[] microbial ecology of the rhizosphere(bookfi.org)

2Microbial ~cology of theRhizosphereHARVEY BOLTON, Jr., and JAMES K. FREDRICKSONBalteile Pacifie .\'orl/1I"cSI La60ralories, Richland, WashinglonLLOYD F. ElLIOTTAgricultllral Research Sen'icc, U.S, Department of Agricu/llIre,Con'ailis, Oregon

I. INTRODUCTIONMicrobial ecology of the rhizosphere refers to the study of the interactions ofmicroorganisms with each other and the em'ironment surrounding the plant rool.The rhizosphere is generally defined as the \'olume of soil that is adjacent to andinfluenced by the plant root (Hiltner, 1904), The term comes 'from the Gre'ek",ord for root (rhizo or rhiza) and includes both the area of 'influence and thephysical localion around lhe root (sphcre). The rhizosphere has been furthersubdivided by some researchers into the eClorhizosphcre, or an outer rhizosphere,and the endorhizosphere, ar an inner rhizosphere, where invasion and coloniza-tion of root cortical cells by soil microorganisms occurs (Balandreau and Knowles,1978; Dommergues, 1978). Portions of lhe rhizosphere can also be called lhemycorrhizo.\;7he:e ,;'hen lhere are mycorrhizal fungi associaled with /OOIS (Linder-man, 1988) or lhe acrinorhizosphcre or acrinorhiza (Torrey and Tjepkema, 1979)when aClinomycetes (i.e., Frankia spp.) are associated with nodules on the rool.The dislinct boundary of the root surface \\ilh the soil has been 'called the rhiz-plane (Clark, 1949), It is often functionally or experimentally difficult to distin-guish lhe rhizoplane from the rhizosphere. ln this re\'iew, lhe term rhizospherewill encompass bOlh the rhizosphere (endorhizosphere and ectorhizosphere) andlhe rhizoplane.

The rhizosphere is lhe physical location in soil where plants and microorgan-isms interacl. II has been estimated that one ",heat plant (Triricum aesl\'III1l) canproduce a total rool length of 71,000 m, which canstitutes a large surface area ",hendispersed throughout the soil (Pa\lychenko. 1937), The interest in rhizospheremicrobiology derives from the abilily af the sil microbiot,a to influence plantgrowth and vice versa. The study of rhizosphere processes requires a multidisciplin-ary approach and is extremely challenging because of the complexity of this system,

27

28 Bollon el aJ.

Schroth and Weinhold (1986) stated that "... those who enjo)' studying orderlysystems amenable to quantitative analysis are likely to consider rhizosphere investi-gations as a masochist"s delight:"

A general definition of ecology is lhe study of both ecosystem structure andfunction (Odum. 1971). Ecosystem structure involves (1) the composition of thebiological communit)'. including species. nllmbers. biomass. life history. and spatial'distribution of populations: (2) the quantity and distribution of abiotic materiaIs,such as nutrients and water; and (3) the range. or gradient, of conditions of exis-tence, sllch as temperature and light. Ecosystem function involves (1) energy flowthrough the ecosystem and biogeochemical cycling and (2) biological or ecologicalregulation, including both regulalion of organisms by environment and regulationof environment by organisms (Odum. 1962). The ecology of microorganisms intherhizosphere is also the study of stru911re and fllnction. An unerstanding of thebasic principies of rhizosphere microoial ecology. including1he fllnction and diver-sit)' of the microorganisms that reside there, is necessary before soil microbialtcchnologies can be applied to the rhizosphere.

The purpose of this chapter is to introdllee the reader to some general princi-pies and processes that occur in the rhizosphere. The reader is also directed toseveral olher excellent reviews on rhizosphere microbial ecology (Balandreau andKnowlcs, 1978; Clark, 19..9; Elliolt el aI., 1984; Foster and Bowen; 1982; Rovira,1979) and the rhizcisphere in general (Curl and Truelove, 1986; Lynch, 1990a).

~i"UIe 1 Microbial growth on the rooI surface. (a) Aggregales of rodshaped cells al-..........~\' begjoDim:. to form in lhe cenler of a

Microbial Ecology of lhe Rhizosphere 29

Next, the mechanisms by which microbial growlh is enhanced in the rhizosphereand how microorganisms can influenee the growth of planls atd other mieroorgan-isms will be diseussed. Finalll', the researeh needed in rhizosphere eeologl' to aidthe development of rhizosphere microbial teehnologies and examples of potentialleehnologies wiU be diseussed.

II. THE RHIZOSPHERE EFFECTA. Introduction

The rhizosphere effeel is a slimulatioll of microbial growlh surrounding the rootbecause of the release of organic compound;, (Fig. 1; ElIiott et aI., 1984). Anunderstanding of the types of organic (ompounds a\'ailable for microbial growth inlhe rhizosphere and ho\\' various phnical, chemical, and bioJogieal factors influ-enee lhe release of these compounds from lhe root is necessarl' both to understandthe stimulation of microbial gro\\'th and acti\ity in lhe rhizosphere and 10 developrhizosphere soil microbial technologies.

A wide variely of organic compo\Jnds of planl origin have been found in therhizo;,phere. A standardization of letms was adopted lo avoid eonfusion whendiscussing lhe sources and namcs of various clas;,cs of organic compounds a\'ailablefor microbial grow th. The organic materiaIs from plant roots were classified byRovira and associates (1979) as follow5:1. Ewdlilcs: low molecular weight cornpound;, (i.e., sugars, amino acids) lhal

leak from intacl ceUs2. Sccrclioll5: compounds lhat are aClively released from rool edis

lh)

30 Bolton el aI.

3. Planr mucilage: there are four sources from various parts Df the rOOl includinga. Secretions by the golgi bodies of the root cap cellsb. Hydrolysates Df the primary cell wall Jocaled between the rooI cap and the

epiderrnisc. Secrelions by epidermal cells and roOl hairs Wilh primary wallsd. Compounds resulting from the microbial degradation and modification of

dead epidermal cells4. Mucigel: gelalinous material on lhe root surface composed of plant mucilage,

bacterial cells, melabolic products, and colloidal organic and minera! malerial5. Lysales: material released lhrough the lysis Df older epidermal cells

Locations on the plant root at which these organic substrales may be released arepresented in Figure 2. .,

There has long been interest ih root-derived organic C in the rhizospherebecause of the enhancemenl of microbial growth on and near the roo!. ln fact,Hillner coined lhe ferm rhizosphere in response to obscrvations oE enhanced micro-bial growth surrounding lhe roaIs of legumes, which was assumed to be caused bythe excretion of organic materiais (Curi and Truelove, 1986). It has been postulatedthal the re!case of organic C from the planl root is in response to injury or microbialattack, or from naturally occurring Ieaky pIdnl mel]lbranes. However, it has aIsobeen suggested Ihal the planl has evolved C leakage to stimulat~ an activerhzosphere microflora. The microflora can, in tum, promole plant growlh byenhancing soi1 organic marrer rransformations, mobilizing inorganic nUlrienls, pr-

3a

~N~;~~~i~fEpldermal and CorticalCeJls lysed and 5lnvaded by BacleriaSloughed Rool Cap Cells

SOII

'i-:d- J 3c':\ Micro Ofganisms

wilh Microbial and 4Plant MucilagesJ5,,,,,,",, '"" .-, "''' }=:: --1&2-.-

J-HI------------- 3b

__ Rool

20 mm

Figure 2 Diagram of a mode! root showing the origin of various organic material that ispresent in lhe rhizosphere. The numbers under lhe nature of the material rerer to thevarious classes described in the texto (Modified from Rovira et aI., 1979.)


ducing growth-promoting substances, acting as antagonists against pathogens, andby other mechanisms treated elsewhere in this book.

The release of organic C compounds TOm the root into the rhizosphere can be,anappreciable proponion of the total C fixed by plants. Manin (1977a) found that 39%of the C that was translocated to wheat roots, or 17% of total plant C, was releasedinto the soi!, presumably from autolysis ofthe root conexo Barley (Hordeum vu/garisL.) grown in solution culture released 60% of the plant roots' dry malter production(Martin, 1977a). Whipps and Lynch (1983) found that between20 and 25% of thetotal "CO, fixed by the plant was lost from the roots of both barley and"wheat grownin nonsterilesand. Native plant gras~ species can also exude significant quantities oftheir fixed C. AgropyrOIl crisrarwll, A. smirhii, and Boute/oua gracilis roots released8, I7, and 15'7c, respeclvely, of the total C fixed by the plant inlO the rhizosphereduring a 90-day growth period (Biondini et aI., 1988).

B. Nature of Organic Carbon in lhe RhizosphereA wide variet)' of soluble organic compounds that are produced by the plant may bereleased into the rhizosphere. The nature of plant-derived compounds found in therhizosphere is dependent on plant species, growth conditions, rooting medium, andstage of plant de\'Clopment. Amino acids, sugars, organic acids, proteins, pol)'saccha-rides, growth-promoting and growth-inhibitingsubstances, ali have been reported asroot exudates (H ale et aI.. 1978). Different classes of compounds ha\'C been identi-fied as root exudates from a "ide variet)' of plant species (Table 1). The diversity ofcompounds that are present in the rhizosphere probably affects the compasition andactivit)' af the microbial population that develops in the rhizosphere. Carboh)'dratesderived from roots are one of the major sources of C and encrg)' for microbial growthand metabolism ir! the rhizosphere (Foster and Bowen, 1982). Glucose is often citedas a major root exudate from various plant species. Corn (Zea mays L.) grown 36days in solution culture released wgars (65%), organic acids (33%), and amino acids(2 (7c) (Kraffczyk et aI., 1984). These authors were able to identify a variet)' of sugars,organic acids, and amino acids (Table 2). The concentration of severa] organic com-pounds were different understerile and nonsterile conditions, dcmonstrating that themicroorganisms present COU Id utilize the organic exudates or alter root exudationpalterns. Twehe different amino acids were detected in ihe root exudates ofaxenicblue grama seedlings (Boll/cfoua graci/is), but onl)' eight could bc identified (Bokhariet aI., 1979). The nature and abundance of organic campounds probably has a majorinfluence on the t)'pes of microorganisms that colonize the rhizosphere. Most of thestudies to date have addressed the gross flux of C from the plant root into therhizosphere at specific stage. of plant growth and for limited periods. Few s)'stematicstudies have been made of the spatial and temporal C flow from roots into therhizosphere. An understanding of microbial stimulation and seJection processes inthe rhizosphere throughou~ the growth cyc1e of the plant will require long-termstudies of C rele ase b)' roots and of lhe temporal and long-term effects of this releaseon the associated microflora.

