Chapter 21

Root Exudates and Soil: Crucialfor Molecular Understandingof Interactions in the Rhizosphere

Nicholas C. UrenDepartment of Agricultural Sciences, La Trobe University, Australia

21.1 INTRODUCTION

Molecular interactions in the rhizosphere between rootexudates and entities in soil are many and varied and,even though they have considerable impact on the micro-bial ecology of soils, it is possible that many of theseinteractions, but not all, are no more than happenstanceand have little impact on plant growth. There are somesuch interactions which are significant such as the inocu-lation of legume roots with rhizobia, but in this context itshould be noted that legumes can grow successfully in theabsence of successful nodulation, and as with all plantsall they need for satisfactory growth are sunlight, nutri-ents, water, and physical support for their root systems.In order for root exudates to have a beneficial impact onplant growth, they need to survive the hazards which arealways present in the rhizosphere.

The roles of root exudates in the rhizosphere havebeen discussed and researched for over the past one hun-dred years or more, and yet, in spite of the extensiveknowledge and understanding that now exists, little ofpractical significance (e.g., improved yields and diseasecontrol) has yet to come of it (For a contrasting view seeChapter 53, 54). The colonization of roots with rhizobia,as mentioned earlier, is significantly more successful inagricultural terms than other examples of root inoculationsuch as with mycorrhizae and the so-called plant growth-promoting bacteria (see Chapter 53); the “success” of thelatter in the laboratory often fails to reoccur in the field

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

(Lugtenberg and Kamilova, 2009; Compant et al., 2010;Dutta and Podile, 2010).

Although the production-oriented view taken by thisauthor of this area of soil and plant sciences is narrow,he does recognize that there are ecosystems, other thanagroecosystems, where nutrient supplies are low and thesuccessful establishment of rhizobial and mycorrhizalsymbioses with respective plants are critical to growthand survival. Perhaps the author is trying to keep thingssimple in an area where the most reliable research hasbeen carried out, to fully understand what is going on,and to determine what is important and what is not.

The emergence of soil metagenomics in recent times(Daniel, 2005) has led to a huge interest in the rhizo-sphere by a wide range of molecular scientists. Unfortu-nately, many are not interested in plant growth in soil,but are looking to mine the microbial biodiversity of soilsfor other purposes; some of these scientists write papersabout the rhizosphere without mentioning soil, its ori-gin, its basic physical and chemical properties, its recenthistory, and so on. Also, it must be stated that “pottingmix” or “a general-purpose horticultural soil” or a “stan-dard substrate mixed with perlite” cannot be regardedas substitutes for field soil; the microbial populations ofthe synthetic soils cannot be assumed to be normal ornatural.

This review attempts to determine why some of theoptimism that has surrounded rhizosphere research byplant pathologists and soil scientists (e.g., Rovira, 1991)

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222 Chapter 21 Crucial for Molecular Understanding of Interactions in the Rhizosphere

may only now become a reality. The author’s skepticismhas developed over 40 years of interest in the rhizosphere,an interest relating generally to plant nutrition and specif-ically to manganese availability in the rhizosphere (Urenand Reisenauer, 1988; Uren, 2007). It has developedout of his own frustration with his own incompleteunderstanding of soils and with the criticism that manyrhizosphere scientists seemingly ignore soil as an integralpart of the rhizosphere. The recent developments inmolecular biology referred to previously have given riseto much optimism (Kiely et al., 2006), rightly so, andalthough cracks may have opened up in the “black boxof soil microbial diversity” (Tiedje et al., 1999), it stilllooks pretty gloomy inside (O’Donnell et al., 2005)—butsix years is a long time in metagenomics! Molecularsurveys of microbial biodiversity provide information onthe microbial communities in soil, but do little that allowsmeaningful interpretation with respect to soil fertility.

