s Physiological Status
Giirlter Neurnann and Volker Riirnheld
The Effect of Root Exudates on Rhizosphere Microbial Populations
Melissu J . Brirnecornbe, F r m s A. De Leij, c r r d J m x s M . Lyr1ch
Direct Versus Indirect Effects of Soil Humic Substances on Plant Growth and Nutrition
Zerw Vnrclniwi and Roberto Pinton
Mineralization and Immobilization in the Rhizosphere Luigi Bndalucco m d Peter J . Kuikrnnrz
1
19
41
9s
141
159
V
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8.
9.
10.
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12.
Organic Signals Between Plants and Microorganisms Dietrich Wert1er
Function of Siderophores i n the Plant Rhizosphere D m i d Crorvlq
Mycorrhizal Fungi: A Fungal Comtnunity at the Interface Between Soil and Roots
Frrrt1ci.y M . Mrrrtitl, Sihirr Pi.rotto, m d Pnolcr Bonfirtlte Functional Ecology of the Rhizobium-Legume Symbiosis Atdrcw S p u r t i t t i
Modeling the Rhizosphere PettJr R. Dcrrrtrh r r m l Tiitla Roose
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
197
223
263
297
327
373
41 l
Luigi Badalucco Dipartimento di Ingegneria e Tecnologie Agro-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 Califor- nia-Riverside, Riverside, California
Peter R. Darrah Department of Plant Sciences, University of Oxford, Oxford, England
Frans A. De Leij School of Biological Sciences, 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 of Surrey, Guildford, Surrey, England
Francis M. Martin Department of Forest Microbiology. INRA Center of Nancy, Champenoux, France
vii
J. Alun W. Morgan Department of Plant Pathology and Microbiology, Horti- culture Research International, Wellesbourne, Warwick, England
Paolo Nannipieri Dipartimento di Scienza Suolo e Nutrizione della Planta, University of Florence, Florence, Italy
Gunter Neumann Institut fur Pflanzenerniihrung, Universitat Hohenheim, Stuttgart, Germany
Silvia Perotto Dipartimento Biologia Vegetale and Centro Micologia del Ter- reno-CNR, University of Torino, Torino, Italy
Roberto Pinton Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine, Udine, Italy
Volker Romheld Institut fur Pflanzenerniihrung, Universitiit Hohenheim, Stutt- gart. Germany
Tiina Roose Centre for Industrial and Applied Mathematics, University of
ox-
ford, Oxford, England
Andrea Squartini Dipartimento di Biotecnologie Agrarie, Universiti degli Studi di Padova, Padova, Italy
Nicholas C. Uren Department of Agricultural Sciences, La Trobe University, Bundoora, Victoria, Australia
Zen0 Varanini Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine. Udine, Italy
Dietrich Werner Department of Biology, Philipps-Universitat Marburg, Mar- burg, Germany
John M. Whipps Department of Plant Pathology and Microbiology, Horticul- ture Research International, Wellesbourne, Warwick, England
The Rhizosphere
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 is the result 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 literature on 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 grown on the complex biochemical interactions in the rhizosphere, although a complete integrated view coming from interdisciplinary approaches has not yet been at-
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, antibiot- ics, and soil stabilizers, can affect the microbial activity and composition of mi- croflora in the rhizosphere, as well as the activity of plant roots (6). Other com- pounds, such as enzymes, siderophores. and molecular signals, produced by 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 dif- ferent. 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 colloidal adsorbing surfaces, such as clay surfaces, in the rhizosphere can markedly decrease the concentration of these substances to very low values- making 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 ex-
tracting humic substances from soil by 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).
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 mi- crobial processes responsible for the mineralization of native organic matter,
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, sid- erophores are iron-chelating compounds secreted by microorganisms and gram- inaceous plants in response to iron deficiencies. Chap. 8 discusses the factors controlling siderophore production, as well 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 by plant roots has been shown (see Chaps. 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 translo- cated to roots, and significant proportion is released into rhizosphere-in quanti- ties 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 so- called “molecular cross talk”) between beneficial microorganisms, such as rhizo- bia 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
manipulation of molecular cross talk could protect crops from damage caused by pathogens, or improve beneficial infections to optimize nutrient uptake 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 activities of soil microorganisms. To be more precise, the soil layer sur- rounding roots should be termed as Pctorhizo.sl)iler-e, whereas the root layer col- onized or potentially colonizable by microorganisms should be indicated by on- clo~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 ~ ~ I ~ c r e is used to 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 hetero- trophical 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 i n the rhizosphere than in the bulk soil (13,16). If the effect on either microbial number or changes in microbial diversity have been detnon- strated, i t has been more difficult to give experimental evidences for the rhizo- sphere effect in terms of carbon and nutrient flows within the rhizosphere environ- ment ( 1 7).