C. Factors lhat Affect Organic Carbon Release in lhe Rhizospherelt is well established that different plant species release different organic com-pounds into their rhizospheres. Ea'rly work by Rovir (1956) demonslrated lhat

32 Bolton el aI.

Table 1 Organic Compounds Detccted a~ Plant Rool E~"datesClass of organic compound Exudle components

Sugars Glucose, fructose, ~ucrose, malto~e, galactose,rhamnose, rihose, xylo~c, arahjno~e, ra(finosc,oligosaccharide

Amino compounds Asparagine, a-alanine, glulamine, aspartic acid,Ieucine.'isolcucine, scrine, aminobulyric acid, gly-cine. c)'srt'ine/c)'~line, mcthion;ne,phenylalaninc, lyrosine, Ihreoninc, Iysinc,prolinc, Iryptophan, Il-alaninc. argininc,homoscrinc, cyslathioninc

Organic acids Tartaric. oxalic, citric, malic, acctic, propionic, hu-Iyric, succinic, fumMic. glycolic, \'alcric, malonic

Fatt)' acids and sterols Palmilic. stcaric, okic, linolcic, ;,nd linok,nic acids;cholc~tcrol. campeslcrol, slibmasterol, sitosterol

Gro ..... th faclors fliotin, thiamine, niacin, panlothenatc, choline,inositoJ. Pl ridoxine, p-amino hcnlOic acid, /l-rnl,thyl nicotinic acid

!'.'uclcotides, fa\'onones, and enzymes F1a\'onone; adeninc, guaninc, uridinelcytidinc;phosphara,c, in\'t:rtasc, amyla~c, pr(llcina~e,polygalacturonase

Miscdlancous All,im. scopolclin, flu",escenl ~uh~lanccs, hydro-cyanic ;,cid. gIYC,tJafl'ral srirnulanls and inhihirors. parasiric wCl'dg{'rrnin"tiorl ~limu1ators

Ihcrc was a substanti:i1 eliffcrcnce iII the root exudalion I'allerns of oals (111'

Microbial Ecology of lhe Rhizosph

Table 2 Soluhlc Root Exudates of Maize Gro",n for 36 Days in SteriJeand :"on'\~rilc l"utricnt Solution Culture

33

Root eXlldatc

SugarsGlucoseArahinnschuclOsoSlIcrosc

Chgallic acidsOxalollcctic acidFumaric acidMalic acid('ilric acidSuccinic JcidIknwic ;,cidAconitic acioTart1rk (KidGlularic ;,d

Amitlo Jcid~GIlIlamic acidr\ Sptlrt ir acielAlanineGI)'cinoy-Aminohulyric acidScrincArginin

34 Bolton el aI.

lhe fixalion of C (Rovira, 1959). A decrease in soil moisture can also increase(Martin, 1977b) or decrease (Reid, 1974) root exudation. Exposing plant roots lOwet and dry cycles in soil can increase rool exudation (Katznelson et aI., 1955).Plant nulrient slatus and root injury can also alter rool exudative patterns. Bowen(1969) found Ihal lhe quantity of root exudales from pine roots (P. radiola) increased under P stress, but decreased under N slress. Mechanical stresses, Dr lhefriction between rools and the porous medium through which lhe rools grow,increase rool exudalion. Barber and Gunn (1974) noled an increase in lhe amounlof amino N-conlaining compounds'and carbohydrates, from 5 lo 9% of lhe tolal drymalter contenl of barle)' rools, when pIanIs were grown in Iiquid cullure containingglass beads when compared with ,liquid culture alone. When agitated in a sandsuspension, wheat and pea roots released, in 1 hr, approximatel)' the sarne amountof amino N compounds that was 'released during a 2-week growth period in quiescent solution culture (Ayers and Tho,rnton, 1968).

Foliar applications of fertilizers nd pesticides can also affct the quantity andcomposition of TOOt exudates (Hale et aI., 1978). Foliar application of N increasedthe amino acid and decreased the sugar content of root exudates, whereas a decrease in the amino acid content and an increase in sugars occurred with a foliar Ptreatment (BaJasubramanian and Rangaswami, 1969). Organic compounds appliedto foliage have been detected in the rooting Solulion and lhe rhizosphere soil,demonstrating that they can be translocated from leaves to the roots and exudated,without alteration,,into the rhizosphere. These compounds ineluded 2,3,6-trichloro:benzoic aeid, a-methoxyphenylacetic acid, and 2-methoxl'-3.6-dichlorobenzoic acid(Linder et aI., 1964); pieloram (4-amino-3,5,6-trichloropicolinic acid) and 2,4,5-T(2,4,S-trichlorophenoxyacetic acid) (Reid and Hurtl, 1970); and streptomyein(Davey and Papavizas, 1961). Streptomycin and a slreptomycin transformationproduct were exuded by the roots of coleus (Co/eus b/umei Benth), indicating thatorganic compounds applied to Jeaves may also be transformed during translocation'and before exudation. Foliar, application of streptomycin did not affeet lhe lotaIquantity of rhizosphere micTOorganisms, but lhe fraction that was gram-negativcbacteria was reduced after the application (Davey and Papavizas, 1961). This dem-onstrated thal chemicals applied to leaves can potcntialll' alter the comrnunitystructure of the rhizosphere, Hormones applied to the leaves can also influencc therhizosphere microflora. Foliar spraying of Phaseo/us aurcus with up to 100 ppmindoleacetic acid increased the rhizosphere microbial population ovcr that of con-trol plants (Singh, 1982). These results suggest lhat the rhizosphere microflora maybe manipulated by the foliar application of chcmicals. Selective stimulation of limicrobial isolate that can utilize a specifie compound mal' be achicvcd' bl' foliarapplication if the compound can be translocated and rcleased into thc rhizosphcrc.Alternatively, jf an organism is resistant to a foliar/y applied chemical that can betranslocated and released into the rhizosphcre. its colonization of the rhizosphcrcmay be enhanced.

The presence of microorganisnis inthe rhizosphere wilf increase root exuda-tion. Barber and Martin (1976) found that 5-10% of the photosynthetically fixed Cwas exudated frombarley roots under sterile conditions, but when microorganismswerc introduced, exudation' increasedto 12-18%.' Agropyron crislatum and A ..smilhii roots, grown for 90 days in the presence of microorganisms in frilled e1ay,released approximalely two and six times the C released under sterile conditions,


respecliveJy (Biondini el aI., 1988). Prikryl and Vancura (1980) fouiJd that theamounl of wheal rool exudale almosl doubJed when Pseudomonas putida .....aspresenl in lhe rooling solution, campared wilh growlh under sterile condilions. Thereason for Ihis slimulalion af rool exudation by rhizosphere microorganisms is notwell underslood. One explanation is that the microorganisms are rapidly metaboliz-ing the available C leaked from the root, thereby creating a concentration gradientleading to funher Jeakage. Microorganisms may also make rools more leaky, eilherby ph)'sically damaging lhe roOts or by producing pIanl hormones or secondarymetaboliles Ihal affecl rooI physiology.

D. Sites af Organic Carbon Release in the RhizosphereThe rooI cap and areas ofaclive growlh are the primary regions where root exudalion .occurs, allhough Ihere is some exudatiori ali along lhe rool. Pearson and Parkinson(1961) assa)'ed ninhydrin-posilive subslances (which refers lO the presence af a-amino groups Ihal reacl wilh lhe ninhydrin lo produce a purple color for free a-aminogroups or a yellow clor for substiluled a-amino groups) excreled from broad bean(Vicia [aha) seedling rools and found specific regions of enhanced exudation. AIfirst, lhe seedling roais were uniformly excreling ninhydrin-positive subslances, bulwith addilional roo I growth, the region behind the rool tip was lhe primary site ofexudalion. Van Egeraat (1975) round similar results wilh pea (P. sarivum) seedlings,where the tips or both lhe main rools and lateral roots were the major areas ofexcrelion ofninhydrinposilive substances. Release of ninhydrin-posilive substancesoccurred along lhe enlire lcnglh of laleral roots as lhey deveIoped.