21.2 ROOT EXUDATES

Rhizodeposition of photosynthetically fixed C into soilleads to the development of the rhizosphere. Estimateshave been made of the amounts and the proportions ofthe amounts which enter soil, but in terms of the num-ber of plant species, and particularly species other thancrop species, the coverage is small. The recent increase inconcentrations of CO2 in the atmosphere has led to fur-ther projections and discussions of what the impact mightbe (Drigo et al., 2008; Jones et al., 2009; Kuzyakov andGavrichkova, 2010).

It is unnecessary here to list the ever-increasing listof root exudates (see Chapter 22), but in keeping with thetheme of this review it should be pointed out that manyof the root exudates in these lists have been identifiedin solution cultures where concentration gradients areobliterated; a situation which is conducive to the leakageof some root exudates that may have no relevance in soilsother than perhaps paddy soils. Be that as it may, Maet al. (2010) investigated the proteome of the mucilagesecreted from the root tip of maize (Zea mays L.) andfound 2,848 distinct extracellular proteins, which com-pares with 124, 52, and 16 cited by the authors (Ma et al.,2010) for mucilage from root cap and border cells forthree dicots, respectively pea, Arabidopsis, and rapeseed;the presence of protein in root tip mucilage has beenknown for some time. Assuming that the border cellswere not physically damaged during collection of themucilage and their separation by centrifugation, this largenumber is quite remarkable in itself, but to this authoreven more significant is the fact that 43 isoforms ofseveral major oxygen-scavenging enzymes, for example,superoxide dismutase, were also found. Such enzymes

may be able to reduce insoluble forms of reactive Fe andMn oxides at the root–soil interface where the contactbetween the mucilage on the root tip and soil providesthe “right set of circumstances” for mobilization of thesetwo nutrients (Uren and Reisenauer, 1988; Uren, 1993).

Quorum sensing, particularly with respect to theinfection of roots with pathogens and pathogen sup-pression in the rhizosphere, has been a popular pursuit(see Section 9). The primary signals, N-acyl homoserinelactones (AHLs), have been detected in intact rhizospheresoils (DeAngelis et al., 2009) and the root exudatesof some plants have been shown to produce similarmolecules (Bais et al., 2006). However, because there aremany bacteria of soil origin which can degrade AHLs(e.g., Molina et al., 2003), it would seem that there isa good chance that they do not persist long enough andat above concentration thresholds to be effective. Wangand Leadbetter (2005) observed the rapid microbialdegradation of several 14C-labelled HSLs in a diverse setof soils, but the results have limited applicability becausethe soils were slurried, mixed, and aerated, a situation ofeven doubtful relevance in slurried paddy soils.

21.3 THE NATURE OF SOIL

The soil is an extremely diverse environment in termsof its biological, chemical, and physical properties. Eachsoil has developed in space and time in equally diverseclimatic regimes such that as far as microbial ecology isconcerned, there are large numbers of niches where indi-vidual organisms and suites of organisms can be found.It is through and between this mosaic of niches that rootsmust find their way and develop a root system which pro-vides nutrients, water, and anchorage.

The activity and distribution of microbes in the soilenvironment are determined by factors such as the avail-ability of substrate (nutrients and energy), the space inwhich the substrates exist and in which microbes can pro-liferate and colonies expand, water potential, aeration, andtemperature. The abundance of substrate and its nature isof prime importance and it is the primary stimulus in thedevelopment of the rhizosphere. But even in the rhizo-sphere access to substrates may be denied to microbesby, for example, other microbes by various means andby physical exclusion in pores too small to accommodatemicrobes.