IV. SOIL ENVIRONMENT, BOUNDARIES, AND MICROBIAL DIVERSITY
The rhizosphere lacks physically precise delimitation ( 1 8). The volume of rhizo- sphere depends on the rate of exudation and impact utilization of rhizodeposits (Chap. 6). The spatial and temporal distribution of exudates as well as their me- tabolism 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 ac- cording to: (a) their chemical properties, such as stability (e.g., hydrolysis and oxidation), volatility, molecular weight, solubility in water, etc. (Chap. 2): (h) the modality of their release (exudates, secreted, or lysates): (c) the way of utilization
by microorganisms; and (d) their function (phytohormones, ectoenzynies, phyto- alexins, etc.). Low-molecular-weight compounds are readily assimilated by mi- crobial biomass, whereas polymeric substrates such as proteins, nucleic acids,
polysaccharides, etc., are first hydrolyzed by 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 classification based 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 parti- cles. 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 because plant tissue maceration is carried out through the activity o f several cell wall-degrading exoenzymes released by the infecting microorgan- isms. 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 prescnt in 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 heterogeneity within m; individual root system might affect rhizo- sphere 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 catabolism of root exudates as well a s differ- ences in root cell activity. Usually, apical root zones are characterized by a higher capacity for release of low-molecular-weight exudates, whereas basal parts of the root system generally show higher microbial activity. The gradients of microbial
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 rhizode- position decreases as the distance from the rhizoplane increases, whereas the opposite generally occurs for the concentration of any plant nutrient in soil. In this context, the role of rhizospheric soil, rather than that of the bulk soil, is crucial for plant nutrition. It has also to be considered that very different situations can occur depending on the type of nutrient (24) and the nutritional status of plants (see Chap. 3); furthermore, different portions of the root system are charac- terized by differential nutrient-specific rates of uptake (25). All the above state- ments point to the necessity of reconsidering the concept of plant nutrient avail- ability giving more importance to the situation occurring in the soil surrounding the root.
Due to differences in rhizodeposition, different plant species but also differ- ent 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 ecosys- tems. This approach, carried out by Biolog plates (Chap. 12), discriminated mi- crobial 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, carbox- ylic acids, and amino acids, suggesting that plants may differ in the exudation of these compounds. It was suggested that the results probably reflected the Pseu- dormna.s carbon utilization profiles (26). Indeed the main disadvantage of the Biolog technique is to be culture dependent. In addition to Pseuclr~nzonns, Flavo- bac.teriurn, Alcaligenes, and A g r o h m t e r i u m species have been shown to be particu- larly stimulated in the rhizosphere due to the presence of 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 iron- stressed 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, A u r eo h a cfer iu m, was present in sites of lateral root emergence
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-thymidine incorporation and radiolabeled leucine incorporation 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 in- creased 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 mecha- nism for most of root microbial infections. Indeed the initial step of any root colonization involves the movement of microbes to the plant root surface; bacte- rial 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.
If the rhizodeposition can affect, as previously stated, the composition of 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 Pseudomo- t1cr.s aenrgit?o.sn (29). Biological control of plant pathogens by rhizobacteria can be based on the production of bacterial metabolites such as siderophores, antibiot- ics, 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 j l ~ r o r e . s c e ~ ~ 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 microorgan- ism 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 le- gume symbiosis (31, Chaps. 7 and IO), mycorrhizal infection (14. Chap. 9), the pathogenic microorganisms as inducers of plant defence response, the nonpatho- genic organisms as inducers of plant defense responses and the Agrohcrcteriutn- plant cell DNA transport.
The most studied molecular cross talk is that between rhizobia and the
, leguminous and nonleguminous host plants (3 I , Chap. 7). Nodulation is a 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 the host plant; this molecular communication is not yet 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 flavonoid has interacted 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 encoded by the /IUC/
genes are responsible for the production and secretion of a family of lipo-chitin- oligosaccharides (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 re- sponse.