Pulse labeling of planls Wilh 14COZ and subsequenl radiographic examinationsof lhe rools hve greatly aided in determining the localions aI which exudalionoccurs (McDougarl'and Ro\'ira, 1970; Rovira, 1973). Determination ar lhe exuda-lionlocation has offered insighl inlo rhizosphere ecology and whelher or not micro-bial colonization of rool surfaces is enhanced aI Ihese "hol SpOIS" of C leakage. Themajor sile of C released from seminal wheal roais inlo lhe soil was lhe zone of roolelongation (Rovira, 1973). Much of lhe I"C-labeled malerial was insoluble poly-saccharide and, presumably, sloughed-off rool cap cells. As sho~'n in Figure 2,sloughed roo I cap cells and mucilaginous malerial along the rool surface can be amajor source of exudate. As lhe number of lilleral wheal rools increases, 50 doesexudation. This suggesl's lhal exudalion is either from laleral roollips or lhe regionat which the lateraIs emerge {rom the main root (McDougall and Rovira, 1970).Occasionally, fixed C can be rapidly Iranslocated and exuded by planl roots. Mc-Dougall and Rovira (1970) found discrete arcas of radioacli\'ily ai lhe apices of lhelaleral rools, 1-2 min after pulsing lhe atmosphere wilh l"COZ' bUl zones of radio-aCli\'ily along lhe primar)' roo I and some of the laleral roais were more diffuse afler2 hrs.

m. THE PHYSICOCHEMICAL ENVIRONMENT Df THERHIZOSPHERE

A. Introduclionln addition 10 C inpuI, there are severa I other planl-induced physical and chemicalalteralians of lhe' rhizosphere that affecl the composilionand aClivities of rhizo-

36 Bolton et aI.

sphere microorganisms. The rhizosphere en~ironment can have a direct inflllenceon the type and number of microorganisms that will colol)ize 'the root, survive,grow, and aUect plant growth. Understanding how microorgimisms are influencedby this environment is an important aspec! of rhizsphere microbial ecology and\ViII assist in identifying soil microbial technologies suitable for manipulating therhizosphere microflora. The reader is referred to Chapter 1 for definitions and adetailed discussion of the physicochemical factors and their influence on soil micro-bial ecology.

B. Physica! FactorsRoots generally grow in soil along the path of least resistance, such as throughpores ar old root channels, but laterlil roots may have to pen'etrate the sai! matrix(Foster, 1986). As they grow through soll, roots displace a volume of soil equivalentto their own volume. The soil displaced by root growth causes a ione of compactinaround the root in which the soil bulk density increases, and particles tend to orientthemselves with their most narrow dimensions parallel to the root (Foster andBowen, 1982). Soil minerais near .lhe root surface can also be altered, comparedwith the bulk soil, as a consequence of increased weathering and disaggregation,Amorphous iron and aluminium oxides can also accumulate,- resulting in smallerpores in the soil nar the root (Sarkar el aI., 1979). The tortuosily, ar the palh thalnutrients and water must follow lo arrive at the root, increases in the rhizospherebecause the average pore sizc or porosity and pore diameters decrcase in therhizospherewhen compared with those of bulk sai I.

The flow of water from the soil to the root creates.a water potential gradientfrom the bulk saiI to the rhizosphere and finally to the roo!. The water potential to'" hich the rhizosphere microbiota is exposed is usually much less (more negative)than in the bulk soi!. Water potentials of -2.00 MPa may 'be present in therhizosphere of mesophytic plan/s, whereas "'ater potentials as 'low as -4.00 MPamay occur inthe rhizosphere ofxerophytes (Foster and Bowen, 1982). Therefor'e,water potential varies fromthe bulk soil to the root surfaee and is a major factOrcontrolling the composition anil activity of rhizosphere microorganisms. Diurnalfluctuations in plant transpiration will create short-term (i.e., hours) fluctuationsmwater potential of the rhizosphere soil as a function of time. Successful rhizospheremicroflora must be able to withsland not only low water potentials, but also widefluctuations that occur over short periods.

C. Chernica! FaclorsPlant cells secrete not onl)' organic compounds that influence microbial growth, butthey also secrete inorganic subslances. Both organic and inorganic compounds canaffect the chemical environment of the rhizosphere, thus indirectly affecting therhizosphere microbiota. Roots can 'selectively absorb and transport ions, therebyaltering the chemical composition of the soil solution in the rhizosphere. The pH,Eh' and concentration 'of nutrients and soluble C will be different ir the rhizospherefrom those in the bulk soi!. Soluble C released from plant roots into the rhizosphereaffecls not only microbial growlh and activity by supplying a C and energy sourc~,but can increase lhe solubilityof cations by complexalin. A gentle percolation ofthe rhizosphere of maize and wbeal recovered unidentified soluble organic materi-

l\1icrobial Ecology of lhe Rhizosphere 37

aIs lha I complexed Co, Zn. and Mn (Merckx el aI., 1986). ln fieldgro .....n barley,thesoluble soil soJution coneentrations of Mn. Zn, and Cuin the rhizosphere changed.during the growing season, with thegreatest mobilization of these cations occurringearl)' in rhizosphere dev'elopmenl (Linehan et aI., 1985).

Changes in rhizosphere pH, eompared with bulk sai!, results from the relcaseof H' ar HC03- ions by roots during ion uptake, by the volution of CO2 from rootand microbial respiralion, and by the release of organic and amino acids b)' roots(Marschner, 1986). As nulrients are absorbed by roots from the soi! soJution, acorresponding ion mUSI be released by the root into the rhizosphere to mainlainionic balance. For the uptake of a cation, H+, and for the uptake of an anion "HC03', are usualIy released (Marschner, 1986). The form of plant-availabJe ~ inthe soil directly affeets rhizosphere pH, since the uptake of ammonium N results ina net excretion of H", whereas nitrate ~ uptake results in HC03- excretion. Smiley(1974) showed differences of up to 2.2 pH units in wheat rhizosphere soi! forammonium N-fertilized versus nitrate Nfertirized plants in the greenhouse and upto 1.2 unils difference in the field. Differences in rhizosphere pH were foundamong wheat varieties and plant genera.

Rhizosphere pH can also influence the activity of plant pathogens. Infection ofwinter wheat and hyphal growth by lhe takeall fungus (Gaellmannomyces gramillis)was reduced in soils lhal were ferlilized with ammonium N compared with nitrate N(Smiley, 1978a,b). Gaemallllomyces graminis was sensitive to the aeidic environ-ment of the rhizosphere caused by ammonium N fertilization (Smile)' and Cook,1973). These sludies showed lhal alleralions of lhe rhizosphere chemical environ-menl direclly affccl microbial growlh and aclion in lhe rhizosphere.

For some plants, it is not possible lo predict the effect that a mineral N formwilI have on rhizosphere pH. Rape (Brassica llapllS var. Emerald) grown aI high-rooting densities in Pdeficienl soi! wilh N supplied as N03 had a decrease inrhizosphere pH from 6.5 lO 4.1 (Grinsted el aI., 1982). The cation!anion balance oflhe plant tissue showed that more cations Ihan anions .....ere laken up during thecxperiment. It was postulaled thal efflux of H+ from lhe rool occurred to mainlainlhe ionic balance across the rool-soil interface and resulted in lhe lowering ofrhizosphere pH (Hedlcy et aI., 1982).

The saiI type in which lhe plants are grown can also influence lhe exlent towhich rhizosphere pH differs from lhe bulk soi!. Hauler and Mengel (1988) foundIhat lhe pH in a sandy soil was 1 unit lower aI the root surface lhan in the bulk soi!,whereas in a calcareous soi!, lhe pH at lhe root surface was the same as that of thebulk soil. The buffering capacilY of the calcareous soil neulralized the pH effect inlhe rhizosphere. Thus. fertilizet, planl species, and soil type ali influence rhizo-sphere pH and the pH aI which lhe rhizosphere microflora musl sur\'i\'e, grow, andfunction.

Rool and microbial respiration in lhe rhizosphere creales a microenvironmentlower in O2 conlent and redox potenliaJ lhan that found in bulk soil. Howe\'er,because of the limiled size of rhizosphere, iI has been difticull to accurately mea-sure the rhizosphere's redox polential. It has been poslulated that anaerobic microsites exist in soil, as demonstraled by denilrificalion Ihat occurs at water c0l}-tents that are Jess lhan saluralion. This phenomenon also occurs in the rhizosphere.Smith and Tiedje (1979) found pOlential denitrificaiion activity was greater in -therhizosphere of com (2. mays) , wilh denilrificalion aClivity decreasing rapidly a few

38 Bolton e! aI.

millimelers from lhe rool surface. These effecls were hypolhesized la be caused bythe ncrease n soluble organc material present in the rhizosphere, which r~sultedin an increase in microbial respiration and a subsequent decrease ~n O2 such thatnitrate N was utilized as an eleqron aceeptor. A rhizosphere mieroorganism that isable to funetion with varying eleetron acceptors (i.e., a faeultative denilrifier) maS'be better adapted to surviving and eompeting under the possible fluctuating O2concenlralions Ihal can occur n lhe rhizosphere. -

IV. MICROBIAL PRESENCE AND GROWTH lN THERHIZOSPHERE

A. InlroductionThe rhizosphere effeet refers to th~ enhanced microbial growth and populaliondensities in the rhizosphere from the increase in soluble C and nutrients, wheneompared wilh those of the bulk soil. Table 3 shows Ihat both higher populationsand a greater dversty of mcroorganisms, as determined by Iransmission eleetronmicroseopy, \Vere found doser to the plant root (Foster, 1986; Foster and Rovira,1978). There can be considerable differences in the relatil'e abundance of V/rioustaxonomie and nutritional groups of microorganisms bet\veen rhizosphere and non-rhizosphere soi! (Ta,ble 4). The ratio of the microbial population in the rhzospherelo that of the bulk soil (the RIS ratio) has been used as a measurement of microbialenhancemenl in the rhizosphere. The enhanced groll'th of microorganisms in lherhizosphere depends on microenvironmental conditions and can extend over 2 mmor more {rom the root surfaee (Foster and Bowen, 1982). .

B. Location of Microbial GrowthAlthough microbial growth is stimulated in the rhizosphere and rhizoplane, therhizoplane is not covered with a continuous layer of micraorganisms. Eleetran anddireel lighl microseopy show thal only 4-10% of the rool surface is eolonized bymicTOorganisms (Rovira et aI., 1974; Rovira, 1979). Alsa, microorgaoisms 00 therool surfaee are nol random]y dislribule. Toe slalislieaJ leeonique deveJoped byGreig-Smith (1961) to deteet patterns af vegetation in terrestrial eeosyslerns wa~

Table 3 Distinct Microbial Types Based on Ultrastructural Morphology andToraI Numbcrs aI Differenr Disrances (ram Subrerranean C/over Roors(Trifolium subterral1eum L.) Determined by Transmission Electron Microscopyof Ultrathin Sections

Distance (iLm)0-10-55-10

10-1515-20

Souree: Foster (1986).