The availability of space and its impact on growthand development of microbes are a neglected area exceptby a few. Hattori and Hattori (1976) state “The size anddistribution of pores in soil are among the most importantfactors determining microbial life since microorganismslive exclusively in pore space, and retention of waterindispensable to microbial life is closely related to the

21.3 The Nature of Soil 223

size of each pore.” The significance of pore sizes and theirdistribution, particularly in the rhizosphere, have been dis-cussed only very rarely in the context of microbial ecologyand investigated even more rarely with respect to thegrowth and proliferation of microbes. Soil pores that arefilled with water between field capacity (pF 2 or −10 kPa)and wilting point (pF 4.2 or −1500 kPa ) have equivalentdiameters of between 30 µm and 0.1 µm, respectively,and so the preferred habitat for most microbes would bein these pores. One can imagine that in soil a continu-ous pore is like a series of caves connected by smallerpores or necks, and each large pore forms a niche wheremicrobes can respond to an influx of root exudates witha burst of growth which could falter because of failingsubstrate supply, limiting space, limiting O2, competitionfrom an uninvited guest, and so on. If the pores are filledwith water, then any aerobic respiration would lead todecreased O2 supplies and thereby curtail the growth ofaerobes. With increasing dryness the microbes would beincreasingly restricted to water films and ultimately con-ditions would suit xerotrophic microbes. Thus the cyclesof wetting and drying alone can cause changes in themicrobial diversity in any pore or set of pores which canaccommodate microbes.

A related issue here is the role and ecology ofmicroaerophilic microbes. It stands to reason that in thepores of the rhizosphere the decreased O2 and increasedCO2 concentrations will determine which organisms arebest suited, and yet there is little mention of it in theliterature other than with respect to wetland soils, partic-ularly N-fixing diazotrophs. Højberg and Sørensen (1993)measured the radial distribution of O2 concentrationsand O2 consumption rates with microelectrodes in therhizosphere of barley roots in a model system; they foundlow concentrations and high consumption rates near theroot surface relative to more remote distances from theroot surface. Further, Lugtenberg and Dekkers (1999)rather cautiously suggested “it is tempting to speculatethat the rhizosphere environment is microaerophilic” onthe basis of investigations relating to the rhizospherecompetence of Pseudomonas species. Because soil CO2concentrations are normally about 10 times those of theatmosphere, then all culturing of soil microbes should bewith such a concentration and not uncontrolled ambientconcentrations in an incubator; O2 concentrations shouldbe adjusted accordingly.

The movement of root exudates through soil dependson many factors, primarily the physicochemical propertiesof the exudates and those of the rhizosphere soil and thediverse array of organisms present. Chemical properties ofthe root exudates such as molecular size, their net charge(either permanent or pH dependent), hydrophobicity, etc.affect their propensity to move. The larger molecules arerestricted in their movement and most are most likely

found close to the rhizoplane, in mucilage and in poresin the rhizosphere, some of which would be too small toaccommodate microbes and thereby protected from degra-dation. At the other extreme, low molecular weight sugarsand organic acids are relatively mobile and diffuse easilyinto what becomes the rhizophere and are rapidly utilizedby the microbes therein. One can imagine a flood of sol-uble exudates diffusing into soil pores of all sizes frompoints of high concentration at the rhizoplane, but micro-bial utilization stems the tide and eventually utilizes ahigh proportion of the root exudates even including thosethat diffused into pores too small to accommodate themicrobes, but then diffused back in response to the con-centration gradient created by microbial utilization. Someof the small molecules may get stuck in small pores forphysicochemical reasons and only be released when thesoil is cultivated. A recent simulation model has beenpublished which illustrates the importance of soil proper-ties in the diffusion of root exudates (Raynaud, 2010). Bynecessity, out of lack of knowledge and complexity, theRaynaud’s model structure of the rhizosphere is depictedsimply as a small root section surrounded by a series ofconcentric cylinders. A further development of the modelcould treat each of the longitudinal physiological and mor-phological zones (i.e., root cap, root meristem, zone ofdifferentiation and elongation, root hair zone, and maturezone) separately to give a more realistic simulation of thediffusion patterns in the rhizosphere.