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 nodula- tion due to either temperature or pH stress was almost completely annulled in bean and soybean roots by 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 Btwfwhizo- h i l r r r z and Rhi:obiur?l population of IO4 and l o 6 cell g
'
of soil, respectively (31).A successful competition can also depend on the ability of the rhizobium to utilize
specific compounds of plant exudates as nutrient sources. Mimosine, a nonpro- teinogenic amino acid present in large quantities in the leguminous trees and 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 some of them have been recognized
;IS 1 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 has resulted in a network of sensor mechanisms that transduce cnvironmental stimuli into gene expression and hence a phenotype complementary to the prevail-
9
ing environmental conditions. Often these stimuli are mediated by molecular sig- nalling. Thus, the so-called “Quorum sensing” involves the extracellular accu- mulation 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 exam- ple of quorum sensing is the regulation of bioluminescence in PhotoBrrctrrilrrlI jischrri. The bacterium is dark when present at low cell density, 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 de- fense, 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 elicit lux!?-dependent transcription 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 operon and thus the bioluminescence. Several N-acyl-L-homoserine lactones
(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 ( 3 5 ) . The AHL signals are involved in various phenotype expressions including synthesis and release of extracellular enzymes or antibiotics, conjugation, etc. ( 3 5 ) . The synthesis of plant cell-wall degrading exoenzymes and the carbapenem antibiotics by a plant 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)-hexa- noyl-L-homoserine lactone (OHHL) (36). The activities of plant wall-degrading enzymes synthesized and released by E. ccrrotovortr are responsible for the macer- ation 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 pro- duction by E. car(mwwo is tightly regulated so as to evade and overcome defense reactions from the plant host. In addition to the quorum component, a few compo- nents of the plant extracts can also induce exoenzyme biosynthesis (38). Specific components of the plant extracts can also coactivate the carbopenem biosynthesis.
The coordinated production of carbopenem antibiotics by E. carotovorn is essen- tial 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
II CH,
CH2 CH2
I
IIH 0
N-(3-oxohexanoyl)-L-hornoserine lactone (OHHL) Photobacteriwnjscheri Erwinia carotovora
bioluminescence 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 homoserine lactone nucleotides produced by some bacterial species
and their phenotype function (From Refs. 34 and 3 5 ) .
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 molecular signals can interact with surface active soil particles
through electrostatic interactions, hydrogen, and van der Waals bonds. In addi- tion. molecular signals with anionic groups can be adsorbed by inorganic soil colloids by a ligand exchange mechanism, unless steric hinderance avoids the contact of the anions groups with the exchangeable sites. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) but not adenosine monophosphate (AMP) were strongly adsorbed by clay minerals (39). I t was suggested that the bulky adenosine group of AMP affected the anion exchange by sterically hinder- ing close contacts between exchangeable sites and the phosphate groups. The sterical hindrance did not occur in ADP and ATP molecules because the terminal phosphate group exchanging the OH groups was further from the adenosine group.
In conclusion. the behavior of the molecular signals can be markedly differ- ent 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 molec- ular signals in the presence of clay and/or humic barriers.
VI. ROOT SENSING OF ENVIRONMENTAL SIGNALS
It is 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 i n the rhizosphere surrounding each root (or even root portion), it has been unequivocally demonstrated that plants have evolved mechanisms to cope with the 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 pro- posed 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 changes in root architecture and also in the capacity for nutrient acquisition (46,47). Recent results indicate that this behavior is de- pendent not only on internal (metabolic) signals but also on the capacity t o sense
the external nutrient. Zhang and Forde (48) demonstrated that roots of ArtrDi- t 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 tran- scription factor; however, studies are required to know the molecular mecha- nism(s) of nitrate-sensing in higher plants. Together with 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. Con- sequently it can be hypothesized the existence of a finely tuned adjustment of root structure and physiology to environmental signal(s) coming from the rhizo- sphere.
Sensing nutrients in the rhizosphere may be further complicated by the
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 inte- grated 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 coloni- zation 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 organized in a way that would promote one-way trans- fer 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 rhizo- sphere can have a specific role in plant-micro-organism-soil interactions. More- over, it starts to be elucidated:
1. Why plants release exudates
2 . What environmental factors affect exudation
3. How the release is regulated and affected by changes in microbial ac- 4. How changes in soil microbiota affect the root exudation
tivity and composition
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
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 rhizo- sphere 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 experi- mental approaches, we still do not have the complete picture that takes into ac- count the relative weight of each factor.
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