Morphologically distinctmicrobial l)'pes

811633

120964134,13

Microbial Ecology af the Rhizosphere 39

Table 4 Wheat Rhizosphere and Nonrhizosphere Popularions of Some Major Taxonomicand Nulrilional Groups of Soil Microorganisms as Delermined by Plale Counls .

Rhizosphere Conlrol soilPopulalions (Iog CFU/g) (log CFU/g) RiS ralio'Taxonomic groups

Bacleria 9.08 7.7(J> 24.0Acrinomyceles 7.66 6.85b 6.6Fungi 6.08 5.0C!' 12.0Prolozoa 3.38 3.0C!' 2.4Microalgae 3.70 4.43' 0.2

NUlrirional groupsAmmonifiers 8.70 6.60b 125.0Gas-producing anaerobes 5.59 4.48' 13.0Anaerobes 7.08 6.78' 2.0Denilrifiers 8.10 5.oob 1260.0Aerobic ceBulose degraders 5.85 5.00' 7.0Anaerobic ceBulose degraders 3.95 3.48NSd 3.0Spore formers 5.97 5.76NS 1.6Azolobacler

40 Bolton et aI.

C. Microbial Colonizalion of Plant Roots and Growth Rates

Colonization of planl rools by microorganisms is nol well underslood. Severalfactors have been implicated as influencing colonization, including the ability of amicroorganism lo adhere lo the rool. Polysacchardes on the microbial cell surface .appear to be important for several microbial-plant associations such as crown galby AgrobaclCriwll wmcfacicns (Douglas el aI., 1982, 1985; Matthysse el aI., 1981;Thomashow el aI., 1987) and lhe nodulation of legumes by Rhizobillm specLs(Cangelosi el aI., 1987; Dazzo el aI., 1984; Leigh et aI., 1985; Smil el aI., 1987). APscl/domonas pl/lida slrain aggressively colonized kidney bean (Phascollls mlgaris)rools and was agglulinated by a glycoprolein from kidn-ey bean rools (Anderson elaI., 1988). Two transposon mutants of P. plIlida, which were not agglulinated by lheglycoprolein, colonized bean rools lo a lesser degree than the wld type. Thesemutants adhered to the root at levels.;20- 10 30-fold less than the wild t~'pe, sugge~ting Ihat glycoproten binding has a role n their attachmenllo bean roais (Andersonet aI., 1988). Piri (fimbriae), surface proteinaceous strctures ema-nating from themicrobial cell, have been mplcated in the attachmenl of Klcbsiella and En-Icrobaeler spp. (Haahtela and Korhonen, 1985; Haahtela et aI., 1985; Korhonen etaI., 1983, 1986) and for Bradyrhizobiunl japollielllll (Vesper and Bauer, 1986; Ves-per et aI., 1987) to roots. Transposon mutants of B. japolliel/nl Ihat produced twiceas many pilia!ed edIs allached at a 2.5-fold higher amount to soybean rool seg-menls and colonized I~ese roots at about twice that of the wild type (Vesper et aI.,1987).

Irrevcrsible binding of rhizobacteria to radish (Raphalll/s satil'lIs) was rapid,with one-half of lhe maximum number binding reached in 5 min followed by long-term (i.e., 25 days) colonization ofradish roots under gnotobiotie conditions (Jameset aI., 1985). Binding was no! reJa!ed to ell hydiophobicity, bu! was enhaneed inthe prescnce of divalent cations (Ca" and Mg"), whereas monovalent catiom (Na'and K') had little effeel. Jaf!les c! aI. (1985) suggcsted that eleelrostalic forces maybe responsiblc for shorl-lerm adhesion 'and for long-Ierm colonization. However,these studies were conducted in gnotobiolic s}'slems in the absence of competition.

Microorganisms predol11inaling in lhe rhizosphere are short, gram-negaliverods, including Psel/doll/onas, Flal'Obaelcril/m, and A/caligenes spp. (lcxa,nder:1977). !nitia! rooI colonizers are often associated wilh soil organic malteT. Pseudo-rnonads are frequent rhizosphere colonizers becausc the)' are associaled with or-ganic malte r, are a nUlrilionally Cliverse group, and are a group wilh a rapid growthrale (Bowen, 1980). Vancura and Kunc(I977) selectil'ely inhibited bacteria andfungi with antibioties and measured soil respiration in and outside the rhizosphere;lhe}' found lhat baclerial acti\'ily in lhe rhizospherc was greater than fungaI. Rcspi-ralion of rhizosphere soi\ \Vas dereased 6-18'7i and 20-45'1< in lhe presencc ofcycloheximide (Actidione; fungaI inhibitor) and streptornycin (bacterial inhibitor),respectivcly (Vancura and Kunc, 1977).

The abilit)' lo grow on both rools and residues was demonstrated wilh root-colonizing Pselldomonas spp. which are delelerious to wheal rool growlh (Elliottand Lynch, 1984, 1985; Fredrickson and Ellioll, 1985a). These organisms produce atoxin lhat inhibils wheal root growlh (Bolton and ElIiott, 1989; Bolton ct aI., 1989;Fredrickson and ElIiolt, 1985b). The organisms were initially isoJaled from whealrools, bUI were able lo colonize the rools of a wide variety of crop planls and crop


re~idues (Fredrickson el aI.. 1gS7). 'Howe\"er, rOOI growlh inhibitlon was somewhalplant-speciftc. The organisms were able lo maintain high popuJalions on nonsteriJe\\hear and varIe)" residues for 40 days io lhe laboratory and accounled for a majorpartion of the lotaI bacterial papulation on lhe residue. The population of a deleteri-ous Pselldol1l0llaS sp. inoculated into soi! increased 100- and lOOO-fold when soilwas amended with 0.23 and 2.3'k ground wheat straw, compared with unamendedsoi! (Fredrickson et aI.. 1987). Stroo and co-wor~ers (1988) showed that a deleteri-ous Pselldol1l0llaS sp. inoculated onto nonsterile wheat straw in the laboratoryconstituted over 80% of lhe total bacterial population. The introduced Pseudo-monas sp. also sUn'h'ed lhtoughout lhe winter in hiEh numpers on b.arley residuesin lhe field [i.e., lO colon, fo'rming units (CFU)fg strawl (Stroo et aI., 1988).Populalions of both the introduced pseudomonad and total hacleria were higher 00rcsidues under no-till management than in tilled plots. The Pseudomol1Gs sp. intro-'dueed onlo lhe barle)' residuealso colonized lhe rots of the winter wheilt erop,demonstrating that bacteria that c100

-COUnlS on l-cm segmenrs 1-2 Cm {rom root apex.SouTce: Bnw," and Ro,,;ra (1976).

42 Bolton et aI.

Bo"",'t:n (1979) calculated generation times 01'1 roots for total microorganismsand Psuedomonas spp. and found that generation timesafter 2 days were 7.5,9.1,and 6.6 hi" for the apical, fifth, and tenlh cenlimeter, respeclively. Growlh afler Ihislime was much slower, and when ali the data were eompared, growth curves similarto the cIassie sigmoidal baclerial growlh curves were obtained (Fig. 3), Bowen(1979)divided Ihese curves inlo two phases. The first phase (see a in Fig. 3) had aninitial rapid growth, referred lO as an 'inlensity factor, ieflecling the amount arrichness of C avaiJabJe for growth 01'1 lhe root surfate. The second phase' (see b Fig,3) was a periodof slower growth, referred to as a eapaeity factor, during whieh the

. supply of subslrale to 'lhe bacterial cells balanced mainlenance energy and grovhslowed, These seclions of the CUT\'es can also be regarded as exponential growth (a)and initial slationary phase (b),

Allhough the growth kinetics of bacteria in liquid eulture and 01'1 plant roOls issimilar, there is a distincI difference, The rool surface 01'1 which the bacteria aregrowing is simultaneously changing ':~nd growing, which creates spccial probJemsfor calcuJaling microbial growlh rales, There are three approachcs for reportingmicrobial populations 01'1 roots. Microbial populalions are reportcd 01'1 the basis ofper grams' dr)' weighl of root, per centimetcrs length of root, or as per surfaee areaof rool. ln each method, the plant root is growing during the experimenl. If totalrool lenglh, weighl. or surface area is used to compare microbial colonization, thenthe increase in these variables as a function of time must be taken. into consider-ation for calculating or comparing microbial gro"':th for the total plant root sy~tem,

There are three misconceptions about microbial growth in the rhizmphere(Bowen. ]980). The first is that growlh, physiology, and interactions of microorgan-

4

5 ..

O..

E~::>u.()C)O

..J

3

2

o Total BacteriaO Pseudomonas spp.

4,03,02.0

Time (days)1.0O~-~--.-~--r----"""------,-0,0

""""'-ui total bacteria and Psrudomollos spp. 01'1 Pillus radialo roots 0.5--lanted in nonsterile sol: a, ntensty factor;

Microbial Ecolog)' of the Rhizosphere

isms in laboratory media or plant solution culture are directly related to gro....1hinlhe rhizosphere. Thes methods do not mimic the soil physical and cht:mical rela-tions outlined earlier that can dramatically influence rhizosphere relationships a~dlhe resultant microbial responses. Researche.rs~ultimatelymust employ nonsterilesoil for evaluating rhizosphere technologies, tX;cause the rhizosphere is opration-ally defined as a volume of soi!. In'addition, competition for space and nutrients .....jllbe much greater in the presence of an indigenous soi! microbial population. HO\\-ever, experiments insimpler systems that are void of soi! are essential f6r under-slanding the mechanisms of rhizosphere microbial dynamics. A second misconcep-lion is that microbial growth and population dynamiCs in culture media are dirctlyapplicable lO the rhizosphere. Although the rhizosphere is an enriched environ-menl, compared with the bulk soi!, it is not comparable wlth a rich laboratorymedium. Third is the notion that natural selection will fa\'or microbial'strains lhatbenefil planl growlh. The traiis that allow an organism'to survive, compete, 'repro-duce, and function in ihe rhizosrhcre are more important Ihan any beneficialeffecls on plan! growth.