A driving force in the development of the currentinterest in soil biodiversity has been that many moremicrobes could be observed in soil than counted bytraditional culturing methods as CFUs. Molecular biologyhas since developed and although the results confirm thatthere are many more microbes in soil, it has thrown upa few issues that relate to molecular interactions in therhizosphere. An important question is whether or notthe “uncultured majority” of the microbial communitiesare important in rhizosphere processes (da Rocha et al.,2009), a question to which we have no definitive answerexcept that some recent work has indicated that rhizo-sphere microbes are not the gluttons they were previouslythought to be and preferred less concentrated substratesources (e.g., Erneberg and Kishony, 2012). On theother hand, Fierer et al. (2007) bemoan the fact that thenewly found microbial diversity of soils has done littlefor ecological interpretation of important issues such aswhich bacteria are most abundant in different soils andwhy. The most sense they can make of metagenomicdata from 71 widely sampled soils in the United Statesis that differentiation can be best made into copiotrophicand oligotrophic categories. Rhizotrophs may be a usefulterm to use to distinguish rhizosphere microbes fromcopiotrophs and oligotrophs. Only further studies ofmicrobial ecology in the rhizosphere which identify the

224 Chapter 21 Crucial for Molecular Understanding of Interactions in the Rhizosphere

different groups of microbes, their location, and theirtrophic habits and preferences can resolve these issues.

The methods used to extract intracellular DNAare many and vary considerably, no doubt because ofthe uncertainty surrounding the fate and behavior ofDNA, RNA, and nuclease in soil and during extractionprocedures and problems associated the co-extractionof substances which interfere with clean-up proceduresand decrease sensitivity and albeit decrease apparentbiodiversity (Pietramellara et al., 2009; see Chapter 5).Research into the biodiversity of the rhizosphere, andthereby the fate and behavior of root exudates therein,relies very heavily upon the extraction of DNA and so itis important that not only are the procedures sound butalso give reproducible results; standardization of proce-dures is essential to allow realistic comparisons to bemade between different soils from different environmentsand between laboratories (see Chapters 5, 6, 9, 10).

21.4 ROOT GROWTHAND DEVELOPMENTOF THE RHIZOSPHERE

Radial and longitudinal gradients that develop over timeas plant roots grow through soil have received graduallyincreasing attention over the years (Foster, 1986; Urenand Reisenauer, 1988; Neumann and Romheld, 2002;Hinsinger et al., 2005; Watt et al., 2006a, 2006b; Walteret al., 2009; Dennis et al., 2010; Marschner et al., 2011).It is a welcome trend and will necessitate an extensionof the classification of terms to subdivide the rhizosphereinto more than just the radial descriptors: rhizodermis,rhizoplane, and rhizosphere; endorhizosphere and ectorhi-zosphere have been used at times. The longitudinaldescriptors should reflect the rhizosphere associated withthe root tip, the zone of differentiation and elongation,the root hair zone and the mature root.

The rhizosphere is first established ahead of the roottip which causes initially some deformation due to theexpansion of root tip cells. Concomitant secretion ofmucilage occurs and then “sloughing off” of root bordercells in those plant species which produce them takesplace. Separation may be a better term than sloughingoff since the current understanding of the process is notknown and “sloughing off” has implications of wasteand dead tissue which may not be apt. Root border cellshave been accredited with a wide range of functions,but probably there are too many to be sensible andfurther investigations of their fate and behavior in soilare urgently required.

Other root products include a mixture of secretions,excretions, lysates, and diffusates which includes CO2from root respiration. A population of microbes, mainly

bacteria, grows rapidly from the small resident, but mostlydormant cells awoken by the influx of root exudates. Theradial extent of this altered volume of soil has proba-bly been studied by many but few as purposefully asMcCully (1999). The impact which the occupation ofpores in the rhizosphere by this rapidly expanding pop-ulation has on soil properties in this volume of soil hashad some attention. As the root cap cells differentiate andexpand, increasing physical forces compact the soil in therhizosphere and may thereby restrict the activity of themicrobes in the newly developed rhizosphere. It is pos-sible that the extrusion of mucilage into pores may evenrestrict the proliferation of microbes in such pores.