The type of root also influences microbial growth. Seminal roots of whealsupporl a larger rhizosphere population of bacteria, actinomycetes, and fungi thannodal roots (Svasilhamparam el aI., 1979): These differences were poseulaced to becauscd by analomical differences in the cortical root tissue. The seminal roolstended lo lose Iheir epidermaJ cells and some cortical cells near the rool surface.The nodal rools lended to retain thcir cortical cells, with little loss of cell material.Through cell Iysis, the seminal roots presumabfy supplied more C to the rhizo-sphere for microbial growth. Also, the numbers of bacteria, actinomycetes, andfungi in the rhizosphere decreased, whereas populations on and in ehc root eissueincreascd with plant age (Sivasithamparam et aI.. 1979).

The microbal ecology of the rhizosphere is also dependent on planl genotypes.Spring wheat lines Cadct and Rescue and two homologous chromosomal substitu-lion \ines, C-R5B and C-R5D, which ",ere identical with Cadel excert for lhesubSlitulion of chromosome 513 and 5D fTOm Rescue, respeclively. were choscn forstudy (Neal el aI., 1973). The substituton of chromosome 5B from Rescue intoCadel significaml)' altered the populalion size of lolal bacleria and several physio-Jogcal groups Df rhizospherc microorganisms (Tabie 6). These resuics demonstrarethat manipulalions of a planl gcnolype may be used lO alter lhe composition of tht'

Table 6 Total Bacteria and Selecl Ph)'siologieaJ Groups Df Mieroorganisms Present in lheRhizosphere of Spririg \\'hea! Lines

\\'heal Line Bacleria CelluJol)'tic PeclinoJylie AmyJoJytic Ammonifying(Iog CFLJ'g dr)' soil)

Cadet 8.2b' 3.7b 4.6d 6.4c 7.3eReseue 8.5a 5.la 6.8a 7.6b 8.1bCR5B' 8.5. 5.2. 6.4b 7.8a 8.3.CRSO' 8.3b 3.5b 5.8e 6.6e 7.2c

'DaIa in eaeh cofumn Ihal i. followed by a different letter is significant/y differenl (p:sO.05, n=3).bCR5B and CR5D are Cadel lines wilh Rescue chromosomes 58 and 5D subslilulions, respecli,",~'y.So"ret: Modified from l'eal el aI. (1973).

44 Bolton el aI.

rhizosphere microbiota. Interactive research between rhizosphere microbiologistsand plant breeders and geneticists -ould provide novel methods to alter microbe-plant interactions. These manipulations could provide a more thorough understand-ing of rhizosphere relations and co"uld produce desired results, such as increasedcrop production and enhanced resistance to soil-borne diseases:

The carrying capacity (microbial colonization potential) of barley roots wasdetermined by Bennelt and Lynch (1981) to be 10.7 log CFU/g dry roo!. When aPseudomonas sp., a Mycoplalla sp., and a CurlobaCleriwlI sp. were inoculatedseparately at 6.0, 8.0, or 10.0 log CFU/g dry root on gnotobiotically grown barleyplants, similar maximum population densities developed after abou14 days. Thesedata suggest thatthe absolute.number of bacteria able to colonize the rhizosphere isat least partly independent of laxonomic or physiological groupings in lhe absenceof compelilion.

Most microbial colonization, s\jTvival, and growth studies conducted in therhizosphere use dilulion-plating lecl\niques for lhe enumeralion of microbial popu-lations. But hecause some microhes strongly adhere lo lhe root, lhere is almostnever a complete removal of microorganisms from lhe rhizosphere, 'even wilhrepeated shaking. Rovira and co-workers (1974) reported t~nfold greater baclerialnumbers when eSlimated by direcl microscopy, compared wilh plale counls. Theuse of plate counls lo enumerate rhizosphere microorganisms will undereslimalcaclual numbers. O\her cultural methods may also be used lo selecl for or againslspecifie microbial phenolypes. Some of lhe rhizosphere microorganisms observedwith eleclron microscopy are morphologically uni que and include 10bed and star-shaped cel!s or eells wilh spiral arms or elongated segments (Foster, 1986). TheseceI! morphologies are usually nol detected by standard cultural methods. Thisdemonstrates that certain rhizoplane microorganisms may be obligatory rhizo-sphere colonists lhal cannot be cultured with traditionaltechniques or lhal cellularmorphology diffcrs according to whether cclls arc growing in the rhizospherc or inan artificial medium. ln studying lhe rhizosphere, iI is importanllo realize that notali of lhe microbial participanls can be idenlified or isolaled. Indeed, it has beendemonslrated lhar microorganisms can exisl in a noneulturable slate in lhe environ-ment, bul retain lheir viability (Colwell et aI., 1985). The evidence indicales thattherc may be groups of rhizosphere microorganisms about which we know nOlhing.

Oncc a rhizosphcre-competent microbe corne~ into contacl with lhe root envi-ronment, colonizalion of the roO! can occur. Organisms associatcd with soil organicmaterial are a prime source of inoculum for rhizo~phere colonization. \Vater move-ment through the soil can also be a means whereby microorganisms are lransportedto the vicinity of lhe root for rhizos'phere colonization to oceur. This mode may bcespecially imporlant as a delivcry s)'stem for rhizosphere inoculants. Water percolat-ing through a soil column increased the tntllSport of a Rhi:obilllll sp. (Breitcnbeckel aI., 1988; Madsen and Alexander, 1982) and a Pseudolllollas sp. (Madsen andAlexandcr, 1982) in laboratory experiments. An advancing wetting front also cn-hanced the transport of Bradyrhi:obiulll japolliclIIll lhrough soil, indicating Ihalunsalurated waler fiow may transport rhizosphere colonists (Breitenbeck el aI.,1988). Irrigation increased transport through soi! and colonization of the potato(SolallulIl luberosulIl) rhizosphere by a Pselldolllollas sp. in field experiments(Bahme and Schrolh, 1987). PercoJating water enhanced lhe co!onizalion of lhe pea(Pisum salil'lIIn) rhizosphere aI grealer depths by added bacterial (Chao el aI.,


1986; Liddell and Parke. 1989) and fungaI (Chao el aI., 1986) rhizosphere colonists.Although useful for screening purposes, the use of sieved soil to investigate bacte-ria! transport and colonization of the rhizosphere in the field may lead to erroneousconclusions. Smith and co-workers (1985) demonstrated that transport of Escher-ichia coli by percolating water was grealer in intact soil cores than in sieved soilpacked in columos. The relative behaviors of the added bacterium and a 0- tracersuggested Ihat bacterial transporl in the intacI soil cores occurred through soilmacropores. avoiding adsorption to the soil matrix and transporl through smallerpores with a more tortuous path. Sie\'ing the soil destroyed the macroporositypresent in the field. Macropores are Iess resistanl lo root penelration and wouldmost likely allow bOIh an increased root densily in the macropore and the fio\\' ofperco!ating water containing the bacleria to come in conlact with the roo!.

Root growth transporls rhizosphere bacteria verticaJly (Bashan and Levanony,1987; Bolton et aI.. 1991a; Chao et aI., 1986; Howie et aI., 1987; Fredrickson et aI.,1989; Madsen and Alexander. 1982; Tre\'ors et aI., 1990; Weller, 1984) and 1alerally(Bashan and Levony, 1987) along lhe rool system in the soil. 11 has been hypothe-sized that J11O\ement of bacteria in the soil and lo other sections of lhe root iscallsed by downward water flow (Chao et aI., 1986; Parke et aI., 1986) and bybacterial attachment to the root and movemenl as lhe rooI grows (Howie el aI.,1987). ln an experimental system developed by Howie and associales (1987). perco-laling water was virtllal\y eliminated, with water movement occurring only towardthe root. Bacterial movement on the roots occllrred as a function of time, withnonmotile bacterial mulanls colonizing the rool to the sarne extent as the wild type.ln a scraralc study. no rclation was found bel"'een mOlility and root or seedeolonization by 32 bacterial slrains representing Psel/domonas pI/lida, P. fillo-resam. and Serriltia spp. (Scher et aI., 1988). Allhough not conclusive, thesestlldies suggesl that bacterial motility may not be a major factor influencing hacte-rial colonizarion of the roo!. Soil type can abo influence root-mediated microhialtransport in soil (Trevors el aI., 1990). Downward movement of a genetically cngi-neered P. jlllorescells occurred only when whcal roots were present and whenvertical waler fiow was ahsent. However. when percolating ",ater was present,whcal roaIs onl)' slightly enhanced P. fil/orl'scells movemenl in a loamy sand. ln aloam. howc'cr. transpor! was enhanced. cven in lhe presence of percolating water,when compared wilh unplanted soil (Trcvors ct aI., 1990).

D. Microbial Biomass in the Rhizosphere

The abilit)' to quanlitatively determine the mass of microorganisms present in therhizosphere is useflll for rhizospherc ecoJogy and nutrient cycling research. A stan-dard techniqlle for measuring the mass of microorganisms (micrograms of biomassC per gram soil) is \he chloroform fumigation \echnique (Jenkimon and powlson,1976). This technique relies upon Iysing the majority of the sail microorganismswith chloroform vapor and measuring respiration during the mineralization of thereleased soluble compounds when the chloroform is removed. This technique hasbeen used to quantify nutrienl content of the soil microbial biomass in therhizosphere including C (Helal and Sauerheck, 1986; Merckx et aI., 1987; Merckxand Martin. 1987; Norton et aI., 1990) and N (Jackson et aI., 1989; Schimel et aI.,1989). This tcchnique also has also been used for estimating P (Brookes et aI., 1982,

46 Bolton et aI.

1984; Hedley and Stewart, 1982; McLaughlin and Alston, 1985) and S (Chapman,1987; Saggar et aI., 1981) in bulk soi! and may have rhizosphere applications.However, this procedure may give unreliable results for estimating the microbialbiomass in the rhizosphere and rhizoplane in close association with liviog root~because of the disruption of plant cells and the release of soll1ble C and becausebacteria encased in the mucigel may survive the chlorofOrm fumigation (Martin andFoster, 1985). Errors also can result in systems with high soluble C contents, whichJenkinson and Powlson (1976) mention. .

The rate of tritiated thymidine incorporatioo was used to quantify growth ratesof rhizosphere bacte~ia (Christensen et aI., 1989). A fluorescent Pselldomonas sp.was grown gnotobiotically 2-4 days in a sugar beet (Sela vulgaris) rhizosphere.Tritiated thymidine was added and the rate of [3H]thymidine incorporation ioto themicrobial biomass was determined and used to calculate a growth rate. The specificgrowth rates obtained by this method)~'ere 4. 710g CFU hr-

'cm-! root or a geoeration

time of 106 hr, which is eomparable iith other literature values. No comparison withcOO"entional growth rate measurements was eonducted. This technique has theadvantage of providing a measurement of in vivo growth rates without relyiog on theculturability of the rhizosphere microflora. Also, short incubation periods are used(i.e., 30 min) and ooly prokaryotic DNA is labeled. However, there are limit'ltions tothis technique. First, we do not know the efficiency of extracting DNA from therhizosphere and purifying it to quantify the incorporated thymidine. Curreot re-search on the fate and effeets of genetically eogineered microorganisms has madegreat strides in improving the efficiency of DNA extraction from enviroomentalsamples (see Chapo 5; Holben et aI., 1988; Ogram et aI., 1987; Steffan et aI., 1988).These efforts should provide improved DNA extraetion efficiencies. Second, a con-version factor based on four constants is required to ealculate growth rates. Christen-sen and colleagues (1989) used a single organism and literature values to com'ert[3H]thymidine incorporation into a specific growth rate. To do this, the thymidinebase eontent of the DNA, the genomic size (i.e., the DNA content) per ceI!, and thespecific activity of the thymidine incorporated into lhe DNA must be known. Bacte-rial synthesis of thymidine, a thymidine pool in the rhizosphere, or poor thymidineuptake kinetics will lead to isotope pool dilution and a low estimate of the growlhrale. Finally, the technique provides an overall growth rate for lhe enlire rhizosphereand offers no information on spatial dislribulions or individual groups of organisms.Dcspite its limitations, this technique could be a useful tool for determining in vivogrowth and activity of rhizosphere microorganisms and could aid in identifying com-petitive strains for biotechnological applications.

E. Enzymes in lhe Rhizosphere

A wide range of enzymes of both plant and microbial origin may be present in lherhizosphere, including oxidoreductases, hydrolases,lyases, and transferases (Lynch,1990b). Enzymes from the rhizosphere microflora are the main cOneero here. En-zymes catalyze the breakdown of orgamc materiais (e.g., .cellulases, dehydroge-nases), fertilizers (e.g., ureases), and organic nutrients to plant-available forms (e.g.,phosphatases, sulfatases). The competitiveness of a rhizosphere colonisl may beenhanced by its ability to enzymatically deeomposeroot cells and soluble C exudate.

Microbial Ecology of the ~hizosphere 47

Conversely, pIam growth may benefit by a rhizosphere microflora that enzymaticallyenhances the cOJJversion ofnutrients from the organic to inorganic form or from theunavailable form to a fonn available for planl growlh. 11 has been directlv demon-straled that enzymes from individual rhizosphere bacteria can be delec'led. Cal.cinated attapulgite, a nonswelling e1ay mineral, was used by Martin and Foster (1985)to develop a model rhizosphere for wheat. Acid phosphatase and calaJase enzymeswere detected by ultracytochemical tests in individual rhizosphere bacteria. Develop-ments in immunocytochemical techniques should alio\\' demonstration and possiblylocalion of rhizosphere enzymes by boIh transmission and eJectron microscopy(Lynch, 1990b).

II is assumed that enzyme activity is generally greater in lhe rhizosphere than inbulk soil because of the larger microbial populalion and lhe presence of roots. Neal(1973) compared phosphatase activily in unplanted soi! with soil planted withgrasses and forbs representative of dominant, codominanl, increaser, or invaderspecies. Only invader plants had a significant increase in phosphalase activity whencompared wilh .the control soi!. Whelher the increase in phosphalase aClivity was aresult of planl or soil microbial activiiy, or both. was not determined. Therhizosphere of rape (Brassica Ilapus) planted in a sandy loam soi! had a phosphalaseaClivily lenfold higher than the unplanted soil afler 35 days (Hedley et a!., 1982).Speir el aI., (1980) compared sulfatase, urease, and prolcinase in soil planled withryegrass with unplanled soi!. ln general, the sulfatase and urease acti\'ity in theplanted soil did not decrease during 5 monlhs, whereas aClivily in the unplanted soi!did decrease. Proleinase activity was highly variable. II was hypOlhesized thaltheplanted soil continued to release enzymes from plant and microbial origino whereaslhe unplanted soi! suffered denaturation of enzyme aClivity during the study penod.Bccause Ihere is a decrease in microbial growlh as distance from the root increases,a gradalion in enzyme activity in the rhizosphere \\'ould also be expecled. Thephosphalase aClivity in lhe inner and outer rhizosphere of maize, barley, and wheatwas studied by Burns (1985). The outer rhizosphere (soi! removed from rooIs withgenlle agilalion) always had phosphalase aclivilies lower than the inner rhizosphere(soi! removed from roots by vigorous agitation), demonstrating enhanced enzymeaClivity eIoser lo lhe root. Also, the rhizosphere of soybean grown in a sandy loamsoil had higher dehydrogenase, urease, and phosphatase aClivities than an un-planted soil after 40 days (Reddy el aI., 1987)

ln a hydroponic system, Gould et aI. (1979) delennined lhe relative contribu-tion of the plant BOll/e/olla gracilis, an inoculated Pselldomoflas sp., and coinocu-lalion wilh lhe Pseudomollas sp. and an ameba (Acallt/lamoeba sp.) to tolalphosphatase activity. The presence of the bacteria or the bacteria and amebaeincreased the acid phosphatase aClivily in Solulion and aIso increased root phospha-lase activity. These results suggest that rhizosphere microorganisms not only con-lribule enzyme aClivity in the rhizosphere, but aIso slimulale enzyme production byintacl rools. ManipuIalions of rhizosphere microflora lo enhance the reJease of abeneficial enzyme would be one approach to enhance plant growlh. Also, theinoculalion of the rhizosphere with a microbe producing Jarge quantities of anenzyme of interest (e.g., an organic contaminanl.degrading enzyme. see Sec.VII.C) is anolher example of how the rhizosphere may be manipulated to benefi!plant growlh.

48 BoIton et aI.

V. MICROBIAL EFFECTS ON PLANTS

Rhizosphere microorganisms are often of interest because the)' can have beneficial(e.g., N2 fixation, mycorrhizae. biocontrol of plant pathogens, production ofgrowth-promoting substances) or detrimental (e .g., disease, d~leterious rhizobac-teria, immobilization of plant nutrients) effects on plant growth. It is necessar)' tounderstand the mechanisms by which rhizosphere microorganisms influence plantgrowth, to develop lechnologies that enhance their beneficial activilies and reducedetrimentaJ aclivities to crop plants. The converse is true for weed species (Le.;take advantage af thedetrimental activities of rhizosphere microorganisms to limitgrowth of or kill plants; (see Sec. VII.B). It is not the purpose of this chapter to _present a review of Ihese broad research areas. The reader is referred lo othcrchaplers in this book for more delailed information on N2 fixation (sec Chaps. 6 and9), vesicular-arbuscuJar m)'corrhizae (see Chapo 13), ectomycorrhizae (see Chapo14), bioconlroJ of plant pathogens "'th fungi (see Chapo I I) and rhizobacteria (secChapo 10), production of growth-promoting sllbstances (see Chapo 12), disease (sccChapo 7), and immobilization of planl nutrients (sec Chapo 3).

VI. MICROBIAL INTERACTlONS

Microorganisms inoculaled into the rhizosphere can have positive (commensalism,mutualism, protocooperation), negative (amensalism, competition. parasitismo pre-dation), and neutral (neutralsm) interactions with the various members of therhizosphere microbial community. For a more complete definition of these variousinteractions, the reader is referred to Chapter 1. The interactlons among the vari-ous microorganisms in lhe rhizosphcre not only can affcct the specific organisms ofconcern, but also other microorganisms and the plan!. Thc discipline of biologicalcontroJ of plant palhogens is founded on lhe principIes of microbial compelition,amensalism. parasitism, or predation, ar a combination thereof. These interactionsare extremei)' important in the ecological stud)' of the rhizosphere, yet they areamong the least well-understood areas in rhiiosphere ecology (Bowen, 1980). Bac-teria occupy less than 10':'~ of the root surface. with most of this colonizationoccurring at regiom of C e"xUdation and a favorablc microen\'ironment, wbich arelikely zones of enhanced activity alld interactions.