DeAngelis et al. (2009; see also Chapter 78) usedmicrocosms, filled with field soil, to study the succes-sion of bacterial and archaeal communities in the rhi-zosphere of wild oat (Avena fatua) roots. They foundthe rhizosphere stimulated a dynamic subset of a diversearray of bacterial and archaeal communities. Also, theysuggest that some members of the Actinomycetes andα-Proteobacteria are particularly rhizosphere competent.They found, to their surprise, that although the rhizosphereof the root tip, as they called it, was highly colonized rela-tive to the bulk soil, the diversity of taxa present was not.In the older regions of the root, the root hair and matureroot zones the dynamic subset increased in relative abun-dance and diversity. Such a study is to be commended forrecognizing that there are these zones and that they aresignificant in terms of microbial ecology. If any criticismis to be found, it is the fact that what they called the roottip was about 4-cm long and it included not only the rootcap and meristem but also the zone of differentiation andelongation.

In contrast to the approach taken by DeAngelis et al.(2009), Watt et al. (2006a, 2006b) sampled the seminalroots of field-grown wheat (Triticum aestivum L. cv Janz)and investigated the numbers and locations of native bac-teria along the root axes by fluorescence in situ hybridiza-tion (FISH; see Chapters 39, 87). They found that the rootcaps were the most heavily colonized and the elongationzones the least heavily colonized; soil has been observednot to adhere to elongation zones of maize roots (Vermeerand McCully, 1982). As with all methods there are limi-tations and the authors discuss those of FISH in relationto field-grown plants. Nevertheless, if DeAngelis et al.(2009) had been able to look at the roots in the same wayas Watt et al. (2006a, 2006b), then they probably wouldnot have combined the root cap and the elongation zoneinto one. Similarly, a micro-sampling technique developedby Dennis et al. (2008) to investigate the density and dis-tribution of bacterial types colonizing microhabitats suchas the rhizoplane could have been usefully utilized.

The progressive development of the rhizosphere andthe temporal changes which take place in the microbial

21.5 Where to From Here? 225

communities attendant upon the root zones needs closerscrutiny. What happens to the microbial communitieswhich first colonize the root tip and the adjacent soil? Dothey run out of substrate? Do they run out of space? Arethey overtaken by a succession of different communitiesas the root tip rhizosphere becomes the rhizosphereadjacent to the zone of elongation with its relativelymeager supply of root exudates? What then happens inthe root hair zone with a fresh injection of root exudatesinto the pre-existing rhizosphere. Murder and mayhemmost likely. Then in the mature root zone the root hairsand cortical cells begin to senesce and are colonizedwith mobile microbes and mycorrhizae. Then if this trainof events is not complicated enough, lateral roots beginto emerge from the mature root zone proximal to theroot hair zone, a process described as a difficult birth(Peret et al., 2009). The lateral root tip extends throughwhat is left of the rhizosphere from the root hair zone;presumably a new root cap rhizosphere develops, but isit initially colonized by the microbes of the mature rootzone before extending into the bulk soil? Is it of anysignificance whatever it is that happens? A new seriesof questions arise. In what ways does the lateral roottip differ in its attributes from those of say seminal andnodal roots? Are these differences important in terms ofroot exudation and rhizosphere development? Hopefully,the new generation of rhizophere scientists will be ableto answer these questions and others which no doubt willarise.