The reader is referred to more in-dcpth discussions of microbial interactions inthe rhizosphere for more detailed information (Bazin et aI., 1990; Curl and Harper,1990; Curi and Truelovc, 1986). For this charter. IWO model systerns are discussedas exarnples of lhe potentiaJ fate and dfects of an inoculated or inlroduced microor-ganism on the nalive microflora or on coinoclllated slrains. One model s)'stem thalis useful for studying microbial interactions in the rhizosphcre and cffects on plantgrowth is the Rhizobilll1l-legume s)'stem. Rhizosphere microflora lhat do not affeetlegume root growth can have positive (Bolton et aI., 1990; Burns elal., 1981;Grimes and Mount, 1984; Li and Alexander. 1988), negative (Fuhrmann and\Vollum, 1989), or neutral effects (Bollon et aI., 1990; Grimes and Mounl, 1984;Smith and Miller, 1974) on legume nodulation by Rhizobilll1l spp. and on sllbse-quent plant growth. Coinoculation ofAzotobactcr.\'ill/alldii and Rllizobilll1l spp.increased the numbers of nodules on the roots of soybean (C/yeille l1Ia.l") , pea(Viglla ullguiclIlala), and dover (Trifolilll1l repell5) (Burns et aI.. 1981). Increased

Jl,ficrobial Ecologyof lhe Rhizosphere 49

nodulation of soybean alsobccurred in thefield. lt was hypdthesized that this \-.;a,sdue to the produetion of a nonexcnitable protein. Field and greenhouse data indi-cated that inereased nodulation ofbeans (Phaseolus vII/garis) by R. phaseoli oc-curred with coinoculation of Pselldomonas plltda (Grimes and Mount, 1984). How-ever, bean yield and shoot weight were not significantly affeeted by coinoeulation,demonstrating that inereasing nodule number or infection by Rhizobillm spp.'maynot affect plant productivity. This was also demonstraled by Bollon et aI. (1990).They found that increased nodulation Df pea (Pisul/l saril'UfIl), as demonslrated byan increased in nodule number, occurred with coinoculalion Df R. /lIgllminosarwnand a deleterious rhizobacterial PsclIdomonas sp. However, nodule and shoot dr)'weghts were the sarne whether or not the PSClldofllonas sp. was eoinoeulated. Liand Alexander (1988) took a different approach to enhanee eolonization andnodulation by rhizobia. Antibiotic-producing bacteria, which were resistant to \he .antibiotic, were coinoculated with a Rhzobiwll sp. onto legume roots. Coloniza-tion and nodulation of the alfalfa and soybean rhizospheres were enhanced. Li andAlexander (1988) hypothesized that suppression of Rhizobilll/l spp. antagonisis bythe antibiotie produced by on~ of the strains was respon'sible for the enhancednodulation. Coinoculation Df legumes with both Rhi::ohiufIl spp. and antibiotic-produeing microorganisms is an area worthy of further study bccause of its potentialfor allering microbial compelition in the rhizosphere. Fulirmann and Wollum(l9S9) dttected a decrease iI) lhe number of taproot nodules and in seedlillg emer-gente of soybean (G. 'max)nd altcred nodulation competition among B. japonCllfllstrains when coinoculatcd wilh PselJdomolJas spp. lron availabilty was implicatedas a factor involvcd in the plant-B. japonCllm-rhizosphere microflora interactions.

Intact soil core microcosms have also been used as a model system for studyingmicrobial interactions in the rhizosphere. The soil core microcosm design devel-oped by Van Voris (1988) for e\aluating the fate and effects of chemieals wasadapted to study the fate and effccts of genetieally engineercd microorganisms

(GE~ls) (Bentjen.et aI., 1989; Frcdrickson ct aI., 1989) and othcr microorganisms(Bollon ct aI., 1991a,b) inlcndcd for release to the cnvironmenl. Soil core micro-cosms are a viablc option for obtaining preliminary information on the fate andeffeels of introduced strains, inc!uding GE:'-1s. because tests and microorganismscan bc colltained in the laboratory (Cairns and Prall, 1986; Fredrickson elal., 1990;Omenn, 1986; Strauss ct aI.. 1986, Trcvors. 1988). The intact soil core microcosmrepresenls an intacl sample of lhe environm.:-nt and is uscful for delermining the(ate and c((eets o(an intrO

50 Bolton et aI.

(Bolton et aI., 1991a), effects on soil microbial community struclure and activity(Bolton el aI., 1991 b), and comparabilily of rnicrocosm results ""ith field data(Bolton et aI., 1991a,b). RCl was inoculaled ioto the surface 15 cm of soil, andwinler wheat ""as planted. More than 80% of lhe tolal pseudomonad population onthe wheat rhizoplane was the introduced slrain ai the lhree-Ieaf stage of ""heatgrowch (Bolton eC a!., 1991b). The proportion of fluorescenl pseudomonads on lherhizoplane decreased from 24 to less lhan 1%' because of RCl inoculation. Theinlroduced slrain was able to out-compete a significanl portion of the native soi!pseudomonads on lhe rhizoplane and also allered the bacterial composilion of therhizoplane by decreasing lhe percenlage of fluorescenl pseudomonads. The popula-lion of RCl on lhe wheal rhizoplane was calculated to be approximately 40% of thetotal aerobic helerotrophs, indicating that RCl compeled favorably with otherheterotrophic bacleria. Inoculalion wilh RCl also decreased species diversity onche rhizoplane, as measured by species evenness and equilability indices. This wasbecause the introduced strain was prfponderant on the rhizoplane and the distribu-tion of individuaIs among the various species was skewed. This sludy demonstratesthat laboratar)' cullured organisms introduced to the rhizosphere can competefavorabl)' with the native soil microflora for colonization of lhe rhizosphere.

The effecl of the introduced slrain Psclldo/J7onos ReI on various microbialpopulations on the rhizoplane decreased as the planl aged (Bolton el a!., 1991 b).Onl)' 42% of the total pseudomonads and 30/


increasing their populations (Le., establishment of inocula). they may becomeeffective weed conlrol ageors. The use of rhizosphere microorganisms as biologicalconlrol agents to prevent the infection of plants with soil-borne pathogens is an a~eathal has received considerable attenlion and is a technolJgy that is curreritly being.applied to some extenl (Cook, 1985). The use of microorganisms, either pathogensor nonparasitic plant palhogens (exopathogens), has potential applications for thebiological control of weeds (Cherringlon and ElIiott, 1987). Here. the focus is noton bioJogical co'ntrol of plant pathogens, but ralher, on the control of weedy plantsby using microorganisms. Research emphasis has to shift from inhibiling or control-Eng the growth and action of planl pathogens ar deleterious rhizobacleria to pro-moling their rhizosphere colonization, growth, survival, and the expression of theirdeleterious traits. A major advantage of using microorganisms for weed control is'lhal lhey can be considerably more seleclive than herbicides. For example,CherringlOn and ElIiott (1987) isolated several raot-caonizing Pseudomonas spp.from the rhizosphere af downy brome (Bromus tectorum), which severely reducedlhe root growth of this weed, bUl nol of that winter wheat. Also, many soil-borneplanl pathogens are very specific and aflen promole disease af only a single speciesor even cultivar.

Research on lhe biocontrol of weeds mUSl firsl identify candidate microorgan-isms wilh lhe necessary attributes for rhizosphere compelence. These attributesshould include aggressive rhizosphere colonizalion, if they are lo survive and grow.Also, they musl express the inhibilory lrail when the planl is mosl susceptible.Finally, uniquc delivcry syslems mighl he needed to permil lhe organsm to be usedeffeclively at various 'slages of wced growth and developmenl. The microbial strainsmusl also be evalualcd for effecliveness and for lheir effecls on nontargel plantsand major beneficial microbial species.

A variely of potenlialIy phylopalhogenic bacleria and fungican be readilyisolaled from the rools of several different weeds (Kremer et aI.. 1990). It is notalways evident that these organisms are normal components of the rhizospheremicroflora because their effects are often dampenedby competition with nonpatho-genic bacteria and because lhey commonly are present in low numbers. As witholher rhizosphere microbial technologies, the ability of a specific microarganism to'effectively compele is likely to be a key faclor in promating its efficacy. Bacteriaisolaled from lhe rhizosphere of seven economicalIy important weed species werepredominantly gramnegalive (>99'it of alI isolates), consisting of fluorescent andolher pseudomonads, Erll'inia herbicola, Flal'obacterillm spp., and Acaligcnes spp.(Kremer et aI., 1990). These species have also been identified as the dominantmicrobial types present in the rhizosphere of crop plants (Bowen, 1980; Rouatt andKatznelson, 1961). Rclatively high praportions (i.e., 35-65%) of the rhizobacterialisolates from weed species could inhibit seedling growth of the pIant from whichlhey were initially isolated in growth pouch and pot assays (Kremer et aI., 1990).This demonstrates lhat numerous naturally occurring microbes cxist in the rhizo-sphere of weeds lhal can have potenlially detrimental effects on their growth.Krcmcr and co-workers (1990) used both a microbial assay (Gasson, 1980) andseedling bioassays to determine if microbial antimclaboliles inhibitory to weedgrowth were being produced. Conflicting results were sometimes oblained becausethe inhibilion of the indicator organism (E. coli) and stimulalion of weed seedlinggrowlh resulted from the sarne organismo The ulilization ofmicrobial assays is time-

52 Bollon el aI.

and labor-efficient, yet SpOI screening of mcrobes isolaled duringseedling bio-assays musl slill be conducled lO ensure unambiguous results and lo improve lhechance Ihal useful slrains are nol missed.

Approximalely 60 and 75% of lhe bacleria isolaled from lhe roots of severalweeds produced antibiolics effeclive againsl a baclerium and a fungus, respeclively(Kremer el aI., 1990). The addilion of Fe",J reduced lhe inhibilory effecl of thesemicrobial assays, implying Ihal anlimicrobal aClivity may have been caused by asiderophore or thal an antibiol produced was regulaled by Fe concentration.From lhese results, ii was suggesled Ihal lhe rhizosphere microbial populalioncould be manipulated in favor of deleterious rhizobacleria (Kremer el aI., 1990).These investigalors suggesled Ihal successful candidales musl be aggressive roolcolonizers, produce specifie phytotoxins against lhe hosl and not nonlargel planls,be able lo compele wilh other rhizo:;phere colonists, and be able lo synlhesize orutilize other baclerial siderophores.

Anolher sludy investigaled lhe biological conlrol of downy brome by manipulal-ing lhe rhizosphere (Kennedy el aI., 19YI). Downy brome is

Microbial Ecology of lhe Rhizosphere

C. Enhanced Drganic Contaminant Degradation in the Rhizosphere

53

Because of lhe enhanced aClivily and growth of microorganisms in the rhizosphere,there is a considerable potential for enhancing the biodegradalion of organic con-taminants present n soil near the plant roO!. Manipulaton of lhe plant communityor of the associated rhizosphere microflora, or both. has potential as a relativelypassive and inexpensh'e technology for remediating soil contaminated with organ-ics. Ahhough manipulation of the rhizosphere specifically for bioremediation .hasnot been developed as a technology. the results from several studies encouragefunher nvestigation:

Hsu and Bartha (J 979) demonstratcd enhanced minera!ization of two or-ganophosphate insecticides (Diazinon and parathion) in the rhizosphere of bushbean (Phaseolus mlgaris). Increases of approximately S and 10% n the mineraliza-tion of [1'C1Diazinan and ["C)parathion. rcspectively. were found in soil with a bushbean rhizosphere. The viable counts (lf heterotrophic microorganisms in theplanted and control soils werc 'similar, although therc was no distinction betweenbulk and rhizosphere soil. These resll1ts sllggest that the plani enhancel mineraliza-tion of the pesticides eilher through a general enhancemcnt in the activity ofthc soi!microbial community or bccausc of a sc1cction for a spccific microbial communitythat was C3fli1blc of degrading rhcsc pcsticidcs (Hsu ,md Banha. 1979).

The rate of ring c1eavage of parathion was also cnhanccd in the rhizosphere ofrice (Oryza sfII\'a cv. Supriya) compared with unplantcd soil (Reddy and Sethuna-than. 1983). Flooding of lInfllamed sail had little cffect on mineralization, with lessthan 5(i ofthe 1"Clparathion being evolved as "CD: during 15 days.ln soil plantedto rice, lInflocidcd sail e,"oh'ed 97< of the ["Clparathion as "CD!. whereas floodingthe soil resulted in a "CO! evolution of 22%. This latter increase in parathionmineralization in the rice rhizosphere W:IS hypothesized to be caused by the en-hanced growlh of this ri ce variely in flO(llkd soi!. Both root and shoot biomasseswere threcfold higher afler 15 days af grawth, when compared with those undernonfloocled conditions. ln tum. this increase in the biomass af the rooIs and shoolsma)' havc cnhanced lhe rhizosphcrc microtlora.

The extel1t of dcgradation of severa! polycyclic aromatic hydrocarbons (PAHs)lI'ilS aceJerated in soil rlantcd wilh lkcp-roc)(cd prairic grasses over that af anlInplanted soil (Aprill and Sims. 1990). The PAHs in this study exhibited no down-ward mobility in soil cores after II-l days. The greater redllction in extractability ofthe PAHs from lhe planted soil was hypothesized lO be caused by their enhanceddegnldation in the rhizosphere or by Iheir increased incorporalion into hllmic mate-rial. The PAHs were not l'C-labelcd. IhllS measllrements of their mineralization wasnot possiblc.

More reccnlly. the biodegradation of trichloroethylene (TCE) was shown to besignificanlly higher in rhizosphere soi!. clmpared with nonrhizosphere sai! (Waltonand Anderson. 1990). Soil was eollcctcd from a former TCE disposal site. Therhizosflhere soil was colleeled from the rooting. zone of the four dominant plantsflecics prcsenr ar rhe disposal site. and nonrhizosphere soi! was collected fromnon"egetated arcas within and outside the dispasal site. The TCE was lost morequickJy from lhe headspace of rhizosphcre soi! slurries than from nonrhizospheresoils. When ["C)TCE was added to rhizosphere and nonrhizosphere soils, a threefold

54 80110n el aI.

increase in l'C02 occurred after 30 days in the rhizosphere soil compared with thecontrol. Therhizosphere soils had a four- to sevenfold higher microbial biomass thanthe nonrhizosphere soils. 1t was hypothesized that the increased biomass in therhizosphere soi! enhanced TCE mineralization. AIl mechanisms of TCE degradationyel known are forluitous or comelabolic reaclions. No pIanIs were grown in lhe soilsduring the TCE mineralizalion experiments, precluding plant uplake of lhe organicagen!. However, iI is unclear whelher the TCE mineralization noled in lhe soilsample would also occur in lhe field with activeIy growing planls. An obvious neXlslep would be to determine TCE fate in lhe rhizosphere during active pIam groilthboth in conlrolled laboratory condilions and in lhe field.

An addilional advantage of using planl-microorganism combinations for lheremedialion of contaminated soils is lhat lranspiration from lhe plant will enhancelhe movemenl of soluble contamin~.nls lO lhe planl rool where lhey can be de-graded by rhizosphere microflora. lso, lhe planl root may be useful as a deliverysystem lo lranspor! contaminant-degrading microorganisms to lhe compound ofinterest WilhoUl disturbing or mixing lhe soi!. Seed coaling or inoculalion of secd-ling rOaIs may allow an added conlaminanl-degrading slrain lo grow along wilh lherool and conlacl an increasing volume of sai!. One limitalion lo lhis approach is lhatless than 10% of the surface area of the rool is typical\y covered by microorganisms;therefore, some of the contaminant may nol be degraded before it is laken up bylhe plan!. For volalile organic solvenls such as TCE, plant lranslocalion may actu-ally resuIt in release of TCE lo lhe atmosphere, much in the sarne way lhal air-stripping is used lo lransfer volatile organic compounds from an aqueous (i.e.,groundwaler) lo a gaseous (i.e., almosphere) phase. AIso, lhis lechnology wouldprobably be bel ter using plants wilh high-rooling densities, such as grasses.

VIII. SUMMARY

A. Research Needs in Rhizosphere Microbial Ecology

The rhizosphere is a dynamic microbial niche. The success of rhizosphcre soilmicrobial lechnologies will 1epend on isolaling and understanding the mechanismsby which microorganisms influence plant growlh as lI'ell as a basic underslanding ofthe traits lhat eonslitute a compelitive rhizosphere colonizer. A microbial isolalelhal carries out a useful process (\f function in lhe rhizosphere will be useless unlessthe organisms can successfully compele in the field and express the desired Irai!.Inlegraled muItidisciplinary research is needed to undersland lhe complex interac-lions of biotic, chemical, and physical processes that inleract to define lhe environ-ment at lhe root surfaee. There are great varialions in lhe environmenlal conditionsalong lhe root surface and radially from lhe rool surface into lhe bulk soi!.Microbial-plant root interactions must be sludied at smallcr scales lo undcrslandthe numerous concunenl processes in the rhizosphere. There is currently a Jack ofinformalion on the dislribution of microorganisms on root surfaces, lhe ir relalivemelabolic activities, and now Ihey inleract la affeet plant growth and lhe growlhand funclion of other microorganisms. Model s)'stems must be carefully chosen andexperimenls designed lo ansll"er specific fundamental questions on why certainmicroorganisms are effective rhizosphere competitors. Useful traits for a compeli->"'ue rhizosphere colonize r probably include a rapid gro\\'lh rate, lhe abilily lo move

Microbial Ecology of lhe R-hizosphere 55

wilh lhe rool as ii grows, lhe abilily lo exhibil some form of anlibiosis againsl Olhercompelilors, and resislance lO inhibilion by olher pOlenlial rool colonizers. Theabililies lo ulilize a unique organic rool exudale or lo selectively increase lherelease of organic C from rools are other traits that may be imporlant.

Genetic manipulalion of the plant or rhizosphere microorganisms is a po" erful1001, wilh considerable pOlential for exploring lhe microbial factors that influenceIheir ability lo effeclively colonize and funclion in lhe rhizosphere. For example,Tomashaw et a!. (l990) demonslraled lhe in vivo produclion of antibiolics bv a P.jluore5cell5 slrain Ihal suppressed lhe rool disease lake-all. ln Ihis sludy, "non-phenazine-producing mulanls and lhe phenazine-producing wild Iype were used 10show lhal phenazine was produced in lhe rhizosphere and was effeclive in reducingthe disease. Such an approach may be useful for addressing lhe role of anlibiolicproduction in microbial compelilion in lhe rhizosphere. Transposon mUlagenesishas been used lo obtain mulanls of a planl growlh-promoting P. jluore5cens Ihalwere characterized as Agg-, lhe inabilily lo be aggluli,nated by a rool surface-associatcd glycoprolein. The Agg- mulants exhibited significant!y lower leveIs ofrool binding (Anderson el a!., 1988) and colonizalion (Tari and Anderson, 1988)Ihan did lhe parenl slrain. Once trails have been idcnlified Ihal influence thepOlcntial of an organism lo colonize lhe rhizosphere, lhe organisms mighl be geneti-cally manipulaled to enhance these abilities.

Tradilional rcsearch in rhizosphere microbial ecology has focused on increasingIhc producti"ity of crop planls. This research has ob"ious me ri Is and should con-tinue. New approaches to weed conlrol, which use rhizosphere microorganisms andenhanced organic contaminanl degradation in the rhizosphere, have the potentialto become useful technologies for soh'ing several environmental problems.

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