21.5 WHERE TO FROM HERE?

A microhabitat in a rhizosphere of a given plant in a givenecosystem at a given point in time is unique, but it isthe result of an enormous number of circumstances. Thenumber of factors contributing is also enormous and soby necessity it is essential to simplify the setup of exper-iments to control the variability that exists. This authorhas drawn the line at soil-grown plants in a microcosmin a controlled environment which is a long way from anecosystem in a desert or a rain forest, but some believethat it should be drawn in the field where uniformity hasbeen imposed by agricultural activities. Others prefer thesimplicity of solution cultures which have little practi-cal relevance other than to hydroponic systems. Whateversystem is used, it is the investigators’ obligation to beaware of the disparity between simple systems and thelimited usefulness of the latter when it comes to predic-tions of what happens in the rhizosphere of soil-grownplants. Similarly, it is important that field soils or freshsoil samples taken recently are used in experiments andnot soils made up by the local nursery with materials ofunknown provenance.

There is need for greater awareness of the longitudi-nal and radial gradients of any kind and the temporal andspatial changes brought about by root growth, particularlyin this context of microbial communities. The furtherdevelopment of methodology, which can be used toinvestigate in situ these temporal and spatial changesin the rhizosphere of soil-grown plants, needs to beencouraged.

Greater awareness is needed of the fate, behavior, andpersistence of root border cells in soils after separationand the temporal changes in their own exudates and asso-ciated microbial communities. Equally the sources, thelocation along roots, the properties, the roles and the fateof mucilage all need to be explored further. Mucilage, likeroot border cells, has been attributed with multiple roles,only some of which are probably important in terms ofmicrobial ecology and plant growth.

The “right set of circumstances” is a theory developed(Uren and Reisenauer, 1988; Uren, 1993) to rationalizeall that was known about the properties of roots and rootsystems, Mn behavior in soils, Mn uptake and availabil-ity, etc. into a theory which satisfactorily explained howplants derived Mn from soils of neutral pH in whichMn deficiency sometimes occurs. In this case the rightset of circumstances occurs randomly and whether ornot a plant gets sufficient Mn from soil depends on howfrequently the “right set of circumstances” arises—it is aprobability issue of how frequently the root makes contactwith the isolated reactive particles of insoluble oxidesof Mn. Similar situations may apply to other processesthat take place in the rhizosphere, which may be ofsignificance in plant growth; it may be the release fromthe root of a phytoalexin or a quorum-sensing molecule,readily biodegradable molecules which need someprotection.

The importance of pore sizes and continuity in bothsoil and the rhizosphere in microbial ecology needs to bepursued to a greater extent than in the past. That such anapparently crucial soil property in microbial ecology hasbeen ignored is quite surprising since it is as relevant assoil structure is to plant growth.

Root exudates such as phytoalexins, allelochemicals,phytotoxins, phytosiderophores, ectoenzymes, etc. havenot been discussed here, but their presence in the rhi-zosphere still continues to be of interest. Another aspectof interest which has been left alone is the coevolutionof roots and microbial ecology of the rhizosphere (e.g.,Brundett, 2002; Lambers et al., 2009). Also, little refer-ence has been made to soil fungi and no reference hasbeen made at all to the role of invertebrate fauna, proto-zoa, nematodes, and microathropods. Their omission hasbeen only in the need to be brief and to keep life sim-ple, not to demean their potential to influence microbialecology.

226 Chapter 21 Crucial for Molecular Understanding of Interactions in the Rhizosphere

The last item on this wish list is that there needs tobe greater cooperation and collaboration between scien-tists interested in soil microbiology, root growth in soils,the role of soil properties in plant growth, ecology in thebroader sense and microbial ecology. Too often we haveseen a plant pathologist or a soil chemist, for example,only and with nobody in mind, go off on a tangent andpromulgate some idea which is easily refuted by anotherscientist in another discipline related to soil. Unfortu-nately, such utopian examples of cooperation arise onlyrarely, but there is always hope.

REFERENCES

Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM. The role of rootexudates in rhizosphere interactions with plants and other organisms.Ann Rev Plant Biol 2006;57:233–266.

Brundett MC. Coevolution of roots and mycorrhizas of land plants.New Phytologist 2002;154:275–304.

Compant S, Clement C, Sessitsch A. Plant growth-promoting bac-teria in the rhizo- and endosphere of plants: their role, colonization,mechanisms involved and prospects for utilization. Soil Biol Biochem2010;42:669–678.

Daniel R. The metagenomics of soil. Nat. Rev Microbiol2005;3:470–478.

da Rocha UN, van Overbeek L, van Elsas JD. Exploration ofhitherto-uncultured bacteria from the rhizosphere. FEMS MicrobiolEcol 2009;69:313–328.

DeAngelis KM, Brodie EL, DeSantis TZ, Andersen GL, LindowSE, Firestone MK. Selective progressive response of soil microbialcommunity to wild oat roots. ISME J 2009;3:168–178.

Dennis PG, Miller AJ, Hirsch PR. Are root exudates more importantthan other sources of rhizodeposits in structuring rhizosphere bacterialcommunities? FEMS Microbiol Ecol 2010;72:313–327.

Dennis PG, Miller AJ, Clark IM, Taylor RG, Valsami-Jones E,Hirsch PR. A novel method for sampling bacteria on plant rootand soil surfaces at the microhabitat scale. J Microbiol Methods2008;75:12–18.

Drigo B, Kowalchuk GA, van Veen JA. Climate change goesunderground: effects of elevated atmospheric CO2 on microbial com-munity structure and activities in the rhizosphere. Biol Fertil Soils2008;44:667–679.

Dutta S, Podile AR. Plant growth promoting rhizobacteria: the bugsto debug the root zone. Crit Rev Microbiol 2010;36:232–244.

Erneberg M, Kishony R. Distinct growth strategies of soil bacteriaas revealed by large-scale colony tracking. Appl Env Eng 2012;78:1345–1352.

Fierer N, Bradford MA, Jackson RB. Toward an ecological classifi-cation of soil bacteria. Ecology 2007;88:1354–1364.

Foster RC. The Ultrastucture of the rhizoplane and rhizosphere. AnnRev Phytopathol 1986;24:211–234.

Hattori T, Hattori R. The physical environment in soil microbiology:an attempt to extend principles of microbiology to soil microorgan-isms. Crit Rev Microbiol 1976;4:423–461.

Hinsinger P, Gobran G, Gregory PJ, Wenzel WW. Rhizospheregeometry and heterogeneity arising from root-mediated physical andchemical processes. New Phytol 2005;168:293–303.

Højberg O, Sørensen J. Microgradients of microbial oxygen con-sumption in a barley rhizosphere model system. Appl Env Microbiol1993;59:431–437.

Jones DL, Nguyen C, Finlay RD. Carbon flow in the rhizosphere:carbon trading at the root-soil interface. Plant Soil 2009;32:5–33.

Kiely PD, Haynes JM, Higgins CH, Franks A, Mark GL, MorrisseyJP, O’Gara F. Exploiting new systems-based strategies to eluci-date plant-bacterial interactions in the rhizosphere. Microbial Ecol2006;51:257–266.

Kuzyakov Y, Gavrichkova O. Time lag between photosynthesis andcarbon dioxide efflux from soil: a review of mechanisms and controls.Global Change Biol 2010;16:3386–3406.

Lambers H, Mougel C, Jaillard B, Hinsinger P. Plant-microbe-soilinteractions in the rhizosphere: an evolutionary perspective. Plant Soil2009;321:83–115.

Lugtenberg B, Kamilova F. Plant-growth-promoting rhizobacteria.Ann Rev Microbiol 2009;63:541–556.

Lugtenberg B, Dekkers LC. What makes Pseudomonas bacteria rhi-zosphere competent. Env Microbiol 1999;1:9–13.

Ma W, Muthreich N, Liao C, Franz-Wachtel M, Schutz W,Zhang F, Hochholdinger F, Li C. The mucilage proteome of maize(Zea mays L.) primary roots. J Proteome Res 2010;9:2968–2976.

McCully ME. Roots in soil: unearthing the complexities of roots andtheir rhizospheres. Ann Rev Plant Physiol Plant Mol Biol 1999;50:695–718.

Marschner P, Crowley D, Rengel Z. Rhizosphere interactionsbetween microorganisms and plants govern iron and phosphorusacquisition along the root axis – model and research methods. SoilBiol Biochem 2011;43:883–894.

Molina L, Constantinescu F, Michel L, Reimmann C, Duffy B,Defago G. Degradation of pathogen quorum-sensing molecules bysoil bacteria: a preventative and curative biological control mecha-nism. FEMS Microbiol Ecol 2003;45:71–81.

Neumann G, Romheld V. Root-induced changes in the availability ofnutrients in the rhizosphere. In: Waisel Y, Eshel A, Kafkafi U, editors.Plant Roots: The Hidden Half . 3rd ed. New York: Marcel Dekker;2002. p 617–649.

O’Donnell AGPppldftrO’Dea(2005), Colvan SR, Malosso E,Supahol S. Twenty years of molecular analysis of bacterial commu-nities in soils and what have we learned about function. In: BardgettRD, Usher MB, Hopkins DW, editors. Biological Diversity and Func-tion in Soils . Cambridge: Cambridge University Press; 2005. p 44–56.

Pietramellara G, Ascher J, Borgogni F, Ceccherini MT, GuerriG, Nannipieri P. Extracellular DNA in soil and sediments: fate andecological relevance. Biol Fertil Soils 2009;45:219–235.

Peret B, Larrieu A, Bennett MJ. Lateral root emergence: a difficultbirth. J Exp Bot 2009;60:3637–3643.

Raynaud X. Soil properties are key determinants for the developmentof exudates gradients in a rhizosphere simulation model. Soil BiolBiochem 2010;42:210–219.

Rovira AD. Rhizosphere research – 85 years of progress and frustra-tion. In: Keister DL, Cregan PB, editors. The Rhizosphere and PlantGrowth . Dordrecht: Kluwer Academic Publishers; 1991. p 3–13.

Tiedje JM, Asuming-Brempong S, Nusslein K, Marsh TL, FlynnSJ. Opening the black box of soil microbial diversity. Appl Soil Ecol1999;13:109–122.

Uren NC, Reisenauer HM. The role of root exudates in nutrient acqui-sition. Adv Plant Nutrit 1988;3:79–114.

Uren NC. Mucilage secretion and its interaction with soil and contactreduction. Plant Soil 1993;155/156:79–82.

Uren NC. Types, amounts, and possible functions of compoundsreleased into the rhizosphere by soil-grown plants. In: Pinton R,Varanini Z, Nannipeieri P, editors. The Rhizosphere: Biochemistry andOrganic Substances at the Soil-Plant Interface. 2nd ed. Boca Raton(FL): CRC Press; 2007. p 1–21.

Vermeer J, McCully ME. The rhizosphere in Zea: new insight intoits structure and development. Planta 1982;156:45–61.

References 227

Walter A, Silk WK, Schurr U. Environmental effects on spatialand temporal patterns of leaf and root growth. Annu Rev Plant Biol2009;60:279–304.

Wang Y-J, Leadbetter JR. Rapid acyl-homoserine lactone quorum-sensing signal biodegradation in diverse soils. Appl Env Microbiol2005;71:1291–1299.

Watt M, Silk W, Passioura JB. Rates of root and organism growth,soil conditions, and temporal and spatial development of the rhizo-sphere. Ann Bot 2006a;97:839–855.

Watt M, Hugenholtz P, White R, Vinall K. Numbers and locationsof native bacteria on field-grown wheat roots quantified by fluores-cence in situ hybridization (FISH). Env Microbiol 2006b;8:871–884.